Polymer Nanoparticles For Nanomedicines

ChristineVauthier GillesPonchel
Editors
Polymer
Nanoparticles for
Nanomedicines
A Guide for their Design, Preparation
and Development
Polymer Nanoparticles for Nanomedicines
Christine Vauthier Gilles Ponchel

Editors
Polymer Nanoparticles
for Nanomedicines
A Guide for their Design, Preparation
and Development
123
Editors
Christine Vauthier Gilles Ponchel
Institut Galien Paris Sud, Faculty of Institut Galien Paris Sud, Faculty of
Pharmacy Pharmacy
CNRS, University of Paris-Sud, University CNRS, University of Paris-Sud, University
Paris Saclay Paris Saclay
92296, Chtenay-Malabry Cedex 92296, Chtenay-Malabry Cedex
France France
ISBN 978-3-319-41419-5 ISBN 978-3-319-41421-8 (eBook)

DOI 10.1007/978-3-319-41421-8
Library of Congress Control Number: 2016951658
Springer International Publishing Switzerland 2016

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Foreword
Polymers are macromolecules composed of many repeated subunits of different

nature, leading to a broad range of compositions and properties. Both synthetic and
natural polymers play a major role in the life sciences. Whereas natural polymers
(nucleic acids, proteins, peptides) are the building blocks of biological structures
and functions and are the support of genetic and epigenetic events, the polymer-
ization of monomers through various modern synthetic routes (e.g., controlled
anionic or radical polymerization, ring-opening polymerization, etc.) enables the
design of synthetic polymers with unique physicochemical properties, including
robustness, viscoelasticity, and a tendency to form glasses and semicrystalline
structures rather than crystals. They may be combined to form tailor-made
supramolecular architectures. The versatility of these polymer structures and the
resulting properties offer many applications in the medical and pharmaceutical
elds. Smart polymers, designed to undergo reversible physical or chemical
changes in response to environmental stimuli (such as temperature, light, magnetic
or electric eld, pH, ionic strength or enzymes) also hold great promise as drug
delivery systems, tissue engineering scaffolds, cell culture supports, bioseparation
devices, sensors, and even actuators systems. Because of their extraordinary ver-
satility, there is an increased interest to use polymers, either natural or synthetic, as
transporter material for the design of nanomedicines. The encapsulation of a drug
into polymer-based nanoparticles allows it, indeed, to protect the drug from
degradation/metabolization; to defend healthy cells and tissues from drugs even-
tual toxicity; to improve drug bioavailability at the site of action (i.e., diseased
cells); and to allow better intracellular penetration and trafcking for drugs that
cannot cross the cell membrane. The ultimate goal is to increase the drug thera-
peutic index by improving the pharmacological efcacy while also reducing its
toxicity. Of course, the design of polymers for the construction of nanodevices is
key to making safe and efcient nanomedicines. When intravenous administration
is considered, the use of biodegradable polymers is mandatory to avoid intracellular
polymer overloading and thesaurismosis. The possibility to control the degradation
kinetics of a drug subsequently allows tailoring the drug release according to its
v
vi Foreword
therapeutic aim. The surface properties of the polymer when formulated as

nanoparticles is another important issue to monitor and avoid excessive comple-
ment activation, protein aggregation or thromboembolic event after intravenous
infusion. Therefore, surface functionalization of nanoparticles should help to hinder
such events or, to better address the nanomedicine in a very specic way toward the
targeted cells by decoration with specic ligands. Surface functionalization of
polymer-based nanoparticles may also permit the bioadhesion along epitheliums or
endotheliums or even the translocation through biological barriers, including the
bloodbrain barrier. Other approaches, albeit less advanced, include the develop-
ment of polymer nanoparticles combining both therapeutic and imaging function-
alities and even nanodevices containing two or more drugs for synergistic
pharmacological efcacy.
The book edited by Drs. Vauthier and Ponchel, Polymer Nanoparticles for
Nanomedicines: A Guide for their Design, Preparation and Development,
represents a crucial and comprehensive work of information with highly advanced
research about the construction of polymer nanoparticles. The logical succession
of the different chapters runs in the following way.
Part I is devoted to the different methods for manufacturing nanoparticles with
clear explanations about the physicochemical principles allowing their formation.
Nanoparticles may be built using various preparation methodologies. For instance,
the so-called nanoprecipitation technique based on the Ouzo effect, the flash
nanoprecipitation process, and the solvent evaporation methods with their numer-
ous adaptations, are well explained. Apart from being prepared by pre-formed
polymers, nanoparticles may be constructed through the in situ polymerization of
monomers which sometimes allows better drug loading. Thanks to the versatility
of these different preparation processes, the size and the shape of the nanoparticles
may be controlled, which may further influence in vivo pharmacokinetic and
biodistribution after administration.
Therefore, the characterization of the nanoparticles is logically addressed in
Part II of the book. Physicochemical characterization includes polymer character-
ization, nanoparticle size, nanoparticle surface properties, drug loading and release,
nanoparticle stability, and batch-to-batch reproducibility. Electron microscopy, both
transmission and scanning, are also important methodologies for the direct visu-
alization of nanoparticles. The interactions with the immune system, the activation
of the complement at the surface of the nanoparticles, as well as the interaction with
cells and intracellular trafcking are dramatically influenced by the characteristics
of the nanoparticles. These processes are discussed in great detail.
Part III of the book discusses how to adjust the characteristics of polymer
nanoparticles with functionalities needed for specic pharmacological applications.
In this view, the choice of the best polymer, the encapsulation process and the drug
loading, as well as, the control of the drug release are at disposal of the formulation
scientists to construct the more efcient nanomedicines. Of course, the toxicological
aspects have to be taken into great consideration, especially the biodegradation
of the nanoparticle polymer core, the safety of the metabolites, the excretion
pathways, and the interaction with blood proteins which may also dramatically
Foreword vii
influence the nanoparticle biodistribution. A special chapter describes the concep-

tion of theranostic nanoparticles combining therapeutic and imaging properties for
personalized medicine.
The last part of the book discusses why polymer-based nanoparticles have
attracted so much interest, whereas only a few of them have been approved and
have reached the market or even the third phase of clinical trials. Regulatory
developments are also considered in a separate chapter.
I recommend reading this book, which assembles a profuse array of knowledge
on the conception and the development of polymer nanoparticles. It represents an
essential reference for a broad scientic community, including academic
researchers and industrial deciders. It should also attract students pursuing a mas-
ters degree or doctorate in the eld of nanomedicine, whether their background is
in education, pharmaceuticals, chemistry, physico-chemistry, or even physics.
Patrick Couvreur
Membre de lAcadmie des Sciences, Professor
Universit Paris-Sud and Institut Universitaire de France
Facult de Pharmacie, Institut Galien Paris Sud, UMR CNRS
8612, Universit Paris-Sud, Chtenay-Malabry, France
Contents
Part I Methods for the Manufacturing of Nanoparticles: Principles

1 Polymer Nanoparticles for In Vivo Applications:
Progress on Preparation Methods and Future Challenges . . . . . . . . 3
Christine Vauthier
2 Nanoprecipitation Process: From Particle Preparation
to In Vivo Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Karim Miladi, Sana Sfar, Hatem Fessi and Abdelhamid Elaissari
3 Targeted Theragnostic Nanoparticles Via Flash
Nanoprecipitation: Principles of Material Selection . . . . . . . . . . . . . 55
Christina Tang and Robert K. Prudhomme
4 Preparation of Polymer Nanoparticles by the Emulsication-
Solvent Evaporation Method: From Vanderhoffs Pioneer
Approach to Recent Adaptations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Nstor Mendoza-Muoz, Sergio Alcal-Alcal
and David Quintanar-Guerrero
5 Methods for the Preparation of Nanoparticles
by Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Christine Vauthier
6 Shape-Controlled Nanoparticles for Drug Delivery
and Targeting Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Gilles Ponchel and Olivier Cauchois
Part II Characterization of Polymer Nanoparticles

Designed as Nanomedicines
7 Physicochemical Characterization of Polymer Nanoparticles:
Challenges and Present Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Jeffrey D. Clogston, Rachael M. Crist and Scott E. McNeil
ix
x Contents
8 Imaging Polymer Nanoparticles by Means of Transmission

and Scanning Electron Microscopy Techniques . . . . . . . . . . . . . . . . 205
Nicolas Tsapis
9 Evaluating the Interactions Between Proteins and Components
of the Immune System with Polymer Nanoparticles . . . . . . . . . . . . . 221
Silvia Lorenzo-Abalde, Rosana Simn-Vzquez,
Mercedes Peleteiro Olmedo, Tamara Lozano-Fernndez,
Olivia Estvez-Martnez, Andrea Fernndez-Carrera
and frica Gonzlez-Fernndez
10 Investigating Interactions Between Nanoparticles and Cells:
Internalization and Intracellular Trafcking . . . . . . . . . . . . . . . . . . . 291
Herv Hillaireau
Part III Turning Polymer Nanoparticle Technologies

into Nanomedicines
11 Designing Polymer Nanoparticle Nanomedicines: Potential
Applications and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Christine Vauthier
12 Selecting and Designing Polymers Suitable for Nanoparticle
Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Sandrine Cammas-Marion
13 Associating Drugs with Polymer Nanoparticles: A Challenge . . . . . 381
Christelle Zandanel and Christine Charrueau
14 Drug Delivery by Polymer Nanoparticles: The Challenge
of Controlled Release and Evaluation . . . . . . . . . . . . . . . . . . . . . . . . 439
Christine Charrueau and Christelle Zandanel
15 Interaction Between Nanoparticles and Plasma Proteins:
Effects on Nanoparticle Biodistribution and Toxicity . . . . . . . . . . . . 505
Anna N. Ilinskaya and Marina A. Dobrovolskaia
16 Toxicological Aspects of Polymer Nanoparticles . . . . . . . . . . . . . . . . 521
Juan M. Irache, Nekane Martn-Arbella, Patricia Ojer,
Amaya Azqueta and Adela Lopez de Cerain
17 Theranostics: In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Viktorija Herceg, Norbert Lange and Eric Allmann
Part IV From Lab to Prescription Desk

18 NanomedicinesA Scientic Toy or an Emerging Market? . . . . . . 591
Matthias G. Wacker
Contents xi
19 Regulatory Perspective on the Development of Polymer

Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
Xiaoming Xu and Mansoor A. Khan
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
Editors and Contributors
About the Editors
Christine Vauthier received her Ph.D. in polymer

chemistry from the University Louis Pasteur at
Strasbourg, France. She then joined the University of
Paris-South, Faculty of Pharmacy as a research assistant.
Presently, she is Director of Research at the CNRS
(Centre National de la Recherche Scientique) at the
Institut Galien Paris Sud, Universit Paris-Sud,
Chtenay-Malabry, France. She also serves as an editor
for Pharmaceutical Research, an AAPS journal. During
her early career, she was visiting scientist at the Center
for Chemical Controlled Delivery, University of Utah,
USA and at the Federal University of Pernambuco,
Recife, Brazil where she had been teaching every year since then. The focus of her
research is about understanding the influence of the physicochemical characteristics
of nanomedicines and their interactions with biological systems when the
nanomedicines are intended to improve drug delivery after mucosal or intravenous
administration. Based on a multidisciplinary approach, her work includes the
synthesis and characterization of polymer nanoparticles from a physicochemical
standpoint, the development of methods to study their interactions with proteins, the
immune system, cells and the study of the influence of the various physicochemical
characteristics of the nanoparticles on their in vivo fate. She is author and co-author
of more than 120 research papers as well as over 20 review papers and book
chapters on nanoparticle preparation, characterization methods, and on the appli-
cation of nanoparticles as drug delivery systems. She has spoken at many confer-
ences and has presented over 100 communications.
xiii
xiv Editors and Contributors
Gilles Ponchel is full Professor at the University of

Paris-South where he teaches Pharmaceutical Technology
and Biopharmacy. He leads a multidisciplinary research
team that belongs to the Institut Galien Paris Sud,
Universit Paris-Sud and specializes in the eld of drug
delivery. The aim of the team is to conceive and to develop
innovative drug delivery systems that can improve the
crossing of active drugs through physico-chemical and
biological barriers. His main research interests are: (i) the
development and the evaluation of bioadhesive delivery
systems and (ii) the conception of pharmaceutically
acceptable nanomedecines, mainly multifunctionalized
nanoparticles prepared from tailored polymers, polypeptides, cyclodextrins, etc., for
optimizing their biodistribution in the context of drug targeting applications. Some of
Prof. Ponchels specic interests are: (i) the impact of their morphologic and structural
characteristics and their capacity to overcome the barriers between the site of delivery and
the site of activity. (ii) the relationships existing at the molecular level between surface
properties of nanoparticles and their capacities of interacting in the body, such as by
bioadhesion and specic recognition. Prof. Ponchel is the author of over 130 research
papers, more than 170 communications, more than 50 invited lectures. He has been
co-author and contributor to books and many book chapters.
Contributors
Sergio Alcal-Alcal Laboratorio de Posgrado en Tecnologa Farmacutica,

Facultad de Estudios Superiores Cuautitln, Universidad Nacional Autnoma de
Mxico, Cuautitln Izcalli, Estado de Mxico, Mexico
Eric Allmann School of Pharmaceutical Sciences, University of Geneva,
University of Lausanne, Geneva, Switzerland
Amaya Azqueta Department of Pharmacology and Toxicology, University of
Navarra, Pamplona, Spain
Sandrine Cammas-Marion UMR 6226 CNRS, Institut of Chemical Science of
Rennes, Team Organic and Supramolecular Chemistry, Ecole Nationale
Suprieure de Chimie de Rennes (ENSCR), Rennes Cedex, France
Olivier Cauchois Institut Galien Paris Sud, CNRS, Univ. Paris-Sud, Universit
Paris-Saclay, Chtenay-Malabry Cedex, France
Christine Charrueau Facult de Pharmacie de lUniversit Paris Descartes, Unit
de Technologies Chimiques et Biologiques pour la Sant UTCBS, CNRS
UMR8258 Inserm U1022, Paris Cedex 06, France
Editors and Contributors xv
Jeffrey D. Clogston Nanotechnology Characterization Laboratory, Cancer

Research Technology Program, Leidos Biomedical Research, Inc., Frederick
National Laboratory for Cancer Research, Frederick, MD, USA
Rachael M. Crist Nanotechnology Characterization Laboratory, Cancer Research
Technology Program, Leidos Biomedical Research, Inc., Frederick National
Laboratory for Cancer Research, Frederick, MD, USA
Marina A. Dobrovolskaia Cancer Research Technology Program,
Nanotechnology Characterization Laboratory, Frederick National Laboratory for
Cancer Research, Leidos Biomedical Research Inc., Frederick, MD, USA
Abdelhamid Elaissari Universit de Lyon, Lyon, France; UMR 5007, Laboratoire
DAutomatique et de Gnie Des Procds, LAGEP-CPE-308G, Universit Lyon 1,
Villeurbanne, CNRS, Villeurbanne, France
Olivia Estvez-Martnez Immunology, Biomedical Research Center (CINBIO),
Institute of Biomedical Research of Vigo (IBIV), Universidad de Vigo, Vigo, Spain
Andrea Fernndez-Carrera Immunology, Biomedical Research Center
(CINBIO), Institute of Biomedical Research of Vigo (IBIV), Universidad de Vigo,
Vigo, Spain
Hatem Fessi Universit de Lyon, Lyon, France; UMR 5007, Laboratoire
Villeurbanne, CNRS, Villeurbanne, France
frica Gonzlez-Fernndez Immunology, Biomedical Research Center
Vigo, Spain
Viktorija Herceg School of Pharmaceutical Sciences, University of Geneva,
Herv Hillaireau Institut Galien Paris Sud, Faculty of Pharmacy, UMR CNRS,
Univ. Paris-Sud, Universit Paris Saclay, Chtenay-Malabry, France
Anna N. Ilinskaya Cancer Research Technology Program, Nanotechnology
Characterization Laboratory, Frederick National Laboratory for Cancer Research,
Leidos Biomedical Research Inc., Frederick, MD, USA
Juan M. Irache Department of Pharmacy and Pharmaceutical Technology,
University of Navarra, Pamplona, Spain
Mansoor A. Khan Formulations Design and Development Core Laboratory,
Texas A&M Health Science Center, Irma Lerma Rangel College of Pharmacy,
College Station, TX, USA
Norbert Lange School of Pharmaceutical Sciences, University of Geneva,
xvi Editors and Contributors
Adela Lopez de Cerain Department of Pharmacology and Toxicology, University

of Navarra, Pamplona, Spain
Silvia Lorenzo-Abalde Immunology, Biomedical Research Center (CINBIO),
Institute of Biomedical Research of Vigo (IBIV), Universidad de Vigo, Vigo, Spain
Tamara Lozano-Fernndez Immunology, Biomedical Research Center
Vigo, Spain
Nekane Martn-Arbella Department of Pharmacy and Pharmaceutical
Technology, University of Navarra, Pamplona, Spain
Scott E. McNeil Nanotechnology Characterization Laboratory, Cancer Research
Technology Program, Leidos Biomedical Research, Inc., Frederick National
Laboratory for Cancer Research, Frederick, MD, USA
Nstor Mendoza-Muoz Laboratorio de Farmacia, Facultad de Ciencias
Qumicas, Universidad de Colima, Coquimatln, Colima, Mexico
Karim Miladi Universit de Lyon, Lyon, France; UMR 5007, Laboratoire
Villeurbanne, CNRS, Villeurbanne, France; Laboratoire de Pharmacie Galnique,
Universit de Monastir, Monastir, Tunisia
Patricia Ojer Department of Pharmacy and Pharmaceutical Technology,
Department of Pharmacology and Toxicology, University of Navarra, Pamplona,
Spain
Mercedes Peleteiro Olmedo Immunology, Biomedical Research Center
Vigo, Spain
Gilles Ponchel Institut Galien Paris Sud, Faculty of Pharmacy, CNRS, Univ. of
Paris-Sud, University Paris Saclay, 92296 Chtenay-Malabry Cedex, France
Robert K. Prudhomme Department of Chemical and Biological Engineering,
Princeton University, Princeton, NJ, USA
David Quintanar-Guerrero Laboratorio de Posgrado en Tecnologa
Farmacutica, Facultad de Estudios Superiores Cuautitln, Universidad Nacional
Autnoma de Mxico, Cuautitln Izcalli, Estado de Mxico, Mexico
Sana Sfar Laboratoire de Pharmacie Galnique, Universit de Monastir, Monastir,
Tunisia
Rosana Simn-Vzquez Immunology, Biomedical Research Center (CINBIO),
Institute of Biomedical Research of Vigo (IBIV), Universidad de Vigo, Vigo,
Spain; Institut Galien Paris Sud, Faculty of Pharmacy, CNRS, Univ. Paris-Sud,
Universit Paris Saclay, Chtenay-Malabry, France
Editors and Contributors xvii
Christina Tang Department of Chemical and Biological Engineering, Princeton

University, Princeton, NJ, USA
Nicolas Tsapis Institut Galien Paris Sud, UMR CNRS, Univ. Paris-Sud,
Universit Paris Saclay, Chtenay-Malabry, France
Christine Vauthier Institut Galien Paris Sud, Faculty of Pharmacy, UMR CNRS,
University of Paris-Sud, University Paris Saclay, 92296 Chtenay-Malabry Cedex,
France
Matthias G. Wacker Project Group for Translational Medicine and
Pharmacology (TMP), Department of Pharmaceutical Technology and
Nanosciences, Fraunhofer-Institute for Molecular Biology and Applied Ecology
(IME), Frankfurt/Main, Germany
Xiaoming Xu Division of Product Quality Research, Ofce of Testing and
Research, Ofce of Pharmaceutical Quality, Center for Drug Evaluation and
Research, Food and Drug Administration, Silver Spring, MD, USA
Christelle Zandanel Institut Galien Paris Sud, UMR CNRS, Univ. Paris-Sud,
Universit Paris Saclay, 92296 Chtenay-Malabry, France
Abbreviations
c-CDC6 c-cyclodextrine modied with carbon chain in C6

c-PGA-NPs poly(c-glutamic acid)
Intrinsic Viscosity
q Density
smix Time scale of mixing
sNP Assembly Time scale of nanoparticle assembly
snucleation and growth Time scale of nucleation and growth of the precipitating core
material
sself-assembly Time scale of block copolymer self-assembly
2CTA GFLGKGFG peptide
3D HFF 3D hydrodynamic flow focusing
A Aggregation ratio
A Adsorption
ABC Accelerated blood clearance
ABCPA 4-4-azobis(4-cyanopentanoic acid)
ACA Alkylcyanoacrylate(s)
AEP Anionic emulsion polymerization
aFFFF Asymmetric flow eld-flow fractionation
AFM Atomic force microscopy
ag Antigen
Ag Silver
AH50 test Hemolytic assay to measure the alternative pathway of
complement activation
AIBN Azobis(isobutyronitrile)
AIDS Aquired immune deciency syndrome
Alum Aluminium salts used as adjuvant
AmB Amphothericin B
ANDA Abbreviated new drug application
APC Antigen-presenting cells
API Active pharmaceutical ingredient
xix
xx Abbreviations
APS Ammonium persulfate

AS03 Oil-in-water emulsion
AS04 Oil-in-water emulsion (composed of monophosphoryl lipid
A adsorbed to Alum)
AUC Area under the curve
AuNPs Gold nanoparticles
AuNRs Gold nanorods
AZT AZidoThymidine
BBB Bloodbrain barrier
BCA Bicinchronic acid
BCO Block co-oligomers
BCR B cell receptor
BCS Biopharmaceutical classication system
BHEM N,N-bis(2-hydroxyethyl)-N-methyl
BLA Biological license application
BMPO 5,6-benzo-2-methylene-1,3-dioxepane
BSA Bovine serum albumin
c Concentration
C3 Complement factor 3
CAD Charged aerosol detector
CAP Cellulose Acetate Phthalate
CARPA Complement Activation Related Pseudoallergy
CCD Charge-coupled device
CD Cluster of differentiation
CDAN N1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine
CDER Center of drug evaluation and research
cDNA Complementary deoxiribonucleic acid
CF Chloroform
CFEG-HRSEM Cold eld-emission gun high-resolution scanning electron
microscope
CFF Cross-Flow Filtration
CFR Code of federal regulations
cGMP Current good manufacturing practices
CH50 test Hemolytic assay to measure the classical pathway of
complement activation
CIJ Conned impinging jet mixer
CL e-caprolactone
clogP Calculated octanol-water partition coefcient
CMC Chemistry, Manufacturing, and Controls
CM-CS O-carboxymethyl chitosan
CME Clathrin-mediated endocytosis
CNS Central nervous system
CPI Catastrophic Phase Inversion
CPT Camptothecin
CQA Critical quality attribute
Abbreviations xxi
CR Complement receptor
CS-ab-GP chitosan-ab-glycerophosphate
CS Chitosan
Core-shell-NPs Coreshell nanoparticles
CT X-ray computed tomography
CTAB Cetyl trimethylammonium bromide
CTL Cytotoxic T lymphocytes
Cu(I) Copper I
CuAAc Cu(I) catalyzed azide-alkyne cycloaddition
CUR Curcumin
CvME Caveolae-mediated endocytosis
CyA Cyclosporine A
Da Dalton
DC Dendritic cells
DCC dicyclohexylcarbodiimide
DC-FCCS Dual-Color Fluorescence Cross-Correlation Spectroscopy
DCM Dicyanomethylene-4H-pyran
DCs Dendritic cells
DCU Dicyclohexyl urea
DDrop Average diameter of the nanodroplets
DEAE Diethylaminoethyl
DL Drug loading
DLS Dynamic light scattering
DMAEMA N,N-dimethylaminoethyl methacrylate
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
dn/dc Change in refractive index with change in concentration
DNA Deoxyribonucleic acid
DNP Average diameter of the nanoparticles
DOPC 1,2-distearoyl-sn-glycero-3-phosphocholine
Dot blot Semiqualitative method for rapid screening without
electrophoresis
DOTA Tertraazacyclododecane tetraacetic acid
Dox Doxorubicin
DOX Doxorubicin
DPI Dual polarization interferometry
DPPC Dipalmitoylphosphatidylcholine
DSC Differential scanning calorimetry
DTT-SH Dithiothritol
DTX Docetaxel
e.g. For example
E Entrapment
EA Ethyl Acetate
EC Ethylcellulose
EE Encapsulation efciency
xxii Abbreviations
EEM EmulsicationEvaporation Method

EFSA European Food Safety Authority
EGF Epidermal growth factor
EGFR Epithelial growth factor receptor
EL 14 Copolymer of lactic acid and ethylene glycol
ELISA Enzyme-linked immunosorbent assay
ELISPOT Enzyme-linked immunosorbent spot
ELSD Evaporative light scattering detector
EM Electron microscopy
EMA European Medicines Agency
EPA Environmental Protection Agency
EPR Enhanced permeability and retention
EPS Extrapyramidal side effects
et al. And others
EU European Union
F127 Pluronic F-127
FA Folic acid
FCS Fluorescence Correlation Spectroscopy
FDA Food and Drug Administration in the United States of
America (FDA)
FFF Field flow fractionation
FNP Flash nanoprecipitation
FOXP3+CD4+T T regulatory cell expressing the transcription factor FOXP3
FTIR Fourier transform infrared spectroscopy
g7 Simil-opioid peptide
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GEM Gemcitabine
GI Gastrointestinal Tract
GIT Gastro-intestinal tract
GMP Good manufacturing practice
GPC Gel permeation chromatography
GRAS Generally Recognized as Safe
HA Hyaluronic acid
HA-SLN Hyaluronic acid targeted solid lipid nanoparticles
HBSS Hanks buffered salt solution
HCC Hepatocellular carcinoma
HCE Human corneal epithelial
HDL High density lipoprotein
HEMA 2-hydroxyethyl methacrylate
HER2 Human epidermal growth factor receptor 2
HFIP Hexafluoroisopropanol
HIFU High intensity focused ultrasound
HIV Human immunodeciency virus
HLA Human leukocyte antigen
HLA-DR Human leukocyte antigen, class II molecule DR
Abbreviations xxiii
HLB Hydrophilic-lipophilic balance

HPH High pressure homogenization
HPIMM High pressure interdigital multilamination micromixer
HPLC High Performance Liquid Chromatography
HPMA Hydroxypropyl methacrylate
HPMAm N-(2-hydroxypropyl) methacrylamide
HPbCD Hydropropylbetacyclodextrin
HRP Horse rabbit peroxidase
HSA Human serum albumin
HTCC N-((2-hydroxy-3-trimethylammonium) propyl) chitosan
chloride
Hy-PEI Hyper-branched poly(ethylene imine),
i.e. That is
IBCA isobutylcyanoacrylate
iC3b Inactive complement factor C3
ICAM-1 Intracellular cell adhesion molecule 1
ICG Indocyanine green
ICH International Conference on Harmonization
ICP-MS Inductively-coupled plasma mass spectrometry
IFN Interferon
Ig Immunoglobulin
IHCA Isohexylcyanoacrylate
IL Interleukin
IND Investigational new drug
INF Interferon
iNOS Inducible nitric oxide synthase
INPs Inorganic nanoparticles
IOBA-NHC Human conjunctival epithelial cells
IONPs Iron oxide nanoparticles
IOP Intraocular pressure
Ip Polymolecularity index
IPA IsopropylAcrylamide
ITC Isothermal titration calorimetry
KLH Keyhole limpet hemocyanin
kV Kilovolts
LAL Limulus amebocyte lysate
LbL Layer-by-layer
LC Drug loading content
LC-MS Liquid chromatographymass spectrometry
LCST Lower critical solution temperature
LD Laser diffraction
LDH Lactate dehydrogenase
LDL Low-density lipoprotein
LE Drug loading efciency
Leu L-leucine ethyl ester
xxiv Abbreviations
LLC Lewis lung carcinoma

LNs Lipid nanoparticles
logP Octanolwater partition coefcient used as a measure of
hydrophobicity
LOP Loperamide
LOP-PLGA-g7 Nanoparticles coated with simil-opioid peptide and contain-
ing loperamide
LOP-PLGA-SA-g7 Nanoparticles coated with sialic acid and simil-opioid
peptide
LPS Lipopolysaccharide
LSC Lauryl succinyl
LSPR Localized surface plasmon resonance
LTZ Letrozole
MAA Methacrylate Acid
Mab Monoclonal antibody
MAC Membrane attack complex
MA-GFLG-Dox N-methacryloyl-glycylphenylalanylleucylglycyl-doxorubicin
Mag-NPs Magnetic nanoparticles
MAL Maleimide
MALLS Multi-angle laser light scattering
MAPK Mitogen-activated protein kinase
MC Methylene Chloride
MCP-1 Monocyte chemoattractant protein-1
MDA Malondialdehyde
MDR multiple-drug resistance
MF59 Oil-in-water emulsion
MHC Major histocompatibility complex
MIVM Multi-inlet vortex mixer
MNPs Mesoporous nanoparticles
mp/Drop Mass of the polymer in the droplets
mp/NP Mass of the polymer in the particles
MPE Maximal possible effect
MPEGPTMC Poly(ethylene glycol)poly(trimethylene carbonate)
MPS Mononuclear phagocytic system
MRI Magnetic resonance imaging
MS Mass spectrometry
MTX Mitoxantrone
MUA 11-mercaptoundecanoic acid
MW Molecular weight
MWCO Molecular weight cut-off
NAC1 N-acetyltransferase 1
nBCA n-butylcyaoacrylate
NC Nanocapsules
NCAM Neural cell adhesion molecule
NCE New chemical entity
Abbreviations xxv
NCS Neocarzinostatin
NCs Nanocapsules
NDA New drug application
NF-kB Nuclear factor kappa-light-chain-enhancer of activated B
cells
NG Nanogel
NIR Near-infrared
NK Natural killer cells
nm Nanometer
NMR Nuclear magnetic resonance
NO Nitric Oxide
NPs Nanoparticles
ns Not specied
NTs nanotubes
O/W Oil-in-water emulsion
ODN Oligonucleotide
OEt Ethyl ester
OI Optical imaging
OLZ Olanzapine
OSHA Occupational safety and health administration
P4VP Poly(4-vinylpyridine)
PAA Poly(acrylic acid)
PACA Poly(alkylcyanoacrylate)
PAGE Polyacrylamide gel electrophoresis
PAH Poly(allylamine hydrochloride)
PALM Photo-activated localization microscopy
PAMAM Poly(amido amine)
PAMPs Pathogen-Associated Molecular Patterns
PBCA Poly(ButylCyanoAcrylate)
PBDL Poly(butylene succinate-co-butylene dilinoleate)
PBLG poly(c-benzyl-L-glutamate)
PBMC Peripheral blood mononuclear cells
PBS Phosphate buffered saline
PCC Physicochemical characterization
PCDA 10,12-pentacosydonic acid
PCEP Poly[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethy-
lene) ammonium iodide] ethyl phosphate
PCL Poly (e-Caprolactone)
PCL-b-PEG Poly(e-caprolactone)-block-poly(ethylene glycol)
PCR Polymerase chain reaction
PCS Photon Correlation Spectroscopy
PD Pharmacodynamics
PDI Polydispersity index
PDM 2-(dimethylamino)ethyl methacrylate
PDMAEMA Poly(dimethylamino ethyl methacrylate)
xxvi Abbreviations
PECs Peritoneal exudate cell macrophages

PEC Polyelectrolyte complexes
PEDOT Poly(3,4-ethylenedioxythiophene)
PEG poly(ethylene glycol)
PEG-PCL Poly(e-caprolactone)-poly(ethylene glycol)
PEG-PHDCA Poly(methoxypolyethyleneglycol
cyanoacrylate-co-hexadecyl cyanoacrylate)
PEG-PLA poly(ethylene glycol)poly(lactide)
PEG-PLL poly(ethylene oxide)-poly(lysine)
PEI poly(ethylene imine)
PEO poly(ethylene oxide)
PES Poly(ethyl sebacate)
PES-DOX Poly(ethylene sebacate) nanoparticles loaded with
doxorubicin
PET Positron emission tomography
PEVA Poly(ethylene-co-vinylacetate)
PFC PolyFluoroCarbone
PFPE Perfluoropolyether
PGA Poly(glycolide)
PGGA Poly(c-glutamic acid)
PHB Poly(b-Hydroxybutyrate)
Phe L-phenyl alanine methyl ester
PHPMA poly(2-hydroxypropyl methacrylate)
PHPMAm Poly N-(2-Hydroxypropyl methacrylamide)
PIBCA Poly(isobutylcyanoacrylate)
PIHCA Poly(isohexylcyanoacrylate)
PIPAAN Poly(isopropylacrylamide)
PIT Phase-inversion temperature
PK Pharmacokinetic
PLA Poly(lactide)
PLA-b-PEG Poly(lactide acid)-block-poly(ethylene glycol)
PLA-PEG Poly(lactide)-poly(ethyleneglycol)
PLA-TPGS Poly(lactide)tocopheryl poly(ethylene glycol succinate)
PLGA Poly(lactide-co-glycolide)
PLGA-b-PEG Poly(lactide-co-glycolide)-block-poly(ethylene glycol)
PLGA-PEO poly(lactide-co-glycolide)-poly(ethylene oxide)
PLG-NCA c-propargyl-L-glutamate N-carboxyanhydride
PLL Poly-L-lysine
PLLA Poly(L-lactide)
PLT Platelet
PMA Poly(methyl acrylate)
PMLA Poly(malic acid)
PMLABe Poly(benzyl malate)
PMLABe80H20 Poly(benzyl malate-co-malic acid)
PMLAHe Poly(hexyl malate)
Abbreviations xxvii
PMLAHe90H10 Poly(hexyl malate-co-malic acid)

PMLAMe Poly(methyl malate)
PMLAMexHy Poly(methyl malate-co-malic acid)
PMM Poly(methyl methacrylate)
P-NPs Polymer nanospheres
PPG Poly(propylene glycol)
PPIX Protoporphyrin IX
PPO Poly(propylene oxide)
PRINT Particle Replication IN non-wetting Template
PRP Platelet-rich plasma
PRRs Pattern Recognition Receptors
PhotoS Photosensitizer
PS Poly(styrene)
PS-b-P4VP Poly(styrene)-block-poly(4-vinylpyridine)
PS-b-PEG Poly(styrene)-block-poly(ethylene glycol)
PSD Particle size distribution
PSMA Poly(styrene-co-maleic acid/anhydride)
PSS Poly(4-styrene-sulfonate)
PTMC Poly(trimethylene carbonate)
PTX Paclitaxel
PUL Pullulan
PUL-PES-DOX Poly(ethylene sebacate) nanoparticles loaded with
doxorubicin
PVA Poly(vinyl alcohol)
PVP Poly(N-vinyl-2-pyrrolidone)
QCM-D Quartz crystal microbalance with dissipation monitoring
QDs Quantum dots
QELS Quasi-elastic light scattering
qPCR Quantitative Polymerase chain reaction
R&D Research and Development Department
RA Rheumatoid arthritis
RAFT Reversible Addition Fragmentation Chain Transfer
RBCs Red blood cells
real time-PCR Real time polymerase chain reaction
RES Reticuloendothelial system
Rg radius of gyration
RGD Tripeptide arginine-glycine-aspartic acid
RGDp Tripeptide arginine-glycine-aspartic acid peptidomimetic
RI Refractive index
RIA Radio-immuno-analysis
RIS Risperidone
RIV Rivastigmine tartrate
RME Receptor-mediated endocytosis
rms Root mean square
RNA Ribonucleic acid
xxviii Abbreviations
ROS Reactive Oxygen Species

RP-HPLC Reversed phase high performance liquid chromatography
RREP Redox radical emulsion polymerization
SA Sialic acid
SAB Sodium acetate buffer
SBF Simulated body fluid
SBR Signal-to-background ratio
sCD14 Soluble CD14
SDS Sodium dodecyl sulfate
SEC Size exclusion chromatography
SEM Scanning electron microscopy
SGF Simulated gastric fluid
SIF Simulated intestinal fluid
siRNA small interfering RNA
SLF Simulated lachrymal fluid
SLNs Solid lipid nanoparticles
SLS Sodium lauryl sulfate
SnOct2 Stannous octanoate
SPECT Single photon emission computed tomography
SPION Super paramagnetic iron oxide nanoparticles
SPR Surface plasmon resonance
SQ Squaraine
SR Scavenger receptor
SRBC Sheep red blood cell
ssDNA Single stranded deoxyribonucleic acid
SSF Simulated saliva fluid
STED Stimulated emission depletion
STORM Stochastic optical reconstruction microscopy
TAT Trans-activating transcriptional activator peptide
Tc T cytotoxic cell
TCR T cell receptor
T-CS Chitosan-glutathione conjugate
TDAR T cell Antibody Response
TDCN Thermo-responsive di-block copolymer nanoparticles
TEA Triethanolamine
TEM Transmission electron microscopy
Tf Transferrin
TfR Transferrin receptor
TGA Thermogravimetric analysis
Th T helper cell
THF Tetrahydrofuran
Thr Na-(methacryloyl)-threonine
TLR Toll-like receptor
TMC TriMethylChitosan
TMT-Cys Trimethyl chitosan-cysteine conjugate
Abbreviations xxix
TNF Tumor necrosis factor

TPGS d-a-tocopheryl poly(ethylene glycol) 1000 succinate
TPI Transitional Phase Inversion
TPP TriPhenylPhosphate
T-PS Photosensitizer prodrug
TRA All trans retinoic acid
Treg T regulatory cell
TRPS Tunable resistive pulse sensing
TSLs Thermosensitive liposomes
U.S. United States
UCNP Up-converting nanophosphors
uPA Urokinase plasminogen activator
uPAR Urokinase plasminogen activator receptor
UPS United state pharmacopoeia
US Ultrasound
USA United States of America
UV Ultraviolet
UVA Ultraviolet A
UVB Ultraviolet B
v/v Volume/volume proportion
VPTT Volume phase transition temperature
W/O/W Water-in-oil-in-water emulsion, double or multiple emulsion
W/O Water-in-oil emulsion
w/v Weight/volume proportion
WGA Wheat germ agglutinin
WPM Wet pearl milling
XRPD X-ray powder diffraction
Z-Avg. Z-average
ZnO Zinc oxide
Part I
Methods for the Manufacturing of
Nanoparticles: Principles
Chapter 1
Polymer Nanoparticles for In Vivo
Applications: Progress on Preparation
Methods and Future Challenges
Christine Vauthier
Abstract Polymer nanoparticles are one type of the arsenal of nanomedicines that
are developed to improve efcacy and specicity of drug delivery and to design
new contrast agents enhancing the performance of diagnostic methods based on
imaging techniques. To answer the various challenges, it has lead the way to
development of suitable nanoparticles. Many types of methods of preparation were
proposed designing nanoparticles taking different structures and integrating various
functions. The purpose of the introduction to the part I of the book devoted to the
methods of preparation of polymer nanoparticles to be used as nanomedicines is to
present the different types of polymer nanoparticles that were designed so far and to
give an overview on their methods of preparation. It is also important to place these
methodologies in a prospective view raising future challenges and bottlenecks.
Keywords Methods
Micelles
Polymer nanoparticles
Nanocapsules
Nanospheres
Nanogel
Polyelectrolyte complex
Self-assembling

Precipitation Polymerization Emulsion Polymer solution Layer-by-layer
Print Microfluidic
Self-assembling
Complex
Spherical particles

Nonspherical nanoparticles Multifunctional nanoparticles
1 Introduction
In the 1970s, polymer nanoparticles were found to be suitable materials thanks to

their small size to serve the purpose of the magic bullet born behind the concept
of drug targeting that was inspired by Paul Ehrlich, an imminent bacteriologist and
immunologist who received the Nobel Prize in Physiology and Medicine in 1908.
However, to be used as drug carriers, polymer nanoparticles need to comply with
C. Vauthier (&)
Institut Galien Paris Sud, Faculty of Pharmacy, UMR CNRS, University of Paris-Sud,
University Paris Saclay, 92296 Chtenay-Malabry Cedex, France
e-mail: christine.vauthier@u-psud.fr
Springer International Publishing Switzerland 2016 3

C. Vauthier and G. Ponchel (eds.), Polymer Nanoparticles for Nanomedicines,
DOI 10.1007/978-3-319-41421-8_1
4 C. Vauthier
regulatory registration and fulll stringent specications. Besides, they must

integrate all functionalities that are needed to complete a specic medical appli-
cation. Among others, this includes a composition made of suitable materials for
in vivo use and preparation conditions that are compatible with the production of
pharmaceutical grade compounds.
2 Development of Methods of Preparation

of Nanoparticles Made of Polymers: Progresses
By the time polymer nanoparticles were rst introduced to be used as drug carriers,
they were produced by polymerization methods (Birrenbach and Speiser 1976; See
the historical perspective by Kreuter 2007; Couvreur 2013). In addition to regu-
latory constraints that are an important limitation for the choice of the polymer
composing the nanoparticles, nanoparticles designed to become nanomedicines
need to fulll various types of functions. Drugs should be associated efciently with
nanoparticles while protection against degradation should be insured in storage
conditions and in vivo during transportation of the nanomedicine toward the target
site of delivery of the drug. This implies that the drug remains associated with the
nanoparticles during transportation. However, the association needs to become
unstable once the nanoparticle has reached the target site, where the drug should be
available to express its biological activity. Behind mechanisms controlling the
stability of the association of the drug with the nanoparticles, other functionalities
are needed to help the nanoparticles to reach the delivery site. The requested
properties, which are contradictory for some of them, can be associated customizing
the design of new polymers. The number of suitable polymers that can compose
nanoparticles developed to be used as nanomedicine produced by polymerization
methods is extremely low being a bottleneck for an extensive development of the
polymerization methods to prepare polymer-based nanomedicines. Other limita-
tions of these methods include the use of organic solvents and sometimes of large
amounts of surfactants, while the majority of polymer nanoparticles synthesized by
polymerization methods are nonbiodegradable. Nevertheless, the rst rapidly
biodegradable nanoparticles were synthesized by emulsion polymerization using
alkylcyanoacrylate monomers (Couvreur et al. 1979). A broad range of nanopar-
ticles composed of poly(alkylcyanoacrylate) were synthesized since then and are
used to develop innovative therapeutic strategies with many types of drugs with
interests for developing treatments of serious diseases (Vauthier et al. 2003a, b,
2007; Andrieux and Couvreur 2009; Nicolas and Couvreur 2009). Today, poly
(alkylcyanoacrylate) nanoparticles prepared by polymerization methods continue to
generate interest on the international scene (Murthy and Harivardhan Reddy 2006;
Vauthier et al. 2007; Graf et al. 2009; Nicolas and Couvreur 2009; Yordanov 2012;
Sulheim et al. 2016). Polymerization methods were successful to provide with
nanoparticles of interest that were translating to clinics being evaluated in clinical
1 Polymer Nanoparticles for In Vivo Applications 5
trial phase II/III for the treatment of hepatocellular carcinoma (primary liver cancer)
(Zhou et al. 2009; Soma et al. 2012; Onxeo 2016). However, all nanoparticles
developed as nanomedicines and prepared by polymerization methods were syn-
thesized with monomers of the alkylcyanoacrylate family limiting the choice of
intrinsic properties that can be given to the particles although some flexibilities are
allowed tuning conditions of polymerization (Chap. 5 from Vauthier).
To enlarge the choice of polymers composing nanoparticles to be used as
nanomedicines, a series of methods were developed based on the use of polymers
that were synthesized independently of the nanoparticles. Obtaining polymer
nanoparticles from already prepared polymers was a challenge. The rst series of
attempts was based on the use of matrices formed by thin emulsions in which the
polymer was dissolved in the tiny droplets composing the dispersed phase of the
emulsion. The polymer was then forced to precipitate using various artifacts in
order to obtain nanoparticles. Evaporation of the solvent contained in the droplets
was the approach proposed in the pioneer work in the early 1980s (Gurny et al.
1981). The development of this emulsication-solvent evaporation method was
applied rst to the production of nanoparticles made of poly(lactide) (PLA), the
most used polymer composing medical devices for parenteral administration. Since
then, the method has been applied to a large choice of polymers. This method
brought a real breakthrough. It was the rst time nanoparticles were obtained
directly from polymers while they were all obtained before by polymerization
methods. It was an important milestone for the development of methods for the
preparation of nanomedicines occurring as polymer nanoparticles. In a derived
method also based on the precipitation of a polymer dissolved in the emulsion
droplets, the polymer solvent is extracted from the droplets diluting the emulsion
with a third solvent in which both the continuous and the dispersed phases of the
parent emulsion are miscible. This operation causes the immediate precipitation of
the polymer contained in the emulsion droplets that compose the dispersed phase of
the emulsion. In general, both the emulsication-solvent evaporation method and
the emulsication-solvent extraction method can be applied with polymers that are
soluble in organic solvents (Chap. 4 from Mendoza-Muoz et al.). Instead of
precipitation, the polymer contained in the droplets of the emulsion can be gelied.
This method was addressed to produce nanoparticles composed of hydrogels to
associate hydrosoluble drugs with nanoparticles that was challenging with previous
methods. The main difculty with methods based on the use of emulsions is to
prepare emulsion with a small size of the emulsion droplets. While the majority of
works were based on the use of mechanical techniques to produce the thin emulsion
required, several authors have suggested the formulation of miniemulsions and
microemulsions as matrices to produce the nanoparticles. More recently, mi-
crofluidic techniques have been introduced. Droplets hence nanoparticles are
formed one by one in a very well controlled manner (Karnik et al. 2008; Valencia
et al. 2012; Pedro et al. 2013; Lim et al. 2014). To avoid the use of organic solvents,
supercritical fluid technologies were envisaged (Sun et al. 2005; Meziani et al.
2006; Elizondo et al. 2012; Sheth et al. 2012; Girotra et al. 2013).
6 C. Vauthier
In another series of methods, nanoparticles are prepared directly from polymer

solutions. Nanoparticles form by causing a rapid change of the physicochemical
conditions that induces the nucleation of particles of small size. In general, they
form by mixing the initial polymer solution with a second medium with which it is
fully miscible. Mechanisms behind nucleation of nanoparticles include precipitation
of the polymer, self-assembling of macromolecules providing that they were
selected with the required architecture or specic properties, formation of com-
plexes and gelation. Figure 1 illustrates the formation of nanoparticles based on the
induction of nucleation from two examples of methods: the formation of polymer
micelles resulting from self-assembling of amphiphilic polymers assisted by solvent
diffusion (Fig. 1a) (Chap. 2 from Miladi et al. and Chap. 3 from Tang and
Prudhomme), and the formation of nanogels triggered by self-assembling of two
polymers having complemental groups to form inclusion complexes between alkyl
chains grafted on one polymer and cyclodextrins grafted on a second polymer (Fig.
1b) (Gref et al. 2006; Hassani et al. 2012).
In some cases, the nucleated nanoparticles are stabilized in a second step that can
be performed in the same vessels. For instance, after nucleation of polymer particles
by precipitation, it is generally necessary to remove the solvent of the polymer from
the dispersing medium. The so-called nanoprecipitation method in which
nanoparticle nucleation is induced by a solvent shift belongs to this category of
method (Fessi et al. 1989; Ganachaud and Katz 2005; Minost et al. 2012; Chap. 2
from Miladi et al. and Chap. 3 from Tang and Prudhomme). Nanoparticles
obtained by gelation are sometimes stabilized by complexation with another
polymer that sticks on the surface to stabilize the particle (Oh et al. 2008; Kabanov
and Vinogradov 2009; Maya et al. 2013; Wu and Delair 2015). Interesting features
with these methods are their rapidity and scalability because production can be
performed with a continuous-based process as demonstrated with the nanoprecip-
itation method. These methods of preparation can be achieved with a large panel of
polymers. Although precipitation methods and methods based on self-assembling
of amphiphilic polymers generally require the use of organic solvents (Fig. 1a)
(Chap. 2 from Miladi et al. Chap. 3 from Tang and Prudhomme, Weber 1998;
Torchilin 2007; Kabanov and Vinogradov 2009; Rowan 2009; Guan et al. 2015;
Fuks et al. 2011; Pearson et al. 2013; Robertson et al. 2013), self-assembling
methods based on the formation of polymer complexes and those based on a
gelation process can be performed in aqueous media avoiding totally the use of
organic solvent (Fig. 1b) (Vauthier and Couvreur 2000; Janes et al. 2001; Gref et al.
2006; Kabanov and Vinogradov 2009; Daoud-Mahammed et al. 2009; Delair 2011;
Hassani et al. 2012; Maya et al. 2013; Eckmann et al. 2014). Another marked
advantage of the last category of method is given by the fact that nanoparticles form
in gentle conditions that are suitable to associate very fragile hydrosoluble mole-
cules with the nanoparticles. For instance, the methods based on the formation of
complexes and nanogels can be used to associate biologically active peptides,
proteins, and nucleic acids with nanoparticles. With methods based on the com-
plexation of polyelectrolytes of opposite charges, peptides, and nucleic acids may
compose one of the polyelectrolyte involved in the formation of the complex
(a) Amphiphilic Hydrophobic drugs entrapped in the

polymer core of the micelle
Nucleaon of parcles
(b)
Hydrophobized Polycyclodextrin
Entrapment of hydrophobic drugs in
dextran
remaining free cyclodextrins
Fig. 1 Preparation of nanoparticles by nucleation of particles thanks to self-assembling of soluble

polymers. Nucleation of polymer particles occurs while mixing two miscible solutions.
a Formation of polymer micelles assisted by solvent diffusion. This can be applied with
amphiphilic polymers. b Formation of nanogels by self-assembling of neutral hydrosoluble
polymers including a polycyclodextrin and a hydrophobized dextran. The nanogels form, thanks to
the formation of inclusion complexes between the cyclodextrins grafted on one of the polymers
and alkyl chains grafted on the second polymer (hydrophobised dextran shown on the gure)
included in the nal nanoparticles (Kabanov and Vinogradov 2009; Delair 2011;
Kataoka et al. 2001; Mukhopadhyaya et al. 2012; Osada 2014; Bekale et al. 2015;
Shiraki et al. 2016). All these techniques of preparation of polymer nanoparticles
allow production of nanoparticles with a wide range of properties thanks to the
nature of polymers that can be used to produce them.
8 C. Vauthier
3 Producing Polymer Nanoparticles with Different

Structures and Characteristics
A broad range of methods of preparation of polymer nanoparticles was requested to

permit association of drugs having various biological activities and physicochem-
ical properties. In general, molecules are associated with the nanoparticles while
they are solubilized in an appropriate solvent. Solubility properties of drug mole-
cules are important factors to consider and that contribute for the success of drug to
nanoparticle association. Although soluble molecules are the majority of com-
pounds that were associated with nanoparticles so far, metal nanoparticles were
interesting ingredients to associate with polymer nanoparticles designing a new
generation of contrast agents for application in diagnostic based on imaging tech-
niques (Khemtong et al. 2009; Maya et al. 2013; Cormode et al. 2014; See Chap. 17
from Herceg et al.). The solvent in which the drug molecule is soluble or metal
nanoparticles occur as a stable dispersion is a key for the choice of the method of
preparation. However, in general, methods of preparation need to be customized on
a case-by-case basis to design each new nanomedicine. Existing methods can be
used to inspire the development of new methods. They were applied to make
nanoparticles with polymers of various nature and to produce nanoparticles having
different structures to resolve many different challenges found to achieve efcient
drug association and releasing issues (Fig. 2) (Chap. 13 from Zandanel and
Charrueau, Chap. 14 from Charrueau and Zandanel).
Methods based on general principles that were described above are all suitable to
prepare matrix-like-type nanoparticles. Reservoir-type nanoparticles, i.e.,
nanocapsules could be obtained modifying and adapting protocols of most of the
previous methods (Couvreur et al. 2002; Mora-Huertas et al. 2010). Figure 3
summarizes the different methods of production of polymer nanoparticles and gives
the type of nanoparticle produced.
Size and shape of nanoparticles are important characteristics to consider as they
both influence the pharmacokinetic and cell uptake; hence, they can dramatically
affect the efcacy of the nanomedicine (Truong et al. 2015). In general, size can be
well controlled by experimental conditions used preparing the nanoparticles.
Nanoparticles with a spherical shape are generally prepared by the above-mentioned
methods. The obtaining of nanoparticles with a shape that differed from a sphere was
reported only in a few cases producing nanoparticles by self-assembling of polymers
and amphiphilic materials (Lee et al. 2010; Cauchois et al. 2013; Chap. 6 from
Ponchel and Cauchois). New methods were specically introduced to design
nanoparticles with well-controlled nonspherical shapes (Chap. 6 from Ponchel and
Cauchois). For instance, rod-like nanoparticles can be produced stretching spherical
particles embedded in a stretchable matrix (Mitragotri 2009; Wang et al. 2011a).
Print methods were introduced to design polymer nanoparticles with a wide range of
shapes (Oh et al. 2008; Wang et al. 2011b; Perry et al. 2011; Sultana et al. 2013)
(Fig. 4).
Matrix-like type nanoparcles Reservoir type nanoparcles

Nanospheres Nanocapsules
Diameter 50 300 nm Diameter 100 300 nm
nanospheres core-corona aqueous reservoir

nanospheres
nanogel polyelectrolyte oil reservoir

complex
Polymer micelles Polymersomes
Diameter down to 20 nm Diameter 60 to 500 nm
polyelectrolyte lipid core

complex core
Fig. 2 Different types of polymer nanoparticles showing the structures
4 Future Challenges
In the infant age of their development, polymer nanoparticles were designed as very
simple particles based on the association of a drug with a nanosized-scale particle
made of biodegradable polymer. The evolution is to design multifunctional
nanoparticles that may include diagnostic and therapeutic elements together with
equipments controlling the pharmacokinetic and biodistribution hence improving
targeting efciency of the carrier and its drug releasing properties. Table 1 sum-
marizes the different functionalities that are desired to associate with nanoparticles
and gives examples of items found in the corresponding toolbox to achieve each
function.
The possibility to design very precise nanoparticles with polymers by tuning
nanoparticle properties to optimize the benet of the treatment for each patient
taking into account the individual variability while the safety prole will be high is
10 C. Vauthier
POLYMERIZATION METHODS
Emulsion/miniemulsion/microemulsion Interfacial polymerization
polymerization
Nanosphere Core-corona nanosphere Oil-containing nanocapsule Water-containing nanocapsule
POLYMERS
EMULSIONS SOLUTIONS
Precipitation Gelation Precipitation Gelation Self - assembling
Solvent Solvent Solvent Complex Amphiphilic
evaporation extraction diffusion formation copolymers
Nanosphere Nanosphere Nanogel Nanosphere Nanogel Nanogel
Polymersome
Core-corona Core-corona Core-corona Polyelectrolyte
nanosphere nanosphere nanosphere complex
Oil-containing Oil-containing Oil-containing Micelle-polyelectrolyte Micelle-

nanocapsule nanocapsule nanocapsule complex core hydrophobic core
Fig. 3 Summary of general principles of methods of preparation of nanoparticles from

polymerization procedure and protocols based on the use of polymers either included in the
dispersed phase of an emulsion/miniemulsion/microemulsion or occurring as a polymer solution.
This summary indicates the type of nanoparticles that are produced from these methods illustrating
the spherical species
(a) (b) (c)
100 nm
Fig. 4 Example of polymer nanoparticles obtained with different shapes as shown by scanning
electron micrograph. a spherical nanoparticles obtained from anionic emulsion polymerization of
isobutylcyanoacrylate (C. Vauthier, personal collection), b rod-like nanoparticles obtained by
nanoprecipitation of poly(-benzyl-l-glutamate) (Mw:70 kDa) (Adapted from Cauchois et al.
2013, reproduced with permission), and c 200 200 nm cylindrical nanoparticles made of poly
(lactide-co-glycolide) prepared by a print method (Adapted from Wang et al. 2011b, reproduced
with permission)
Table 1 Functionalities that may be associated with polymer nanoparticles designed as

nanomedicines
Function Tool Tool occurrence* Additional information
Therapeutic Therapeutic Drug molecule Small chemical molecules
agents and macromolecules from
biology, peptides,
proteins, and nucleic acids
Metal nanoparticles Gold nanoparticles
enhancing efcacy of
radiotherapy
Metal nanoparticles (gold
nanoparticles, magnetic
nanoparticles) enhancing
treatment based on
hyperthermia
Nanocrystals Upconverting
nanoparticles (UCNP) for
photodynamic therapy
Drug releasing Stimuli responsive Chemical stimuli: pH,
control system polymer oxidant, reductant
Physical stimuli: light,
temperature, and
ultrasound waves
Biochemical stimuli:
enzymatic degradation
Diagnostic Contrast agent for Ultrasmall paramagnetic Magnetic resonance
imaging iron oxide nanoparticles imaging
techniques (USPIO)
Perfluorocarbone Ultrasound imaging
Fluorescent tracers Optical imaging
occurring as molecular
compounds or metal
nanoparticles (quantum
dots)
Guidance Controlling Macromolecules arranged Stealthiness,
general at the nanoparticle mucoadhesion, diffusion
interactions with surface in tissues
tissues and the
immune system
Cellular and Molecular ligand highly Antibody or other types of
molecular specic to a well-dened proteins
targeting cell receptor Small molecule (folic acid
for instance)
External guidance Magnetic particles Targeting from the
application of an external
magnetic eld
*Examples of items of the toolbox to achieve each function
12 C. Vauthier
seen as an opportunity to elaborate personalized therapeutic protocols (Mura and

Couvreur 2012). To permit the development of suitable nanoparticles to be used in
personalized nanomedicine, nanoparticles should be tailor-made with a great flex-
ibility. Methods for the preparation need to satisfy all exigencies that are required to
make the nanoparticles a pharmaceutical compound having a given activity. In the
same time, there will be a need for methods fullling reproducible preparation of
nanoparticles with customized properties. Emerging approaches are based on the
development of platforms that allow preparation of nanoparticles which properties
can be tuned easily. For instance, those based on self-assembly of polymers are
progressing as they can be applied to assemble a family of polymers in which each
is bearing a different functionality to be included into one nanoparticle (De Miguel
et al. 2015; Bao et al. 2013). Another suitable method that offers possibilities to
integrate different functionality in a single nanoparticle is based on the superim-
position of polymer layers forming the nal nanoparticles layer-by-layer (Caruso
2001; Bao et al. 2013; Yan et al. 2014). These strategies allow the building of
multifunctional nanoparticles with high precision. Preparation of multifunctional
nanoparticles is also accessible by most of the other described methods providing
that the polymer that gives the structure of the nanoparticle also shows the required
properties. In general, this can be achieved customizing the design of polymers to
give them all desired features as explained in the Chap. 12 from Cammas. For
instance, this approach can be used to conceive stimuli responsive nanoparticles
delivering their cargo in well-controlled conditions (Mura et al. 2013). Surface
functionalization can be adjusted introducing postsynthesis modications. This is
often used to equip the nanoparticle surface with a targeting moiety to optimize
precision of the delivery method at the target site (Nicolas et al. 2013).
Postsynthesis modications are predominantly achieved by chemical methods but
the layer-by-layer approach is another option to achieve surface modication of
nanoparticles (Labouta and Schneider 2010; Poon et al. 2011; Bao et al. 2013;
Ejima et al. 2013; Nicolas et al. 2013; Yan et al. 2014). In the movement which
tends to increase the number of functionality to associate with nanoparticles, it is
nevertheless important to keep in mind that the complexity should not compromise
translation to clinic. Bottlenecks to development of highly sophisticated
nanomedicines may arise from their method of preparation among other factors.
Whatever will be the functions to associate with the nanoparticles, the method of
preparation needs to be scalable producing large amount of nanoparticles. It should
also be robust to insure the reproducible production of the nanoparticles and to
comply with the high rate of quality requested for pharmaceutical grade compounds
to insure their safety.
This part of the book was aimed to describe methods that can be used to produce
polymer nanoparticles that are interested to develop nanomedicines. Choice has
been made to illustrate methods from each group. Chapters 2 from Miladia et al.
and 3 from Tang and Prudhomme focus on methods based on nanoprecipitation
that are using polymer solutions while the precipitation of the polymer is induced
by a solvent shift. Chapter 4 proposed by Alcala-Alcala et al. described methods
based on the use of emulsions. Methods based on polymerization are described in

Chap. 5 proposed by Vauthier. The obtaining of nonspherical nanoparticles is the
subject of Chap. 6 proposed by Ponchel. All these chapters were written to provide
with basic and practical information to inspire the development of nanomedicines
made of polymer sharing the authors expertise on the key methods of preparation.
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Chapter 2
Nanoprecipitation Process: From Particle
Preparation to In Vivo Applications
Karim Miladi, Sana Sfar, Hatem Fessi and Abdelhamid Elaissari
Abstract Nanoparticles have been widely prepared during the past decades. In
fact, encapsulation could provide several advantages over conventional pharma-
ceutical forms (Miladi et al. in Int J Pharm 445(12):181195, 2013; Campos et al.
in J Colloid Sci Biotechnol 2(2):106111, 2013; Grando et al. in J Colloid Sci
Biotechnol 2(2):140145, 2013; De Melo et al. in J Colloid Sci Biotechnol 2
(2):146152, 2013; Mazzaferro et al. in J Colloid Sci Biotechnol 1(2):210217,
2012; Lira et al. in J Colloid Sci Biotechnol 2(2):123129, 2013; Wang et al. in J
Colloid Sci Biotechnol 1(2):192200, 2012). Although, several techniques have
been used for the preparation of submicron particles from preformed polymers,
nanoprecipitation is regarded as a quite simple and reproducible technique that
allows the obtaining of submicron-sized polymer particles. Additionally, many
research works have focused on the enhancement of the reproducibility of the
technique in order to render it more suitable for industrial applications.
Nanoprecipitation is still widely used to prepare particulate carriers which are based
on various polymers. Biomedical applications of such drug delivery systems are
multiple (Rosset et al. in J Colloid Sci Biotechnol 1(2):218224, 2012; Khan et al.
in J Colloid Sci Biotechnol 1(1):122128, 2012).
Keywords Supersaturation Nucleation Encapsulation Hydrophilic

molecules PLGA particles Microfluidics Bilamination Anticancer agents
K. Miladi H. Fessi (&) A. Elaissari (&)

Universit de Lyon, 69622 Lyon, France
e-mail: fessi@lagep.univ-lyon1.fr
A. Elaissari
e-mail: elaissari@lagep.univ-lyon1.fr
K. Miladi H. Fessi A. Elaissari
UMR 5007, Laboratoire DAutomatique et de Gnie Des Procds, LAGEP-CPE-308G,
Universit Lyon 1, Villeurbanne, CNRS, 43 bd. du 11 Nov.1918, 69622 Villeurbanne, France
K. Miladi S. Sfar
Laboratoire de Pharmacie Galnique, Universit de Monastir, Rue Avicenne,
5000 Monastir, Tunisia

DOI 10.1007/978-3-319-41421-8_2
18 K. Miladi et al.

Nanoprecipitation Flash nanoprecipitation Solvent displacement Interfacial

deposition Nanocapsules Nanospheres
1 Introduction
Nanoprecipitation is also called solvent displacement or interfacial deposition. It is

considered as one of the rst developed techniques used for the encapsulation of drug
molecules. This technique was developed by Fessi et al. (1989). Since its develop-
ment, the technique has been widely used for the encapsulation of mainly,
hydrophobic drugs in either nanocapsules or nanospheres. Many polymers were used
for this purpose, especially, biodegradable polyesters such as, poly(lactide) (PLA),
poly(lactide-co-glycolide) (PLGA), and poly(e-caprolactone) (PCL). Nanocapsules
are vesicular forms that exhibit core-shell structure in which the drug is mainly
conned to a reservoir or within a cavity surrounded by a polymer membrane.
Nanospheres are, however, matrix-type colloidal particles in which the drug is dis-
solved or dispersed within the polymer matrix. The drug molecule could be also
adsorbed on the surface of the nanocarrier (Mora-Huertas et al. 2010; Letchford and
Burt 2007). Nanoprecipitation is based on the interfacial deposition of polymers
following the displacement of a semi-polar solvent miscible with water from a
lipophilic solution (Fessi et al. 1989). It is an easy and reproducible technique that has
been widely used in the preparation of nanoparticles. Nanoprecipitation has many
advantages over other encapsulation techniques: (1) Simplicity (2) ease of scalability
(3) good reproducibility (4) large amounts of toxic solvents are avoided (5) obtaining
of submicron particle sizes with narrow size distribution, and (6) no need for using of
high energy input (Lassalle and Ferreira 2007). In 2005, Bilati et al. (2005) developed
a modied nanoprecipitation method designed for the encapsulation of hydrophilic
(a) (b)
Fig. 1 a Scanning electron microscopy (SEM) micrographs of PLGAPEG nanoparticles (Anand

et al. 2010). b SEM micrograph of nanoparticles prepared by nanoprecipitation (Costantino et al.
2005). Source: Elsevier
2 Nanoprecipitation Process: From Particle Preparation to In Vivo Applications 19
molecules. Figure 1a, b show scanning electron microscopy (SEM) images of

nanoparticles prepared by nanoprecipitation.
2 Technical Aspects
2.1 Mechanism of Particle Formation by Nanoprecipitation
Nanoprecipitation is a simple and reproducible technique that produces particles with

narrow size distribution over a wide range of processing parameters (Budhian et al.
2007). It requires two miscible phases: an organic/oil phase and an aqueous phase
(see Fig. 1). Lince et al. (2008) showed that the process of particle formation in the
nanoprecipitation method includes three phases: nucleation, growth, and aggrega-
tion. Supersaturation was described as the driving force of all these phenomena. It is
dened by the ratio of polymer concentration to polymer solubility in the organic
solvent. Supersaturation is crucial because it also determines the nucleation rate.
Here, fluid dynamics and mixing of phases play an important role. In fact, they
influence supersaturation and owing to the rapidity of particle formation process,
they determine also the nucleation rate. Consequently, poor mixing produce few big
nanoparticles (low nucleation rate) while good mixing conditions give birth to high
nucleation rates, i.e., larger population of smaller nanoparticles (Lince et al. 2008).
Quintanar-Guerrero et al. (1998), however, explained nanoparticles formation as a
result of differences in surface tension. This nding was based on research carried out
by Davies on mass transfer between two liquids and on the GibbsMarangoni effect
(McManamey et al. 1973; Davies 1975). In fact, a liquid with a high surface tension
(aqueous phase) pulls more strongly on the surrounding liquid than one with a low
surface tension (organic phase solvent). This difference between surface tensions of
the aqueous and the oil phase causes interfacial turbulence and thermal inequalities in
the system. This leads to the continuous formation of vortices of solvent at the
interface of both liquids (Fig. 2). The organic solvent diffuses from regions of low
surface tension which causes gradual precipitation of the polymer on the oil surface
and forms nanocapsules (Mora-Huertas et al. 2010).
2.2 Drugs
Nanoprecipitation technique is essentially used to encapsulate hydrophobic mole-

cules. However, some good results were also obtained with hydrophilic molecules.
Table 1 contains some examples of drugs encapsulated by nanoprecipitation and
their corresponding nature. More examples will be given in the Chap. 13 by
Zandanel and Charrueau. Most of the drug encapsulation studies focused either on
poorly water-soluble or amphiphilic compounds that are highly soluble in water
20 K. Miladi et al.
Drug
Polymer
Organic
solvent Solvent evaporation
Aqueous Drug encapsulated
phase
in nanoparticles
Magnetic
stirrer
Interfacial deposition Formation of the

of the polymer nanoparticles
Nanoprecipitation technique
Fig. 2 The nanoprecipitation technique
miscible organic solvents. However, many studies used other approaches to allow
the encapsulation of hydrophilic molecules. Three main approaches have been
investigated: (1) The dissolving of the hydrophilic molecule in the external aqueous
phase, (2) the use of a cosolvent, or (3) the dissolution of small amounts of the
molecule in the organic phase. Bilensoy et al. (2009) encapsulated mitomycin C in
PCL-based nanoparticles coated with chitosan by dissolving the hydrophilic drug in
the aqueous phase. Peltonen et al. (2004) used ethanol and methanol as cosolvents
and added them to an aqueous solution of cromoglucate to allow drug dissolution in
the organic phase. Govender et al. used nanoprecipitation to prepare PLGA
nanoparticles containing the water-soluble molecule, procaine hydrochloride.
Experimental procedure consisted on the dissolution of PLGA and a specied
quantity of the drug in acetonitrile (Govender et al. 1999).
2.3 Oil Phase
The oil phase consists on an organic solvent which is miscible to water such as,
ethanol or acetone. The organic phase contains also the polymer and the
hydrophobic drug. Other compounds could be added to the solvent such as
triglycerides, mineral or vegetable oils, or hydrophobic surfactants. Addition of
mineral or vegetable oils allow obtaining nanocapsules rather than nanospheres.
Surfactants hamper the aggregation of the particulate carriers. Table 2 shows some
examples of oil phases that could be used in nanoprecipitation. One can notice that
acetone is the most commonly used organic solvent in nanoprecipitation.
Table 1 Examples of drugs encapsulated in polymer nanoparticles by nanoprecipitation

Hydrophilic molecules References Hydrophobic References
molecules
Cromoglucate Peltonen et al. (2004) Olanzapine Seju et al.
(2011)
Doxorubicin Sanson et al. (2010), Paclitaxel Wang et al.
Han et al. (2013) (2013)
Bovine Serum Albumin Gao et al. (2006) Amphotericin-B Van de Ven
et al. (2012)
Levofloxacin Cheow and Hadinoto Aceclofenac Katara and
(2010) Majumdar
(2013)
10-Hydroxycamptothecin Zhang et al. (2007) Curcumin Mazzarino et al.
(2012)
Mitomycin C Bilensoy et al. (2009) Retinoic acid Almouazen
et al. (2012)
Heparin Eidi et al. (2010) Naringenin Krishnakumar
et al. (2011)
Stevioside Barwal et al. (2013) Efavirenz Seremeta et al.
(2013)
Salbutamol Hyvnen et al. (2005) Naproxen Rosset et al.
(2012)
Procaine Govender et al. Chloroaluminum Siqueira-Moura
(1999) phthalocyanine et al. (2013)
2.4 Water Phase
The aqueous phase is usually water but some other excipients such as hydrophilic
surfactants could be added to avoid particles aggregation. These surfactants could
be natural or synthetic. Likely, some polymers could be added to aqueous phase as
coating materials. Hydrophilic drugs could be dissolved in the aqueous phase.
Table 3 shows some examples of aqueous phases that could be used in the nano-
precipitation method. As it can be seen, the most used aqueous phase is simply
water and the most used surfactant is Pluronic F68.
2.5 Polymers
Numerous polymers have been used to prepare nanoparticles by nanoprecipitation.

To be suitable for in vivo applications, polymers must be biodegradable and bio-
compatible. The most used materials are biodegradable polyesters such as PLGA,
PCL, PLA, and Eudragit. Coating materials could also be grafted or adsorbed to the
initial polymer to confer new surface properties such as, mucoadhesion, protection
from reticuloendothelial system (stealth particles) or to tune hydrophilicity.
22 K. Miladi et al.
Table 2 Examples of organic phases used in nanoprecipitation

Composition of the oil phase References
Oil phases comprising one solvent
Acetone Bazyliska et al. (2013), Bernabeu et al. (2013), Shah
et al. (2014), Siqueira-Moura et al. (2013), Barwal et al.
(2013), Peter Christoper et al., Pavot et al. (2013), Das
et al. (2013a), Crpanl et al. (2011), Gupta et al. (2010),
Liu et al. (2010), Joshi et al. (2010), Cheng et al. (2008),
Muthu et al. (2009), Pertuit et al. (2007), Danhier et al.
(2009a), irpanli et al. (2009), Yuan et al. (2008), Vila
et al. (2004), Fonseca et al. (2002), Leroueil-Le Verger
et al. (1998), Nafee et al. (2013), Zili et al. (2005), Yenice
et al. (2008), Memisoglu-Bilensoy et al. (2005), Ali et al.
(2013), Zhang and Zhuo (2005), Das et al. (2013b),
Kumar et al. (2012), Paul et al. (2013), Musumeci et al.
(2013), Mazzarino et al. (2012), Eidi et al. (2012)
Ethanol Ubrich et al. (2005), Perret et al. (2013a, b)
Ehtylacetate Tao et al. (2013)
Acetonitrile Wang et al. (2010), Dong and Feng (2004, 2007), Leo
et al. (2004)
THF de Miguel et al. (2013), Peracchia et al. (1999),
Kaewprapan et al. (2012)
DMF Suen and Chau (2013)
DMSO Esfandyari-Manesh et al. (2013)
PEG Ali and Lamprecht (2013)
Oil phases comprising solvent mixtures
Acetone/ethanol Noronha et al. (2013), das Neves et al. (2013), Le
Broc-Ryckewaert et al. (2013)
Acetone/methanol Das and Suresh (2011)
Acetone/coconut oil Bazyliska et al. (2013)
Solution of capric/caprylic Moraes et al. (2009)
triglyceride mixture in acetone
Acetone and mixture of Loyer et al. (2013)
chloroform and NEt3
Sorbitan monostearate, mineral oil Rafn Pohlmann et al. (2002)
and acetone
thf/water Kaewprapan et al. (2012)
THF tetrahydrofuran, DMF dimethylformamide, DMSO dimethylsulfoxide, PEG poly(ethylene
glycol)
Copolymers could also be used (Miladi et al. 2014). Table 4 contains some examples
of polymers used for the preparation of nanoparticles by nanoprecipitation.
Table 3 Examples of aqueous phases used in nanoprecipitation

Composition of the water phase References
Water Esfandyari-Manesh et al. (2013), de Miguel et al. (2013),
das Neves et al. (2013), Suen and Chau (2013), Das et al.
(2013a), Le Broc-Ryckewaert et al. (2013), Le
Broc-Ryckewaert et al. (2013), Liu et al. (2010), Danhier
et al. (2009a), Dong and Feng (2007), Yuan et al. (2008),
Nafee et al. (2013), Loyer et al. (2013), Yenice et al.
(2008), Memisoglu-Bilensoy et al. (2005), Peracchia et al.
(1999), Zhang and Zhuo (2005), Perret et al. (2013a, b),
Kaewprapan et al. (2012)
Aqueous solution of Pluronic Noronha et al. (2013), Shah et al. (2014), Siqueira-Moura
F68 et al. (2013), Barwal et al. (2013), Crpanl et al. (2011),
irpanli et al. (2009), Dong and Feng (2004), Leroueil-Le
Verger et al. (1998), Ubrich et al. (2005), Das et al.
(2013b), Kumar et al. (2012), Paul et al. (2013), Eidi et al.
(2012)
Aqueous solution of poloxamer Peter Christoper et al., Muthu et al. (2009)
407
Aqueous PVA solution Ali and Lamprecht (2013), Gupta et al. (2010), Das and
Suresh (2011), Pertuit et al. (2007), Tao et al. (2013),
Aqueous solution of Tween 80 Moraes et al. (2009), Zili et al. (2005)
Aqueous solution of Cremophor Bazyliska et al. (2013)
EL
Water containing TPGS Bernabeu et al. (2013)
Water/ethanol Pavot et al. (2013), Cheng et al. (2008)
Solution of Pluronic F 127 in Joshi et al. (2010)
phosphate buffer (pH 9.0)
PBS (0.01 M, pH 7.4) Letchford et al. (2009)
Ethanol Vila et al. (2004)
Aqueous poloxamer 188 solution Fonseca et al. (2002)
Aqueous sodium cholate solution Leo et al. (2004)
Aqueous solution of polysorbate Rafn Pohlmann et al. (2002)
80
Aqueous sodium taurocholate Ali et al. (2013)
solution
Water/ethanol mixture containing Musumeci et al. (2013)
Tween 80
Aqueous solution of acetic acid Mazzarino et al. (2012)
and poloxamer 188
PVA poly(vinyl alcohol), TPGS alphatocopheryl poly(ethylene glycol) 1000 succinate, PBS
phosphate buffer saline
2.6 Influence of Operating Conditions
The technique is based on the addition of one phase to the other under moderate
magnetic stirring (see Fig. 1). The subsequently obtained suspension of
24 K. Miladi et al.
Table 4 Examples of polymers used in nanoprecipitation

Polymer References
PLGA Bazyliska et al. (2013), Shah et al. (2014), Siqueira-Moura
et al. (2013), Peter Christoper et al., Ali and Lamprecht
(2013), Das et al. (2013a, b), Le Broc-Ryckewaert et al.
(2013), Crpanl et al. (2011), Gupta et al. (2010), Wang
et al. (2010), Joshi et al. (2010), Moraes et al. (2009), Cheng
et al. (2008), Muthu et al. (2009), Pertuit et al. (2007),
Danhier et al. (2009a), irpanli et al. (2009), Fonseca et al.
(2002), Leroueil-Le Verger et al. (1998), Ali et al. (2013),
Paul et al. (2013), Tao et al. (2013), Musumeci et al. (2013)
PCL Noronha et al. (2013), das Neves et al. (2013), Crpanl et al.
(2011), irpanli et al. (2009), Leroueil-Le Verger et al.
(1998), Zili et al. (2005), Yenice et al. (2008), Rafn
Pohlmann et al. (2002), Mazzarino et al. (2012)
PLA Barwal et al. (2013), Pavot et al. (2013), Leroueil-Le Verger
et al. (1998), Leo et al. (2004), Rafn Pohlmann et al. (2002)
Eudragit RL Ali and Lamprecht (2013), Ubrich et al. (2005)
Eudragit RS 100 Das and Suresh (2011)
Eudragit RS Ubrich et al. (2005)
Eudragit RS PO Eidi et al. (2012)
PEG-PLGA Ali and Lamprecht (2013), Liu et al. (2010), Danhier et al.
(2009a), Musumeci et al. (2013)
PEG-b-PCL Suen and Chau (2013), Danhier et al. (2009a), Nafee et al.
(2013)
PEGPCL-PEG Zhang and Zhuo (2005)
PLA-PEG Vila et al. (2004)
PCL conjugated to Pertuit et al. (2007)
5-aminosalicylic acid
PCL-TPGS Bernabeu et al. (2013)
mPEG-PLA Wang et al. (2010), Dong and Feng (2004, 2007)
MePEG-b-PCL Letchford et al. (2009)
PLA and hydrophobically Yuan et al. (2008)
modied Chitosan
PBLG derivatives de Miguel et al. (2013)
Amphiphilic derivatives of Loyer et al. (2013)
poly(benzyl malate)
b-CDC6 Memisoglu-Bilensoy et al. (2005)
b-amphiphilic cyclodextrin Perret et al. (2013a)
PEGylated and non Peracchia et al. (1999)
PEGylated PHDCA polymer
PLGA and DOTAP Kumar et al. (2012)
Dextran decanoate Kaewprapan et al. (2012)
PBLG poly(c-benzyl-l-glutamate), b-CDC6 cyclodextrin modied on the secondary face with
6C aliphatic esters, PHDCA poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl-
cyanoacrylate), DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
nanoparticles is subjected to evaporation of the organic solvent by a rotavapor or at

ambient temperature. The next step consists of the removing of the aqueous phase
either by ultracentrifugation or freeze drying. The obtained nanoparticles are
characterized by the measurement of size, zeta potential, and by transmission
electron microscopy (TEM) or scanning electron microscopy (SEM). Many oper-
ating conditions could exert important effect on the characteristics of the obtained
nanocarriers. Effects of these parameters are summarized in Table 5.
2.6.1 Amount of Polymer
Many studies evaluated the effect of the variation of polymer amount on the
characteristics of the nanoparticles. Table 5 presents some examples for the effect
of polymer amount on nanoparticle characteristics. As it can be seen, an increase of
polymer amount generally increased particle size and encapsulation efciency. This
could be explained by an increase of the viscosity of the oil phase which gives birth
to bigger particles and render drug diffusion more difcult. According to Legrand
et al. (2007), polymer concentration in organic solvent should remain below the
limit between the dilute and semi dilute regime to avoid formation of aggregates.
2.6.2 Molecular Weight of the Polymer
Polymer molecular weight is a crucial parameter that could exert strong influence
on particles properties. Lince et al. evaluated the effect of PCL molecular weight
on particle size. The greater the molecular weight, the smaller the size of the
particles. An increase of polymer molecular weight led to a decrease of particles
size from 144.1 to 93.6 nm. This phenomenon was explained by faster precipitation
of the high molecular weight PCL owing to its more limited solubility in the
acetone/water medium (Lince et al. 2008; Seremeta et al. 2013). Conversely,
Blouza et al. reported an increase of particles size following an increase of polymer
molecular weight. This nding was explained by higher viscosity of the organic
solution in the case of high polymer molecular weight (Limayem Blouza et al.
2006). In another study, Legrand et al. showed no influence of the molecular weight
of PLA on the size of nanoparticles produced in the absence of surfactant. In
contrast, they found that the yield of formation of nanoparticles was greatly
influenced by the molecular weight of the polymer highlighting that there is an
optimal molecular weight of PLA to obtain high production rate of nanoparticles. It
was suggested that all PLA chains with molecular weight outside the optimal range
are precipitating as aggregates and contribute to reduce the yield of production of
nanoparticles (Legrand et al. 2007).
26 K. Miladi et al.
Table 5 Influence of operating conditions on nanoparticles properties

Operational Action Effect References
parameter
Drug amount Increase No signicant Chorny et al. (2002)
effect on particles
size
Increase of Govender et al. (1999), Khayata et al.
particles size (2012a)
No signicant Chorny et al. (2002)
effect on drug
loading
Polymer Increase Increase of Chorny et al. (2002), Limayem Blouza et al.
amount particles size (2006), Simek et al. (2013), Dong and Feng
(2004), Ali et al. (2013), Bazyliska et al.
(2013), Khayata et al. (2012a), Lince et al.
(2008), Plasari et al. (1997), Nehilla et al.
(2008) and Guhagarkar et al. (2009)
Increase of drug Chorny et al. (2002), Dong and Feng (2004)
loading
Polymer Increase Increase of Limayem Blouza et al. (2006), Holgado et al.
molecular particles size (2012)
weightb Decrease of Seremeta et al. (2013)
particles size
No signicant Budhian et al. (2007)
effect on particles
size
effect on drug
loading
Oil to water Decrease Decrease of Budhian et al. (2007), Bazyliska et al.
phase ratio particles size (2013) and Fonseca et al. (2002)
Increase of Limayem Blouza et al. (2006), Nehilla et al.
particles size (2008)
No signicant Chorny et al. (2002)
effect on particles
size
Increase Decrease of drug Budhian et al. (2007), Limayem Blouza et al.
loading (2006) and Guhagarkar et al. (2009)
Decrease of Dong and Feng (2004)
particles size
Increase of Stainmesse et al. (1995)
particles size
Organic phase Increase Decrease of the Lince et al. (2008)
addition rate particles size
(continued)
Table 5 (continued)
Operational Action Effect References
parameter
Surfactant Increase Decrease of the Contado et al. (2013), Siqueira-Moura et al.
amount particles size (2013) and Guhagarkar et al. (2009)
Decrease then Budhian et al. (2007), Limayem Blouza et al.
increase in (2006) and Khayata et al. (2012a)
particles size
No signicant Dong and Feng (2004)
effect on particles
size
effect on drug
loading
Stirring rate Increase Decrease Asadi et al. (2011)
Organic Increase No signicant Chorny et al. (2002)
solvent effect on particles
evaporation size
rate No signicant Chorny et al. (2002)
effect on drug
loading
a
Yield of nanoparticle formation increases while concentration of polymer remains in the dilute
regime (Legrand et al. 2007)
b
Yield of nanoparticle production decrease when polymer molecular weight diverge from the
optimal value (Legrand et al. 2007)
2.6.3 Amount of Surfactant
Stabilizer amount influence on particle properties has been largely studied. An

increase in size of PLGA nanoparticles at high poly(vinyl alcohol)
(PVA) concentrations (510 %) has been reported by Zweers et al. (2003) and
Arica and Lamprecht (2005), while Allemann et al. (1992) reported a continuous
decrease in particle size. Lamprecht et al. (2001) noticed also that an increased
sodium cholate concentration led to a particle size reduction. In order to explain this
contradiction, Budhian et al. (2007) and Arica and Lamprecht (2005) proposed the
presence of two competing effects at high PVA concentrations: an enhanced
interfacial stabilization that caused a size decrease and an increased viscosity of the
aqueous phase which led to a less favorable mixing efciency and thus, to a size
increase. The concentration of PVA at which one effect starts dominating over the
other depends on the system and processing parameters. For PLGA nanoparticles,
the size rst decreased due to better stabilization and then increased at higher PVA
concentrations due to high aqueous phase viscosity (Arica and Lamprecht 2005;
Budhian et al. 2007). Guhagarkar et al. noticed a sharp decrease in particle size
from greater than 1000 nm to around 300 nm as PVA concentration increased from
0.1 to 0.5 %. Further increase in PVA concentration to 4 % resulted in an increase
in particle size. In fact, the subsequent increase in viscosity of external aqueous
28 K. Miladi et al.
phase hampered effective diffusion of organic phase leading to larger droplet for-
mation and thus, an increase of mean size (Guhagarkar et al. 2009). Similar results
at higher PVA concentrations have been reported (Quintanar-Guerrero et al. 1996;
Moinard-Chcot et al. 2008; Murakami et al. 1997).
Stabilizer nature is another crucial parameter that could have an impact on
particle size. For instance, Van de Ven et al. (2012) showed that smaller
nanoparticles were prepared using Poloxamer 188 in combination with sodium
cholate, whereas the largest ones were obtained with PVA. Likely, studies per-
formed by Limayem Blouza et al. (2006) and Khayata et al. (2012a) showed that
surfactant type changed the size of vitamin E-loaded nanocapsules as Tween 80
gave the smallest particles.
2.6.4 Oil to Water Phase Ratio
Fonseca et al. (2002) reported that doubling the aqueous phase volume resulted in a
signicant decrease in the size of PLGA nanoparticles. In fact, in nanoprecipitation,
the nanoparticles are formed due to rapid solvent diffusion to the aqueous phase
(Quintanar-Guerrero et al. 1997). Consequently, as the volume of the aqueous
phase increases, the diffusion of the organic solvent in the aqueous phase increases
which decreases particle size. Additionally, an increase of the aqueous phase vol-
ume increases the drug amount that can be dissolved in the aqueous phase, which
causes more drug loss into the aqueous phase (Budhian et al. 2007).
2.6.5 Solvents Nature and Order of Phases Addition
Choice of solvents depends on requirements of the method and physicochemical

properties of the polymer. In fact, organic solvent must respond to three criteria:
(1) dissolving capacity toward polymer (2) miscibility with water, and (3) low
boiling point in order to facilitate evaporation. Aqueous phase consists, however, of
a nonsolvent for the polymer. This phase would thus cause polymer precipitation to
form nanoparticles. It was shown that theta solvent (a solvent in which polymer
coils act like ideal chains) tends to give smaller nanoparticles than other solvents
(Flory 1969; Legrand et al. 2007). The nature of the aqueous and oil phase and the
order of phases addition could strongly influence nanoparticles properties. For
instance, influence of aqueous phase pH was described by Govender et al. who
reported an increasing drug entrapment and drug content trend due to an increase of
aqueous phase pH from 5.8 to 9.3. In fact, aqueous phase pH influenced the
ionization of the encapsulated drug, procaine hydrochloride and hence, its solu-
bility. Consequently, an increase of the aqueous phase pH decreased the solubility
of procaine hydrochloride and enhanced drug entrapment into nanoparticles
(Govender et al. 1999). The effect of oil nature was also evaluated by (Khayata
et al. 2012a) who noticed that nanoparticles prepared with castor oil were the largest
ones. This was explained by the higher viscosity of this oil. In fact, it was shown
that as oil viscosity was higher, dispersed phase viscosity increased. Polydispersity
index (PDI) also augmented when the oil viscosity increased. This nding was
similar to results reported by Rafn Pohlmann et al. who noticed an increase in
particle diameter and PDI with an increase of oil viscosity (Rafn Pohlmann et al.
2002; Khayata et al. 2012a). Effect of organic solvent nature was evaluated by other
studies that had shown that solvents of high polarity like acetone gave birth to small
nanoparticles by promoting rapid diffusion to the aqueous phase (Legrand et al.
2007; Thioune et al. 1997). It was shown that a lower dielectric constant of the
organic solvent resulted in larger particles size (Bilati et al. 2005). Guhagarkar et al.
compared particles size and entrapment efciency of poly(ethylene sebacate) (PES)-
based nanoparticles. Particle size decreased signicantly when tetrahydrofuran
(THF) and acetone were used in combination as solvent compared to THF alone at
all polymer concentrations. This was explained by more rapid diffusion of the more
polar solvent acetone into the nonsolvent phase that favored the formation of
smaller nanoparticles. In fact, the dielectric constant of THF/acetone (1:1) was
found to be 14.5 compared to 7.5 for THF alone. In addition, increased diffusivity
of the organic solvent due to addition of acetone could cause leaching of the drug
into the aqueous phase thus, decreasing encapsulation efciency (Guhagarkar et al.
2009). The order of phases addition seems also to exert an effect on particles
characteristics. The effect of adding the aqueous phase into the organic phase versus
adding the organic phase into the aqueous phase was determined by Khayata et al.
who prepared vitamin E-loaded nanocapsules. Obvious aggregation between par-
ticles was observed when the aqueous phase was added to the organic phase. This
was explained by the presence of the stabilizer in the aqueous phase that plays an
important role in stabilizing the nanocapsule formed. This aggregation disappeared
when organic phase was added to the aqueous phase (Khayata et al. 2012a). Bilati
et al. used proposed a nanoprecipitaion technique which is intended to hydrophilic
drugs encapsulation. Used solvents consisted of polar aprotic solvents, ketones, or
esters. Dimethylsulfoxide was described as an interesting solvent especially for
protein dissolution. Nonsolvent was chosen on the basis of its polarity in order to
enhance nal drug loading. Here, alcohols were shown to be suitable nonsolvents
that could provide nanoparticles with different sizes. The same mechanism
described previously for the particles formation is involved in particles formation as
miscible solvents are always used (Bilati et al. 2005).
2.6.6 Stirring Rate
In nanoprecipitation, the most commonly used stirring method is magnetic stirring.

An increase of the stirring rate generally results in a decrease in the particles size.
This is explained by more efcient shear mixing and thus, more rapid diffusion of
the organic solvent to the water phase (Asadi et al. 2011).
One can conclude that many operating parameters have to be managed to obtain
nanoparticles bearing good characteristics. Table 6 contains some approaches to be
followed to monitor major particles properties.
30 K. Miladi et al.
Table 6 Principles and parameters that control particle size and drug content for nanoparticles
prepared by nanoprecipitation (from Budhian et al. 2007) with modications)
Principles Parameters
Decrease Increase shear stress Increase stirring rate
particle size Increase volume of aqueous phase
Decrease polymer concentration in
organic phase
Increase surfactant concentration in
aqueous phase
Decrease polymer molecular weight
Increase Increase shear stress Decrease stirring rate
particle size Decrease volume of aqueous phase
Increase polymer concentration in
organic phase
Decrease surfactant concentration in
aqueous phase
Increase polymer molecular weight
Increase Inhibit drug diffusion during Increase particle size
drug organic solvent evaporation Decrease relative volume of organic
loading Increase drug-polymer interaction solvent
Increase polymer concentration in
organic phase
Intermediate polymer molecular weight
Select organic solvent with intermediate
drug-solvent interactions
Reduce drug solubility in the aqueous
phase (alter pH)
Include specic interactions between
drug and polymer end groups
3 Innovative Approaches Using Nanoprecipitation
Since the rst discovery of the technique, many efforts have been made to improve its
reproducibility, scalability, and safety. Enhancement of reproducibility could mini-
mize inter-batch variations while improvement of scalability allows the obtaining of
formulations which are easily applicable in the pharmaceutical industry. Safety could
be provided by avoiding the use of toxic organic solvents. Most common approaches
are presented in Table 7. They consisted of the use of innovative mixing devices such
as, T-shape mixer (Briancon et al. 1999), membrane contactor (Khayata et al.
2012b), microfluidics (Bally et al. 2012) or flash nanoprecipitation technique
(DAddio and Prudhomme 2011).
3.1 Membrane Emulsication
Scalability is one of the major encountered limitations in the manufacture of

nanoparticles. Conventional nanoprecipitation did not allow the production of large
Table 7 Applications of innovative approaches to obtain nanoparticles based on nanoprecipitation carried out with a mixing device
Technique Drug Polymer Oil phase Water phase Size Zeta potential References
(nm) (mV)
T shape mixer Eudragit Acetone/isopropanol Aqueous 100500 Briancon et al.
mixture solution of (1999)
surfactant
Membrane Vitamin E PCL Acetone Aqueous 250353 20(15) Khayata et al.
contactor solution of (2012b)
Tween80
Membrane Vitamin E PCL Acetone Aqueous 170393 19.4(12.4) Khayata et al.
contactor solution of (2012a)
Tween80
Microfluidics Linear polymers are poly THF containing a Water 76217 Bally et al.
(methyl methacrylate)s and nonionic surfactant (2012)
branched polymers (Cremophor ELP)
Flash b-carotene Poly(styrene)-block-poly THF Water 801000 Johnson and
nanoprecipitation (ethylene oxide) Prudhomme
(2003a)
Flash PMMAs* with coumarin side THF Water 140320 Chung et al.
nanoprecipitation functionality (PCM) (2013)
Flash Poly(MePEGCA-co-HDCA))* Acetone Water 100300 50(8) Valente et al.
nanoprecipitation (2012)
2 Nanoprecipitation Process: From Particle Preparation to In Vivo Applications
*PMMAs poly(methyl-methacrylic acids), poly(MePEGCA-co-HDCA) poly(methoxy poly(ethylene glycol) cyanoacrylate-co-hexadecyl-cyanoacrylate)

31
32 K. Miladi et al.
scale batches. Membrane contactor could be an interesting alternative in such cases.

The technique is relatively simple and could be used to produce large volumes of
colloidal dispersions (Yedomon et al. 2013). It has also been shown to be suitable for
the preparation of polymer nanoparticles (Charcosset and Fessi 2005; Limayem
Blouza et al. 2006; Khayata et al. 2012b). Membrane emulsication involves the
permeation of the dispersed phase through a porous membrane into a tangentially
moving continuous phase (see Fig. 3a, b). The organic phase is pressed through the
membrane pores allowing the formation of small droplets. The precipitation occurs
between the droplets of the organic phase and the aqueous phase flowing tangen-
tially to the membrane surface (Khayata et al. 2012b). Khayata et al. performed
accelerated stability studies on vitamin E-loaded nanocapsules prepared by con-
ventional nanoprecipitaion and by a membrane contactor. These studies showed
good physical and chemical stability for both particles. However, nanocapsules
prepared by conventional nanoprecipitation were stable for a longer time
(Khayata et al. 2012b).
Fig. 3 a Experimental setup

of the membrane contactor
technique (Limayem Blouza
et al. 2006). b The membrane
module (Khayata et al.
2012b). Source: Elsevier
3.2 Microfluidics Device
Nanoprecipitation is usually performed via one-pot pouring of the polymer solution

into the nonsolvent, or by dropwise addition of one phase into the other.
Microfluidic processes, using a hydrodynamic flow-focusing setup (Karnik et al.
2008; Rhee et al. 2011) or a conned impinging jet reactor (Johnson and
Prudhomme 2003b; Lince et al. 2011; Nagasawa et al. 2005) have emerged to
improve the mixing of the two phases. Bally et al. used a continuous-flow nano-
precipitation process in which, a diluted polymer solution and water were separately
pumped and nanoprecipitation occurred within the micromixer. The latter consisted
either of either a T-junction or a High Pressure Interdigital Multilamination
Micromixer (HPIMM) (see Fig. 4 for HPIMM). The obtained suspension of
nanoparticles could be collected at the outlet of the micromixer (Bally et al. 2012).
Effect of the proportion of solvent and nonsolvent which is dened by the
parameter R was investigated by Bally et al.
Volume flow ratewater

R
Volume flow ratepolymer solution
It was shown that R managed the number of formed particles whatever was the
mechanism considered. In nucleation mechanism, increasing R leads to higher
supersaturation and more nuclei, which decrease the nal particle size. In the
mechanical mechanism, a higher value of R increases the potential interface and
more droplets are formed during phase separation. As a consequence, the local
concentration of the polymer is decreased which leads to smaller nanoparticles. It
was shown also that particles size depended both on initial polymer concentration
(C) and on the value of R. At low R value, (R = 3), particle size did not signicantly
change at variable C. This was explained by the presence of two competing
Fig. 4 Overview of HPIMM inner microstructure, used for nanoprecipitation (Bally et al. 2012).
Source: Elsevier
34 K. Miladi et al.
mechanisms which are nucleation and growth mechanism. Nucleation rate was
shown to increase with C which decreased particle size. Conversely, at high
polymer concentrations ( 1 wt%), growth phenomena appeared due to proximity
of polymer chains. It was concluded that higher nucleation rate nally compensated
with higher growth probability when C increases. However, following an increase
of R to 10, size of the particles increased from 106 to 210 nm with C. This sig-
nicant difference was attributed to more aggregation at high polymer concentra-
tion. Aggregation of growing particles also contributed to the increase of particle
size. The effect of the mixing process on the particles size was also studied as it was
previously shown to affect nanoparticles properties (Lince et al. 2008). Bally et al.
compared conventional T-junction, (operating via bilamination mixing) with a
multilamination micromixer. Obtained data showed that bilamination mixing gave
bigger particles with sizes close to ones obtained by conventional nanoprecipitation.
This proves a poor mixing ability. Consequently, ne mixing was described as
crucial to produce small nanoparticles at an initial polymer concentration of 1 wt%.
Additionally, it was shown that micromixer-assisted nanoprecipitation gave small
nanoparticles using less nonsolvent. According to Bally et al, a value of R = 2 led
to nanoparticles lower than 200 nm whereas at least R = 10 is required for con-
ventional nanoprecipitation to obtain the same size. In addition, micromixing allow
nanoprecipitation of polymer solution with concentrations up to 5 wt% which is
impossible in conventional method in which polydisperse samples were obtained
(Bally et al. 2012).
3.3 Flash Nanoprecipitation (FNP)
Simple nanoprecipitation carried out with a conventional process results in

heterogeneous mixing resulting in polydispersed particle sizes. FNP, however, is a
scalable process that could be used to prepare nanoparticles with controlled size
distribution and a high drug loading rate. This technique was rst described by
Johnson and Prudhomme (2003a) to produce nanoparticles encapsulating
hydrophobic drugs. FNP produces nanoparticles with a narrow size distribution
ranging from 80 to 1 m. The nanoparticles are obtained via a rapid precipitation
process. FNP offers also high loading capacity and the ability to encapsulate
multiple drugs in the same nanoparticle. Several successful applications of FNP
have been reported for encapsulation of various hydrophobic drugs, peptides,
imaging agents, or a combination of both therapeutics and inorganic colloids
(Chen et al. 2009; Budijono et al.; Kumar and Adamson 2010; Shi et al. 2012).
More information about the potential of this technique is given in Chap. 3 from
Tang and PrudHomme (Fig. 5).
Fig. 5 A schematic
representation of mutli-inlet
vortex mixer used in FNP
(DAddio and Prudhomme
2011). Source: Elsevier
4 In Vivo Applications of Nanoparticles Designed

by Nanoprecipitation
Nanoparticles designed by the nanoprecipitation technique were intended to various

in vivo applications. Some of these formulations are summarized in Table 8, which
also contains some technical aspects of the formulations such as the used polymers,
the different phases, and the corresponding in vivo application. Only recent for-
mulations that have been assessed in vivo were taken into account.
4.1 Example of Nanoparticles Developed for Cancer

Therapy
Many anticancer agents were encapsulated by the use of the nanoprecipitation

technique. Nanoparticles may target cancer cells by passive and active way. Passive
way is related to the reduced particles size which allows nanocarriers to cross
through fenestrations of endothelial cells and reach tumors. Thanks to the leaky
vasculature and the poor lymphatic drainage, Enhanced Permeability and Retention
effect (EPR) appears, which enhances the uptake of drugs. Active targeting, how-
ever, permits the delivery of the drug to a well-dened tissue or cell by the help of a
molecular recognition which occurs between a ligand grafted on the nanoparticles
and a receptor exposed on the outside of target cell surface membrane.
Table 8 Examples of nanoparticles prepared by the nanoprecipiation technique and assessed in vivo
36
Drug Material Organic phase Non organic phase Size (nm) Zeta potential In vivo Reference
(mV) application
Doxorubicin Gelatin-co-PLA-DPPE Acetone Water 131.5 Cancer Han et al.
(2013)
Aceclofenac Eudragit RL100 Acetone 0.02 % (w/v) 75.52184.36 22.532.6 Ocular Katara and
Tween 80 in water inflammation Majumdar
(2013)
Melatonin PLGA and PLGA Acetone water/ethanol Intraocular Musumeci
PEG mixture (1:1 v/v), pressure et al. (2013)
containing 0.5 %
(w/v) of Tween 80
Retinoic acid PLA 0.75 % Miglyol in 0.05 % of 153.6229.8 10.4(29.4) Glioma Almouazen
acetone Montanox VG 80 et al. (2012)
in water
Paclitaxel Hydrophobized Acetone Water 154.6 Cancer Lee et al.
pullulan Dimethylformamide 253 nm (2012)
DMSO 132.6 nm
Dimethylacetamide 140.5 nm
127.6 nm
Docetaxel mPEGPCL Acetone Water About 70 Hepatocellular Liu et al.
carcinoma (2012)
Amphotericin-B PLGA DMSO/ Solution of a 86153 31.4(9.1) Invasive fungal Van de Ven
acetone (1:1) stabilizer in water infections et al. (2012)
Insulin (p DMSO/H2O (1:2) Water 181.1220.9 37.8(17.5) Diabetes Zhang et al.
(AAPBA-r-MAGA)) v/v (2012)
(continued)
K. Miladi et al.
Table 8 (continued)
(mV) application
Camptothecin beta-cyclodextrin Ethanol Water 281 13 Cancer Crpanl
PLGA Acetone Water 187 0.06 et al. (2011)
PCL Acetone Water 274 19
Olanzapine PLGA Acetonitrile 0.25 % (w/v) 91.2 -23.7 Schizophrenia Seju et al.
Poloxamer 407 (2011)
solution in water
Curcumin PLGAPEG Acetonitrile 0.1 % pluronic F-68 80.9 Cancer Anand et al.
in water (2010)
Amphotericin-B Eudragit RL 100 Acetone/methanol 1 % (w/v) PVA 134.2290 22.742 Fungal Das et al.
(3:1) adjusted to solution in water keratitis (2010)
pH4
Doxorubicin Poly(ethylene THF/acetone (1:1) Solution of 10 % of 102.8334.5 25(18) Hepatic cancer Guhagarkar
sebacate) Tween 80 (v/v) in et al. (2010)
water
Sparfloxacin PLGA Acetone 1.5 % (w/v) PVA in 181232 22.8(22.2) Bacterial (Gupta et al.
water conjunctivitis (2010)
Rivastigmine PLGA Acetone Pluronic F 127 in 135.6 23.7 Alzheimers Joshi et al.
phosphate buffer pH disease (2010)
9
Letrozole PLGA Acetone 0.51 % (w/v) 15100 12 Breast cancer Mondal
poloxamer-188 in (19.5) et al. (2010)
water
2 Nanoprecipitation Process: From Particle Preparation to In Vivo Applications
Loperamide SAGPPLGA Acetone Poloxamer 188 in 180 22.8 Chronic Tosi et al.
water neuro-diseases (2010)
(continued)
37
Table 8 (continued)
38
(mV) application
Paclitaxel PLGAPCLPEG Acetone Water 114 0.36 Ovarian and Danhier
PLGAPCLPEG 138 0.09 breast cancers et al.
RGD 146 0.12 (2009b)
PLGAPCLPEG
RGDp
Risperidone PLGA Acetone 0.5 % Poloxamer 84.1219.1 Psychiatric Muthu et al.
407 in water disorders (2009)
Cyclosporin A Hyaluronic acid Acetone Water Ocular Yenice et al.
adsorbed to PCL immune (2008)
disorders
Gelatin-co-PLA-DPPE: gelatin-co-PLA-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, PVA poly(vinyl alcohol), THF tetrahydrofuran, DMSO
dimethylsulfoxide, PLGA poly(lactide-co-glycolide), PLGAPEG pegylated poly(lactide-co-glycolide), SAGPPLGA sialic acid and glycopeptides
conjugated PLGA, (p(AAPBA-r-MAGA)) poly(3-acrylamidophenylboronic acid-ran-N-maleated glucosamine)
K. Miladi et al.
4.1.1 Intravenous Administration
Han et al. formulated Doxorubicin-loaded gelatin-co-PLA-dipalmitoyl-sn-glycero-

3-phosphoethanolamine nanoparticles. In vivo experiments showed decreased
toxicity of the drug formulated in the developed nanoparticles compared to free
Doxorubicin (DOX). In addition, it was shown that developed nanoparticles bore
smaller tumor volumes than free doxorubicin when administered to mice.
Nanoparticles were then more efcient and less toxic than the free drug (Han et al.
2013). Another alternative was assessed to improve DOX efcacy in liver cancer by
enhancing liver targeting. In spite of being a drug of choice for hepatic carcinoma
treatment, DOX hydrochloride presents major drawbacks such as the obtaining of
low concentrations in the liver. Other limitations consist of cardiotoxicity,
nephrotoxicity, myelosuppression, and multiple drug resistance due to
P-glycoprotein efflux. To circumvent those shortcomings, the authors aimed to
develop long circulating nanocarriers targeted to the liver. The objective was to
target Asialoglycoprotein receptor (ASGPR) which is predominantly present in
large numbers in the hepatocyte membrane. Polysaccharide including pullulan
(PUL), was chosen as a ligand. In fact, pullulan was described to be internalized by
hepatocytes via ASGPR mediated endocytosis. Poly(ethylene sebacate) (PES) was
used to encapsulate the drug. This polymer presents some advantages such as its
ease of synthesis, its good hydrolytic stability, and low cost. In vivo biodistribution
studies were performed on healthy female Sprague Dawley rats. Three formulations
were assessed: a DOX solution, PES nanoparticles loaded with doxorubicin (PES
DOX), and PES nanoparticles coated with PUL and containing doxorubicin (PUL
PESDOX). It was shown that PESDOX and DOX provided higher concentra-
tions of the drug molecule in the liver. Conversely, PULPESDOX gave higher
blood concentrations of the drug. These results were explained by a higher uptake
of PULPESDOX nanoparticles by Kupffer cells and by the prolonged circulation
provided by pulluan. The authors explained lower liver concentration of PES
DOXPUL by a bypass of kuppfer cells. High blood concentrations of PESDOX
PUL were explained, however, by long circulating property and stealth effect
conferred by pullulan. Moreover, PESDOX and PULPESDOX nanocarriers
gave signicantly lower heart concentration of DOX which could be interesting to
reduce cardiac toxicity (Guhagarkar et al. 2010).
Lee et al. prepared nanoparticles based on hydrophobized pullulan (pullulan
acetate) and containing paclitaxel (PTX). An in vivo study using HCT116 human
colon carcinoma-bearing mice showed that nanoparticles reduced tumor growth
more than free PTX. Efcient accumulation of nanoparticles in tumors was
explained by EPR effect and the passive targeting function, although the
nanoparticles did not have an active targeting ligand (Lee et al. 2012). Danhier et al.
prepared PTX loaded and PEGylated PLGA-based nanoparticles. Tripeptide
arginine-glycine-aspartic acid (RGD) has been shown to bind preferentially to
particular integrin avb3 which is highly expressed on tumor cells and neighboring
endothelium. RGD pepetidomimetic (RGDp) was developed to mimic the activity
RGD. Prepared nanoparticles were grafted either with RGD or RGDp in order to
40 K. Miladi et al.
target tumor endothelium and thus, enhance the antitumor efcacy of PTX. Both of
the ligands were grafted on PCLPEG chains included in the nanoparticles. The
used polymers were shown to be safe as drug-free nanoparticles resulted in the
same tumor growth prole as Phosphate Buffer Saline solution. In vivo targeting of
tumor endothelium was assessed by fluorescence studies. It was shown that
fluorescence obtained following the administration RGD conjugated nanoparticles
was higher than the fluorescence obtained with RGDp conjugated nanoparticles and
nonconjugated nanoparticles. RGDp was, however, higher than in nonconjugated
nanoparticles. Furthermore, in vivo antitumor efcacy was evaluated in transplanted
liver tumor bearing mice. Obtained data showed that RGD conjugated nanoparticles
were more efcient to inhibit tumor growth than RGDp conjugated nanoparticles
and nonconjugated nanoparticles. In addition, survival rate provided by RGD
conjugated nanoparticles was signicantly higher than RGDp conjugated
nanoparticles and nontargeted nanocarriers (Danhier et al. 2009b).
Docetaxel (DTX), which is a taxane, possesses an anticancer activity. This drug
may cause several side effects due to its nonspecic action. Bone marrow
depression, hypersensitivity reactions, and febrile neutropenia are among those
toxicological manifestations. PEGylation of carriers has emerged as a smart alter-
native to prolong circulation time of nanoparticles which facilitates their accumu-
lation in tumors. In fact, stealth surface hampers binding to serum proteins and thus,
recognition by reticuloendothelial system. Poly(e-caprolactone)poly(ethylene
glycol) (PEGPCL) has the advantage of being approved by the Federal Drug
Administration to be used clinically. Efciency of nanoparticles was assessed in
H22 tumor bearing mice (a model of hepatic cancer) and compared to the com-
mercialized formulation of DTX Taxotere and DTX solution. Obtained results
indicated that nanocarriers signicantly reduced tumor growth compared to the
other formulations. In addition to enhanced uptake by cancer cells and prolonged
circulating time, it was shown by in vivo near-infrared fluorescence imaging that
nanocarriers were also eliminated from other normal cells which diminished their
toxicity. Penetration studies showed a passive penetration of the nanoparticles
through leaky vessels surrounding cancer cells thanks to their submicron size (Liu
et al. 2012).
Letrozole (LTZ) is an oral nonsteroidal aromatase inhibitor indicated for the
treatment of breast cancer. Mondal et al. prepared PLGA nanoparticles and eval-
uated them in vivo to see if nanocarriers would provide better tumor targeting. In
vivo studies were conducted in normal mice and Ehrlich Ascites tumor-bearing
mice by injection in tail vein. The blood concentration of drug-loaded nanocarriers
at 24 h post-injection was threefold higher than that of free LTZ. This was
explained by a slower blood clearance of the nanoparticles. The tumor uptake of the
nanoparticles was signicantly higher than the free drug (1.99 % of initial dose/g
compared to 0.43 % of initial dose/g) (Mondal et al. 2010).
4.1.2 Local Administration
Another anticancer agent, all trans retinoic acid (TRA), was encapsulated in
PLA-based nanocapsules prepared by nanoprecipitation. Retinoic acid is an active
derivative of vitamin A which can inhibit the macrophage production of inflam-
matory cytokines and can, thus, be indicated for some tumors where macrophages
play a major role. However, TRA possesses some drawbacks such as poor water
solubility and low stability. It was found that nanoparticles injected intratumorally
were efciently phagocytized by glioma inltrating macrophages (Almouazen et al.
2012). Camptothecin (CPT) is also an efcient anticancer agent. This drug presents,
however, some drawbacks such as its extremely high insolubility in water and its
chemical instability even in physiological pH which may lead to a loss of the
pharmacological activity and cause toxic effects. Cirpanli et al. aimed to develop
beta-cyclodextrin nanoparticles and polymer nanoparticles (PLGA and PCL) loaded
with CPT for brain cancer treatment. Antitumor efcacy of nanoparticles was
assessed on a 9L rat brain tumor model. Cyclodextrin nanoparticles gave the best
results (33 and 27 days as median survival time compared to 23.5 and 25.5 days for
PLGA and PCL nanoparticles). This signicant improvement of survival was
explained by the high loading efciency exhibited by these nanocarriers compared
to other formulations (Crpanl et al. 2011).
4.2 Example of Nanoparticles Developed for Brain Delivery
Brain delivery could be alternative to treat central nervous system disorders but
passage could be poor because of the presence of the Blood Brain Barrier (BBB).
Many nanocarriers have been prepared to circumvent this concern and improve
brain targeting. Olanzapine (OLZ), for example, is a second generation antipsy-
chotic which is effective on the associated negative symptoms of schizophrenia.
The drug, has, however, low bioavailability due to an important hepatic rst-pass
metabolism. In addition, OLZ presents low penetration through BBB because of an
efflux by P-glycoproteins. Moreover, many side effects may appear such as
hypotension, dry mouth, tremor, akathisia and somnolence. Seju et al. assessed nose
to brain drug delivery. In vivo efciency of the prepared PLGA nanoparticles was
evaluated versus a drug solution. It was shown that after 3 h of nasal administration,
nanoparticles provided a tenfold much higher accumulation of OLZ in the brain
compared to the solution form. PLGA nanocarriers showed also no signicant
toxicity on nasal mucosa, indicating their suitability as carriers for nasal delivery of
drugs (Seju et al. 2011). Joshi et al. prepared PLGA nanoparticles loaded with
rivastigmine tartrate (RIV) and indicated for the management of Alzheimer disease.
Clinical use of RIV has shown a poor entry to the brain from blood circulation due
to its hydrophilic nature. In vivo studies were performed in scopolamine-induced
amnesic mice. An increase in learning and memory capacities was obtained for RIV
solution as well as for the nanocarriers but this improvement was slower in the case
42 K. Miladi et al.
of RIV solution. This was explained by better brain targeting provided by

nanoparticles which could present an interesting alternative for better management
of Alzheimer disease (Joshi et al. 2010). Loperamide (LOP), an opioid drug, is
known to cross bloodbrain barrier (BBB) but also to be immediately pumped back
out due to the action of the P-gp. The possibility to cross the BBB and to be retained
in the brain tissue may make LOP able to exert some opioid effects such as the
antinociceptive activity. Tosi et al. prepared LOP loaded nanoparticles in order to
target the brain. PLGA nanoparticles were decorated with sialic acid (SA) and/or
simil-opioid peptide (g7). Two properties were then allocated to the prepared
nanocarriers: First, the ability to cross the BBB due to the presence of g7, (a
BBB-penetrating peptide) and second, the capacity to interact with SA receptors in
the brain which prolongs the time of residence of the nanoparticles in the brain.
This ensured a sustained pharmacological action of the encapsulated drug. In vivo
nociceptive study was performed on male albino rats to determine the Maximal
Possible Effect (MPE) to measure the intensity of the opioid effect. Two doses of
nanoparticles coated with g7 (LOP-PLGA-g7) and nanocarriers coated with SA and
g7 (LOP-PLGA-SA-g7) nanoparticles were assessed. It was concluded that, at both
doses, nanocarriers reached rapidly the brain (15 min after the injection). After 30
60 min, MPE decreased then increased after 6 h. Obtained values remained then
constant for about 15 h but diminished subsequently after 24 h. It was shown also
that pharmacological activity of LOP was prolonged compared to other formula-
tions (Tosi et al. 2007). Moreover, LOP-PLGA-SA-g7 nanoparticles exhibited more
prolonged pharmacological activity than LOP-PLGA-g7 nanoparticles. In fact,
conjugation of SA modied the surface characteristics of the nanoparticles which
resulted in a prolongation of the pharmacological action (Tosi et al. 2010).
Risperidone (RIS) is an atypical antipsychotic agent which may cause
dose-dependent extrapyramidal side effects (EPS). Consequently, the use of low
doses is necessary to avoid such manifestations. RIS is practically insoluble in
water and undergoes important rst-pass metabolism. Long-acting injectable for-
mulations have been already developed but presented poor initial drug release
which implied initial oral supplementation. Prepared nanoparticles were assessed
in vivo by studying the antagonism of apomorphine-induced climbing and snifng
(antipsychotic activity) in Swiss albino mice. It was shown that PLGA nanoparti-
cles signicantly inhibited apomorphine-induced climbing and snifng up to 72 h
while the RIS solution exhibited inhibition up to only 12 h. Furthermore, the
incorporation of the nanocarriers in an in situ gel system controlled the initial rapid
release of RIS from nanoparticles and showed the maximum inhibition in the
apomorphine-induced climbing and snifng. This was explained by a control of the
initial burst by the incorporation in the in situ gel. It was show also that
nanoparticles signicantly reduced catalepsy which is an EPS (Muthu et al. 2009).
4.3 Example of Nanoparticles Developed to Treat Ocular

Diseases
The delivery of drugs into the eye must challenge poor drug ocular bioavailability
which is principally caused by precorneal loss. In fact, it was reported that barely
90 % of the applied drug undergoes a precorneal loss by lacrimation and drainage.
Precorneal loss ways include rapid tear turnover, nonproductive absorption, tran-
sient residence time in the cul-de-sac, and the relative impermeability of the drugs
to the corneal epithelial membrane (Katara and Majumdar 2013). Nanoparticles
have several advantages over conventional drug delivery systems intended to ocular
delivery. In fact, they have slower ocular elimination and they could provide sus-
tained release of drugs. While ocular delivery of poly(alkylcyanoacrylate)
nanoparticles was described to cause disruption to the corneal epithelium cell
membrane, other polymers were shown to be safe such as, PCL and Eudragit RL.
The latter has a positive charge which allows a better adhesion to eye tissue and
thus, more prolonged residence time in the cornea (Das et al. 2010).
Encapsulation of melatonin in PLGA and PLGAPEG nanoparticles was
assessed for glaucoma (an optic neuropathy characterized by elevation of intraoc-
ular pressure: IOP) treatment by Musumeci et al. Obtained nanoparticles showed
ocular tolerability in rabbit eyes. Furthermore, both formulations provided pro-
longed decrease in IOP but PLGAPEG-based nanoparticles were more efcient by
providing greater decrease. These results were explained by the higher mucoad-
hesion of the PLGAPEG nanoparticles thanks to the PEG groups. In addition, the
cornea and conjunctiva have a net negative charge. Thus, the lower negative zeta
potential of PLGAPEG nanocarriers allowed a better and more prolonged inter-
action with the eye (Musumeci et al. 2013). Particulate nanocarriers would be then
well-tolerated alternatives to prolong contact with the eye tissue. Eudragit-based
nanoparticles containing the anti-inflammatory drug, aceclofenac were prepared and
their efciency was evaluated in vivo by administration to rabbits. Eudragit
RL100 is a positively charged polymer due to many quaternary ammoniums in its
structure. This property allows mucoadhesion to the anionic cornea. Katara and
Majumdar assessed the effect of the prepared nanoparticles versus an aqueous
solution of the drug on arachidonic acid-induced polymorphonuclear leukocytes
migration and lid closure in rabbit eyes. Obtained results showed lower lid dis-
closure for both aceclofenac formulations but nanoparticles provided smaller lid
closure compared to the drug solution. Furthermore, more enhanced
anti-inflammatory effect was exerted by nanoparticles compared to drug solution
(Katara and Majumdar 2013). Das et al. developed Eudragit RL nanoparticles
loaded with amphotericin-B (AmB) which is a polyenene antibiotic indicated in
fungal keratitis. Other formulations consisting mainly of liposomes and colloidal
dispersions were successfully used but presented stability concerns. Stability
studies performed at room temperature and at 26 C showed good stability of the
nanoparticles during 2 months. Eye irritating effects of the formulation was
assessed in vivo in albino rabbits. All the obtained data showed that values of
44 K. Miladi et al.
irritation and opaqueness were almost zero which conrmed the suitability and the
safety of the formulation for ocular delivery. Positive charge of the polymer
facilitated effective adhesion of the nanocarriers to the corneal surface and ensured a
strong interaction with the negatively charged mucosa of the conjonctiva and the
anionic mucin present in the tear lm (Das et al. 2010).
Yenice et al. prepared hyaluronic coated PCL nanospheres containing cyclos-
porine A (CyA). CyA is a neutral hydrophobic peptide which is indicated for
multiple ocular immune disorders. Systemic use of the drug is limited because of
the various signicant side effects that may appear such as, hypertension,
nephrotoxicity, and hepatotoxicity. Diffusion to the ocular tissue is thought to occur
only when the eye is signicantly inflamed. Hyaluronic acid (HA) was used due to
its mucoadhesion properties which may enhance ocular residence time of cyclos-
porine A and thus, enhance its ocular bioavailability and prolongs its activity. In
vivo studies were performed by topical administration of three different formula-
tions to Male albino New Zealand rabbits: a solution of CyA in castor oil, PCL
nanospheres, and PCL nanospheres coated with HA. Obtained corneal concentra-
tion of CyA for nanospheres formulations were 68 fold higher than those of castor
oil solution. HA-coated nanospheres provided signicant increase in CyA corneal
uptake and similar results were obtained for the conjonctival tissue (Yenice et al.
2008). Sparfloxacin is a newer generation hydrophobic fluoroquinolone used in
bacterial conjunctivitis. This drug is poorly water soluble and presents bioavail-
ability concerns. Gupta et al. aimed to enhance sparfloxacin bioavailability by the
preparation of PLGA naoparticles. An in vivo ocular retention study was performed
on Male New Zealand albino rabbits. Developed nanocarriers were compared to a
marketed formulation. A good spreading was observed over the entire precorneal
area for both formulations but the marketed formulation showed rapid clearing from
corneal region. PLGA nanoparticles, however, adhered to the cornea for a longer
duration providing, thus, a more extended release of drug. Particles size seems to be
the key factor to explain this prolonged residence time on the cornea as PLGA is a
negatively charged polymer and is not known to be naturally mucoadhesive (Gupta
et al. 2010).
4.4 Other Applications
AmB is a polyene antibiotic which is commonly indicated for invasive fungal

infections and visceral leishmaniasis. This drug has a poor water solubility which
limits its oral bioavailability. In addition, many side effects were described in
patients receiving AmB such as fever, chills, vomiting, headache, nausea, and renal
malfunctions, especially with the commercialized formulation Fungizone. Newer
lipid-based formulations are more tolerated but their expensiveness and the need of
well-dened daily doses limited their success. Van de Ven et al. aimed to develop a
more potent and cost-effective formulation of AmB. Hemolysis assay showed that
PLGA nanoparticles were less hemolytic than drug solution and some of them were
even not hemolytic at all. A selected formation was evaluated in the acute A.
fumigatus mouse model and its potency was compared to a nanosuspension of
AmB, Fungizone, and Ambiosome. Obtained data revealed that PLGA
nanoparticles reduced A. fumigatus more efciently than Fungizone. In addition,
nanocarriers were about two times more efcient to clear mice organs from the
fungi than Ambiosome. The nanosuspension was, however, four times more
efcient than Ambiosome (Van de Ven et al. 2012).
The nasal route possesses many advantages over the oral and the parenteral
routes in the delivery of biomacromolecules. In fact, it is noninvasive, painless,
does not require sterile preparation, and allow self-administration. However, the
development of drug delivery systems intended to nasal delivery must challenge
poor absorption through the nasal mucosa and eventual enzymatic degradation.
New generation phenylboronic acid-functionalized glycopolymers were developed
to avoid these shortcomings. Their properties are linked to the presence of boronic
acid and its derivatives which could bind to glycoproteins and glycolipids within
cell surfaces. Moreover, boronic acid derivatives could resist to enzymatic degra-
dation because they exert potent inhibition toward serine proteases such as trypsin,
chymotrypsin, elastase, and leucine aminopeptidase. These properties made inter-
esting the use of these special polymers in the development of nanocarriers,
especially in the case of the encapsulation of biomacromolecules. Insulin was
encapsulated in poly(3-acrylamidophenylboronic acid-ran-N-maleated glu-
cosamine) p(AAPBA-r-MAGA) copolymers and administered to mice by the
intranasal route. The potency of the developed nanoparticles was compared to an
insulin solution. It was concluded that the insulin solution was not able to reduce
signicantly glucose blood levels while a signicant decrease was provided by
nanoparticles. This conrmed enhanced nasal absorption of insulin provided by
phenylboronic acid-functionalized glycopolymer nanocarriers (Zhang et al. 2012).
Curcumin (CUR) is a yellow pigment in the spice turmeric (Curcuma longa). This
drug is poorly soluble in water and presents very low oral bioavailability. CUR
exhibits antioxidant, anti-inflammatory, anti-survival, antiproliferative,
anti-invasive, and antiangiogenic activity. The assessment of the bioavailability of
PLGAPEG nanoparticles was performed in Balb/c mice versus pure CUR.
Obtained results showed that serum levels of CUR provided by nanocarriers were
almost two times higher than those provided by CUR solution. Moreover,
nanoparticles insured a sustained release of the drug (Anand et al. 2010).
5 Conclusion
Several hydrophobic or hydrophilic drugs could present bioavailability, stability, or

unpleasant taste concerns. Encapsulation of such molecules in nanoparticles could
be a very interesting alternative to solve these problems in order to enhance the
efcacy of such molecules and promote patient compliance. Nanoprecipitation is a
simple and reproducible technique that has been widely used for the preparation of
46 K. Miladi et al.
polymer nanoparticles intended for several biomedical applications since its rst
discovery. Operating conditions have to be well managed to obtain nanoparticles
with suitable properties for the biomedical applications they are designed for.
Several research works have been made to use nanoprecipitation in a conventional
way while other works focused on the enhancement of its scalability, repro-
ducibility, and safety. Membrane technology, microfluidics, and flash nanoprecip-
itation were introduced to achieve such purposes. Advantages of submicron carriers
prepared by nanoprecipitation in the biomedical eld have been conrmed in vivo
by numerous studies. These achievements include enhanced bioavailability, better
targeting and tolerance, sustained release, and enhanced absorption of the drug
through biological barriers.
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Chapter 3
Targeted Theragnostic Nanoparticles
Via Flash Nanoprecipitation: Principles
of Material Selection
Christina Tang and Robert K. Prudhomme
Abstract Flash NanoPrecipitation is a simple, rapid, and scalable method capable

of continuously processing nanoparticles with sizes tunable between 50 and
500 nm with narrow size distributions and high drug loading capacities. In Flash
NanoPrecipitation, an amphiphilic block copolymer is dissolved in organic solvent
with a desired core material. When rapidly mixed with a miscible antisolvent for the
core material that causes a rapid decrease in solvent quality, the core material
precipitates and the amphiphilic block copolymer self-assembles directing the
formation of a colloidal nanoparticle. Adsorption of the hydrophobic block of the
block copolymer arrests precipitation of the core material and the hydrophilic block
sterically stabilizes the nanoparticle. The assembled nanoparticles are kinetically
frozen. Given appropriate mixing conditions (i.e., time scales of mixing faster than
nanoparticle formation), the rate of nucleation and growth of the precipitating core
material must be appropriately matched with the rate of self-assembly. This
bottom-up approach can be adapted to multiple systems of interest and can be used
to encapsulate hydrophobic drugs and/or imaging agents. Methods to encapsulate a
range of materials including poorly water soluble drugs [active pharmaceutical
ingredients (APIs)], weakly hydrophobic, ionizable APIs as well as peptides,
siRNA, organic, and inorganic imaging agents have been developed. Multiple
components (drugs and/or imaging agents) can be coprecipitated to develop mul-
tifunctional nanoparticles with therapeutic and diagnostic capabilities. Further, the
surface chemistry of the nanoparticle can be tailored to enable active targeting. To
control the surface chemistry, a functionalized block copolymer can be incorporated
into the mixing/assembly process. The functionalized block copolymer can be the
block copolymer conjugated to a small molecule targeting ligand (e.g., mannose,
folate). Alternatively, the PEG end of the block copolymer can be modied with a
reactive end group for conjugation to a targeting ligand after the nanoparticle has
been assembled. Surface modications with small molecules, single stranded DNA,
peptides, and proteins have been performed. Appropriate material selection is
C. Tang R.K. Prudhomme (&)

Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ
08544, USA
e-mail: prudhomm@princeton.edu

DOI 10.1007/978-3-319-41421-8_3
56 C. Tang and R.K. Prudhomme
critical in the formulation of functional and stable nanoparticles dictated by choice

of the core material and stabilizer, respectively. Therefore, we review examples of
how Flash NanoPrecipitation has been used to formulate nanoparticles for potential
applications in targeted delivery of cancer therapeutics, peptides and imaging
agents with a focus on practical aspects of appropriate materials selection for both
the core material and stabilizer.

Keywords Nanomedicine Drug delivery Theragnostic Nanoprecipitation

Active targeting Self-assembly Polymer Colloid
1 Introduction
Flash NanoPrecipitation is a powerful platform for producing functional nanopar-

ticles for a range of applications in nanomedicine. It is a simple and scalable tech-
nique capable of continuous processing of nanoparticles with tunable sizes, narrow
size distributions and high drug loading capacities. In Flash NanoPrecipitation, an
amphiphilic block copolymer is dissolved in organic solvent with desired a core
material such as therapeutic and/or imaging agent. The solvent is rapidly mixed
against a miscible antisolvent for the core material of interest (Fig. 1). Upon mixing
there is a rapid decrease in solvent quality causing precipitation of the core material
as well as self-assembly of the amphiphilic block copolymer which directs
nanoparticle assembly. Precipitation of the core material is arrested by adsorption of
the hydrophobic block of the block copolymer while the hydrophilic block sterically
stabilizes the nanoparticle. Given proper materials selection, the assembled
Fig. 1 Overview of Flash NanoPrecipitation

3 Targeted Theragnostic Nanoparticles Via Flash Nanoprecipitation 57
nanoparticles are kinetically frozen and there is no dynamic exchange of the indi-
vidual block copolymer chains (Johnson et al. 2006; Johnson 2003; DAddio and
Prudhomme 2011; Akbulut et al. 2009a; Gindy 2008).
Given sufciently rapid mixing, nanoparticle assembly involves the precipitating
core material and self-assembly of the block copolymer. Thus, the rate of nucleation
and growth of the precipitating core material must be appropriately matched with
the rate of self-assembly. Fast nucleation and growth rates relative to self-assembly
lead to formation of macroscopic, unstable precipitates whereas fast self-assembly
relative to nucleation and growth can result in a large population of empty micelles.
The size of the resulting nanoparticles can be tuned by adjusting the relative rates of
these two processes. In practice, these rates and resulting nanoparticle size are
affected by the component molecular weight and concentration, and the relative
amount of block copolymer to core materials. Increasing the total solids concen-
tration or the molecular weight of the core material tends to increase particle size
and increasing the amount of block copolymer relative to the core material tends to
reduce particle size. Supersaturation of the core material is also an important
consideration which is affected by the quality of the antisolvent as well as the
amount of organic solvent used during mixing. Increasing the amount of organic
solvent present during mixing can increase supersaturation leading to a decrease in
particle size. These formulation considerations are summarized in Fig. 2 (Johnson
et al. 2006; Johnson 2003; DAddio and Prudhomme 2011; Gindy 2008; Figueroa
2014).
Successful nanoparticle formation occurs when the time required for homoge-
nous mixing is less than nanoparticle assembly (nucleation and growth as well as
self-assembly of the block copolymer), i.e., mixing on the order of milliseconds.
Such rapid mixing has been achieved in microfluidic devices (Karnik et al. 2008;
Kolishetti et al. 2010). However, the micromixing achieved using specialized
mixing geometries such as the multi-inlet vortex mixer (MIVM) or conned
impinging jet (CIJ) mixer enables high-throughput processing. The MIVM is based
Fig. 2 Flash NanoPrecipitation formulation considerations

on tangential flow of incoming streams while the CIJ mixer is based on collision of
the organic stream and antisolvent stream with equal momentum in a conned
geometry, depicted in Fig. 1. The MIVM, in particular, allows for continuous
processing that can readily be scaled up from laboratory to plant. Full descriptions
and characterizations of the mixer types are described elsewhere (Johnson and
Prudhomme 2003b, c; Liu et al. 2008; Liu and Fox 2006; Cheng et al. 2009, 2010;
Cheng and Fox 2010; Shi et al. 2011a, b; Han et al. 2012).
Equipped with an appropriate mixer, Flash NanoPrecipitation is a versatile
method to engineer multifunctional nanoparticles based on encapsulation of mul-
tiple components including therapeutics and/or imaging agents (Gindy 2008).
Furthermore, Flash NanoPrecipitation is a convenient platform to tailor the surface
chemistry of nanoparticles as a functionalized block copolymer can be incorporated
during the mixing process. This functionalized block copolymer can facilitate
conjugation to a range of ligands to enable active targeting of the nanoparticle
(DAddio 2012). Leveraging the simplicity and scalability of this process with
versatile material selection will be instrumental in achieving the full potential of
Flash NanoPrecipitation as a powerful tool in nanomedicine. Therefore, we will
focus on practical aspects of appropriate materials selection for both the core
material and stabilizer. Processing considerations to maximize particle stability will
also be discussed.
2 Core Material Selection
Flash NanoPrecipitation is a versatile tool to encapsulate a wide range of functional

materials including therapeutics and/or imaging agents. Using this bottom-up pre-
cipitation method, the solubility of the core material in the nal dispersion solvent
mixture will determine the quality (i.e., encapsulation efciency and stability) as
well as the functionality of the nanoparticles. For successful encapsulation using
Flash NanoPrecipitation, nucleation and growth of the core material must occur
before self-assembly of the block copolymer. Nucleation and growth of the pre-
cipitating core material is driven by supersaturation, i.e., concentrations higher that
the saturation solubility in the mixed solvent due to introduction of the antisolvent
during mixing. Sufciently high supersaturation and rapid mixing facilitate fast
nucleation relative to growth leading to homogenous nucleation and diffusion
limited growth which results in narrow size distributions. Use of molecularly sol-
uble components with high supersaturation (low solubility in the mixed solvent
relative to the concentration in the organic solvent) is desired for successful
encapsulation. In practice, the ratio of the concentration of core material in the
organic solvent to the equilibrium solubility concentration of the core material in
the solvent mixture is a reasonable measure of supersaturation (DAddio 2012) and
ratios greater than 100 are suggested for successful encapsulation (Johnson and
Prudhomme 2003a). This measure of supersaturation is affected by the solute
concentration in organic solvent, organic solvent concentration used during mixing,
and the properties of the solute (Figueroa 2014). A more detailed explanation of
supersaturation is described elsewhere (DAddio and Prudhomme 2011; Figueroa
2014).
When selecting core materials with suitable solubility, it is helpful to consider
the octanol and water partition coefcient as a measure of hydrophobicity. The logP
or calculated logP (clogP) is the negative logarithm of the partition coefcient of the
solute between octanol and water. A higher logP indicates a more hydrophobic
material. As a general starting point, materials with logP or clogP values above 6
are preferred for direct encapsulation with Flash NanoPrecipitation (Figueroa 2014;
Pustulka et al. 2013; Zhu 2014a). Properties such as ionization and crystallinity
must also be considered. Ionization can enhance solute solubility in the mixed
solvent and reduce the stability of the nanoparticle dispersion (Figueroa 2014).
Crystallinity may also affect particle stability as the non-equilibrium phases initially
encapsulated may solubilize and recrystallize outside the nanoparticle (Figueroa
2014; Liu et al. 2007).
The resulting nanoparticle dispersions are characterized by drug loading and
encapsulation efciency as indications of encapsulation. The drug loading and
encapsulation efciencies are dened as
massdrug
Drug loading % 100 X:1
massNP
Mass drug measured in NP dispersion

Encapsulation efficiency % 100
Mass of drug in stock solution
X:2
The stability of the resulting nanoparticles is also considered. Nanoparticles that

increase in size over time due to Ostwald ripening are not considered stable.
Nanoparticle dispersions in which macroscopic precipitation is observed due to the
core material partitioning out of the nanoparticle core and reprecipitating or
recrystallizing outside of the nanoparticle are also deemed unstable.
Highly hydrophobic APIs including: itraconazole (Kumar et al. 2009a), oda-
nacatib (Kumar et al. 2009a), cycloporin A (DAddio 2012) and nitric oxide pro-
drugs (Kumar et al. 2009b) can be directly encapsulated using Flash
NanoPrecipitation.
The drug loadings achieved were 50 wt% for itraconazole, odanacatib, and
cycloporin A and 35 wt% for nitric oxide prodrugs. Encapsulation efciencies are
above 80 %. Other hydrophobic materials including polymers, e.g., poly(lactide)
(PLA), poly(e-caprolactone) (PCL), poly(styrene) (PS), dyes, e.g., Hostasol Yellow,
and lipids, e.g., a-tocopherol (Vitamin E) have also been encapsulated using Flash
NanoPrecipitation with comparable loadings and encapsulation efciencies
(Figueroa 2014). However, direct encapsulation within stable nanoparticles using
Flash NanoPrecipitation is limited to highly hydrophobic (logP > 6) core materials.
For other core materials of interest, e.g., crystallizable compounds, ionizable com-
pounds, or materials with logP < 6 that do not form stable nanoparticles due to
insufcient supersaturation, alternative strategies are required. Three general

strategies to facilitate encapsulation and/or improve the resulting nanoparticle sta-
bility have been explored: use of prodrugs, coprecipitation, and in situ complexation.
2.1 Prodrug
Prodrugs (active pharmaceutical ingredient (API) chemically conjugated to a

hydrophobic structure) of APIs of interest can be encapsulated when formulation of
the API alone is not possible due to limited supersaturation. This approach requires
that the API of interest contains a reactive moiety to which a hydrophobic structure
can be covalently linked. In this technique, the solubility of the API is decreased
through conjugation to hydrophobic substances to increase supersaturation and
impart stability to the resulting particles. In a successful prodrug strategy, the active
API is regenerated through degradation of the linkage with no toxic by-products
(DAddio and Prudhomme 2011; Figueroa 2014; Saad 2007; Ansell et al. 2008).
This approach increases the hydrophobicity of the API of interest and enables
encapsulation within stable nanoparticles when direct encapsulation of the API is
not possible; however, the maximum drug loading is typically lower than if the drug
were directly encapsulated.
For example, rifampicin, an antibiotic used in the treatment of tuberculosis,
could not be directly encapsulated in stable nanoparticles by Flash
NanoPrecipitation. The rifampicin is not sufciently hydrophobic to be encapsu-
lated in the core of the nanoparticle; it partitions out of the nanoparticle core into the
mixed solvent and rapidly recrystallizes. By conjugating the hydroxyl group of the
rifampicin to the carboxylic acid of vitamin E succinate or PCL (MW 2 kg mol1)
through an ester linkage using a carbodiimide coupling agent to increase the
hydrophobicity of the nanoparticle core, stable 200 nm nanoparticles could be
achieved using Flash NanoPrecipitation. Rifampicin could also be conjugated to
2-ethylhexyl vinyl ether (C8) through an acetal linkage. Nanoparticles containing
this prodrug construct were stable for at least 4 days at pHs between 4 and 7.4.
Despite being encapsulated within a hydrophobic core, the prodrug could be
cleaved via hydrolysis of the acetal linkage and *5 % was released over 120 h
effectively prohibiting the growth of E. coli over 10 days (Liu 2007).
Paclitaxel, a hydrophobic chemotherapeutic, has been similarly formulated into
nanoparticles. Paclitaxel could initially be encapsulated but the nanoparticles were
unstable as but recrystallization was observed within 15 min. To improve the sta-
bility of the nanoparticle, the 2-hydroxyl of the paclitaxel was conjugated to the
terminal carboxylic acid of Vitamin E succinate to increase the hydrophobicity of
the Paclitaxel. The more hydrophobic prodrug particles produced by Flash
NanoPrecipitation were stable over days. The nanoparticles contained 31 wt%
paclitaxel with 100 % encapsulation efciency (Saad 2007). Synthesis and appli-
cation of the prodrug facilitated encapsulation of the paclitaxel within stable
nanoparticles by Flash NanoPrecipitation when direct encapsulation of paclitaxel

was not possible.
Building on this work, Ansell et al. encapsulated a series of paclitaxel prodrug
conjugates. In their approach, paclitaxel was conjugated to a lipophilic anchor using
di-acid cross-linkers. Both the linker and anchor could be varied independently to
tailor the pharmacokinetics and efcacy of the nanoparticle formulations. The lipid
alcohols (such as a-tocopherol, oleyl alcohol, cholesterol, and 1,2,-dimyritosyl-sn-
glycerol were conjugated to the cross-linker (succinic anhydride or diglycolic acid)
through a cyclic anhydride. The linker was then attached to the paclitaxel using
carbodiimide chemistry. Nanoparticles formulated with prodrug and a lipid in the
core was 2030 nm in diameter. After storing the nanoparticles in 300 mM sucrose at
4 C for 11 weeks, less than 5 % of free paclitaxel was found by high performance
liquid chromatography (HPLC), a concentration which showed no antitumor activity.
The particle and the drug were tracked in vivo and experiments indicate that the
nanoparticle formulations are most likely cleared as a result of single polymer chain
partitioning out of the particle over time. Gradual loss of the stabilizer results in
destabilization of the particle and leads to elimination of any payload that had not
already partitioned out. To increase the time before elimination, a relatively low ratio
of drug to stabilizer and use of a stabilizer with a low partitioning rate are preferred to
obtain efcacy dictated by the physical and chemical properties of the prodrug rather
than the delivery vehicle itself. At a given particle composition, the partition rate and
efcacy of the prodrug depended on the hydrophobicity of the lipid anchor indicating
the release was a function of the drug properties rather than degradation of the carrier.
The prodrugs with the longest partitioning half-lives are expected to be capable of
delivery more drug to the tissue of interest (Ansell et al. 2008).
This approach has also been applied to encapsulation of proteins. The primary
amines of a peptide or protein can also be coupled to the terminal carboxylic acid
group of anchor molecules via carbodiimide chemistry. Lysozyme, a model protein
was conjugated to Vitamin E Succinate or PCL-succinate (MW 2.1 kg mol1).
Experimentally, the degree of conjugation increased linearly with ratio the ratio of
carboxylic acid to amine. Thus, the degree of modication can be controlled
through specication of reactant molar ratios. Use of a larger anchor molecule (PCL
compared to Vitamin E) required longer reaction times to obtain the same degree of
modication due to steric hindrance. Lysozyme-Vitamin E conjugates could be
encapsulated within 1 micron particles, which is smaller than unmodied lysozyme
which formed particles on the order of 5 microns. Lysozyme-PCL conjugates with
*50 % degree of modication, i.e., 34 PCL molecules per lysozyme produced
particles *165 nm in diameter with 34.5 wt% protein and 100 % encapsulation
efciency (Gindy 2008).
Encapsulation of prodrugs is a valuable approach for developing therapeutic
nanoparticles. In addition to providing means to formulate stable nanoparticles,
choice of cleavable chemical linker also enables tunable release. Further, multiple
drugs could be attached to various sites of a given cleavable chemical linker to
encapsulate multiple drugs within a single nanoparticle core. Chemical linkage of
multiple drugs would provide well-controlled stoichiometry and simultaneous

release for a synergistic effect and avoiding antagonism (Ansell et al. 2008).
2.2 Coprecipitation
Since direct encapsulation of a desired core material using Flash NanoPrecipitation

is limited to highly hydrophobic materials (logP > 6), strategies to encapsulate less
hydrophobic materials that cannot be directly encapsulated are desired. While
encapsulation of hydrophobic prodrugs is possible as discussed in the above sec-
tion, prodrug formation requires chemically conjugating the API to a hydrophobic
moiety. Chemical conjugation requires reactive sites on the API of interest contain a
reactive moiety to which a hydrophobic structure can be chemically linked. Further,
the API must be regenerated through degradation of the chemical linkage to the
hydrophobic structure and the resulting by-products must be nontoxic. The disad-
vantage of this approach is that conjugation to the hydrophobic structure is con-
sidered a new chemical entity which requires Food and Drug Administration in the
United States of America (FDA) approval.
When conjugation is not possible or desired (to avoid additional FDA approval),
an alternative strategy is coprecipitation. In coprecipitation, Flash NanoPrecipitation
is performed with the API, a hydrophobic cosolute, and an amphiphilic block
copolymer stabilizer. In this approach, hydrophobic solutes or polymers are used as
nucleating agents to seed particle growth via heterogeneous nucleation. The addition
of hydrophobic macromolecules such as poly(styrene) (PS), PLA or PCL
homopolymer, in particular, reduces the activation energy for particle growth,
induces nucleation, and controls the number of nuclei, reducing particle size. When
using cosolutes such as hydrophobic polymers such as PS, PLA, or PCL or lipids
such as cholesterol or Vitamin E, the differences in solubility and supersaturation
alter nucleation kinetics and the material with the higher supersaturation will
nucleate rst inducing heterogeneous nucleation and growth of the active which may
enhance the incorporation of the desired API (Gindy 2008). When successful, the
nanoparticle core contains the API is dispersed or solubilized in the hydrophobic
cosolute (Figueroa 2014). Successful encapsulation of the API using coprecipitation
requires that the API have high afnity for the hydrophobic cosolute.
Although incorporation of a cosolute in the nanoparticle core will reduce the
maximum drug loading, this approach can improve particle stability. When the API
is dispersed or solubilized in the cosolute within the nanoparticle core the driving
force for the API partitioning out of the nanoparticle core and reprecipitating or
recrystallizing is reduced which limits Ostwald ripening and recrystallization.
Further, coprecipitation of multiple components is naturally suited for developing
multifunctional hybrid nanoparticles that contain multiple materials with various
functionalities, e.g., multiple drug cocktails, multimodal imaging, and theragnostic
particles containing a drug and imaging agent. Daddio has veried empirically that
the nanoparticle composition is the same as the composition in the organic solvent;
therefore, coprecipitation can produce nanoparticles with multiple components at

prescribed concentrations (DAddio 2012).
For example, nanoparticles containing organic sunscreen lters, A Plus or T 150
to absorb UVA or UVB, respectively, could be encapsulated by coprecipitating
with PS as a coprecipitant and poly(styrene)-block-poly(ethylene glycol) (PS-b-
PEG) as a stabilizer. The nanoparticle size could be tuned between 100 and 350 nm
and were stable for at least 3 months. Further, inorganic sunscreen lters (titanium
dioxide or zinc oxide) could also be incorporated with PS and PS-b-PEG into a
composite nanoparticle for a synergistic sunscreen effect. The ability to tune the
absorption spectra of the NP formulation provides a powerful platform for
improving sunscreen effectiveness (Shi et al. 2012).
Flash NanoPrecipitation has also been used to encapsulate progesterone to treat
traumatic brain injury. Progesterone has neuroprotective and neuroregenerative
properties but has proven difcult to formulate at the high concentrations needed
due to its hydrophobicity and crystallinity. Using Flash NanoPrecipitation and a
poly(lactide acid)-block-poly(ethylene glycol) (PLA-b-PEG) amphiphilic block
copolymer stabilizer, progesterone yielded a cloudy suspension with visible
aggregates. After ltering through a 5 lm nylon syringe lter, the suspension
contained 37 nm particles with an encapsulation efciency of less than 10 wt%. The
low encapsulation efciency was attributed to the relatively high solubility of the
progesterone in the mixed solvent system. To improve encapsulation efciency, a
hydrophobic cosolute was included in the formulation. Compounds steroidal in
structure that could be encapsulated using Flash NanoPrecipitation such as
cholesterol and prednisone cosanyl diglycolate were considered. Coprecipitation
resulted in translucent suspensions with no visual aggregates; however, removal of
the organic led to macroscopic crystallization and precipitation of the progesterone
out of the nanoparticle. D-a-tocopherol (Vitamin E), oil that is generally recognized
as safe by the FDA, was also considered as the progesterone is soluble in the oil at
concentrations of 25 wt%. Incorporating Vitamin E as a hydrophobic cosolute, the
encapsulation efciency was 56.8 % and there was no signicant change in particle
size after removal of the organic solvent. Increasing the vitamin E concentration
improved encapsulation efciencies, while increasing the stabilizer concentration
had no effect. Using a 1:1:1 wt. ratio of the PLA-b-PEG: Vitamin E: progesterone,
the optimized formulation was a dispersion of 270 nm particles with an encapsu-
lation efciency of 69.4 % and drug loading of 24 % (Figueroa et al. 2012).
In other work, rifampicin could be initially encapsulated using Flash
NanoPrecipitation. However, the drug was soluble in the solvent mixture and
readily partitioned out of the nanoparticle and recrystallized. Rifampicin prodrugs
(rifampicin conjugated to vitamin E succinate or PCL via acetyl linkages) could be
coprecipitated with rifampicin to improve drug loading from 33 % (rifampicin
prodrug encapsulation) to 42 % (rifampicin prodrug and rifampicin encapsulation)
(DAddio 2012). In another example, paclitaxel was coprecipitated with PCL. The
addition of PCL resulted in a slight improvement in the stability of the nanoparticles
as the solubility of the paclitaxel in the nanoparticle core is enhanced and subse-
quent partitioning of the recrystallization is delayed. The polymer matrix also
provides a resistance to drug diffusion, delaying release from the particles.

Similarly, paclitaxel prodrugs were coprecipitated with a hydrophobic solute such
as Vitamin E succinate or PCL to control nanoparticle size. Using PCL, nanopar-
ticles between 80 and 160 nm could be produced at a constant drug loading by
varying the molecular weight of the PCL. With Vitamin E succinate, particle size
could be tuned between 50 and 270 nm. Further, coprecipitation encapsulation of
multiple therapeutics, e.g., paclitaxel and estradiol prodrugs or anti-tuberculosis
drug cocktails including an antibiotic and a P-glycol protein efflux pump inhibitor
was possible (DAddio 2012; Saad 2007).
APIs with logP values between 4.2 and 5.2 can also be formulated into
nanoparticles by coprecipitating with a hydrophobic solute. For example, using
fenobrate (logP 4.86), an antilipemic agent used to lower blood cholesterol and
triglycerides, nanoparticles between 170 and 200 nm were produced using vitamin
E as a cosolute. Because fenobrate is highly soluble in the vitamin E core, high
drug loading (at least 83 %) could be obtained using D-a-tocopheryl poly(ethylene
glycol) 1000 succinate as a stabilizer. The dispersions were stable for at least
1 week at 4 C. Similarly, nanoparticle dispersions of indomethacin, a weakly
acidic, relatively low logP (clogP * 4), non-steroidal anti-inflammatory agent used
to treat rheumatoid arthritis, were produced using D-a-tocopheryl poly(ethylene
glycol) 1000 succinate as a stabilizer, and Vitamin E as a cosolute. In an optimized
formulation, the nanoparticles were stable with a peak mean diameter of 147 nm,
96 % encapsulation efciency and indomethacin loading of 15 % (wt.
indomethacin/wt. nanoparticle). The indomethacin concentration in the NP dis-
persion was 0.8 mg mL1. Cinnarizine, a weakly basic anti-histamine agent for
control of motion sickness, could also be formulated into sub-200 nm particles that
were stable for at least 1 week at 4 C using this approach. However, poor
encapsulation efciencies, i.e., less than 45 % were achieved. Coprecipitating the
cinnarizine with vitamin E succinate in place of vitamin E improved the encap-
sulation efciency to *60 %. The resulting nanoparticles contained 15 % cinnar-
izine (wt. cinnarizine/wt. nanoparticle) and the cinnarizine concentration in the NP
dispersion was 3.14 mg mL1. The improved encapsulation efciencies may be
attributed to a slight afnity between the cinnarizine and vitamin E succinate to
form an ion pair as well as better miscibility of cinnarizine with vitamin E succinate
when compared to vitamin E based on Hansen solubility parameters (Figueroa
2014).
In other work, encapsulation hydrophobic, small molecule cross-linkers copre-
cipitated with a polymer has been used to impart particle stability. Zhang et al.
synthesized aromatic diazides (cross-linker) that could be used to photocrosslink PS
in water at ambient temperatures. Performing Flash NanoPrecipitation with the
cross-linker, PS and PS-b-PEG resulted in 120 nm nanoparticles that encapsulated
one cross-linker per two polymer chains. The resulting aqueous suspension of
particles was exposed to UV light (k * 365 nm) for 15 min and the suspension
transformed from white to bright yellow. The cross-linked particles were stable
under extreme heating. When exposed to thermal cycling at 95 C,
non-cross-linked particles disassemble shrinking from 120 to 30 nm while
cross-linked particles remain stable. Particle stability may be advantageous in

particles used for diagnostic imaging to avoid leakage of the dye or contrast agent
(Zhang et al. 2011a). Photocrosslinking of biocompatible PCL modied with
biocompatible coumarin nanoparticles has also been reported (Chung et al. 2012).
The particles showed minimal cytotoxicity to human embryonic kidney 293 cells
and are attractive for potential in vivo applications.
Coprecipitation may affect the aggregation state of the core materials. In the
context of drug delivery, the aggregation state of a compound can affect the dis-
solution kinetics, bioavailability and chemical stability. Kumar et al. encapsulated
pyrene, hostasol yellow, and amphotericin B (hydrophobic, fluorescent dyes) using
a PS-b-PEG stabilizer and used fluorescence measurements to probe the aggrega-
tion state and dynamics of rearrangement of the compounds in the core of the
nanoparticles. The fluorescence spectra of pyrene is sensitive to the polarity of its
environment due to its aromatic structure, the emission structure of hostasol yellow
red shifts upon molecular aggregation, and the absorption of amphotericin B shifts
upon aggregation resulting in a lower excitation wavelength. Coprecipitating PS
and pyrene, the florescence of the excimer was quenched indicating specic
chemical interactions between the styrene ring of the PS nanoparticle core and the
pyrene. Time dependence of the fluorescence spectrum indicates that the pyrene
redistributes in the core of the nanoparticle over 24 days after Flash
NanoPrecipitation. When coprecipitated with cholesterol or PCL, signicant exci-
mer is evident content suggesting the pyrene nucleates independently from the core
material. Over 4 days, the excimer peak decreases as the pyrene disperses into the
nanoparticle core which is expected to be soluble based on Flory-Huggins theory.
In the case of Hostasol Yellow, the fluorescence spectrum is stable over time
suggesting that the dye is phase-separated from the PS core which agrees with the
FloryHuggins theory. Amphotericin B nanoparticles were produced using PS-b-
PEG as a stabilizer. In the nanoparticle core, the strongest emission is due to the
aggregated species. Over 2 days, the intensity of the peak associated with aggre-
gates increases indicating the core rearranges as hydrogen-bonded-hydroxyl-rings
regions increase to reduce the free energy of the core. If coprecipitated with vitamin
E, the dominant fluorescence is associated with the monomeric form of
Amphotericin B and the fluorescence of the aggregates is stable over 2 days
indicating no rearrangement (Kumar et al. 2010).
3 Imaging Agents
Coprecipitation is also a useful tool for producing multifunctional nanoparticles.

For example, nanoparticles for biomedical imaging can also be producing using
Flash NanoPrecipitation as a variety of imaging agents (organic and inorganic) can
be encapsulated. Further, the imaging agents can be coprecipitated with
hydrophobic therapeutics providing ample opportunity for the development of
theragnostic agents (Pansare et al. 2012, 2014).
For example, Akubulut et al. (2009b) report preparation of nanoparticles

encapsulating pyrene and Vitamin E with 58 wt% pyrene loading as well as
nanoparticles containing multiple fluorescent dyes which were used for in vitro
breast tissue imaging. Nanoparticles for fluorescence-based biomedical imaging
have also been developed using a new family of highly hydrophobic fluorescent
dyes. These dyes are based on pentacene structures with bulky silicon-containing
substituents and orthogonally disposed side groups that impose steric hindrance
which prevent molecular stacking and reduce self-quenching. Coprecipitating the
dye with PS and PS-b-PEG for stabilization, the emission spectra of the dye is
consistent with the dye in solution at innite dilution. Because this class of dye is
highly hydrophobic (logP * 10), no partitioning of the dye out of the nanoparticle
over time was observed. The maximum fluorescence intensity per particle occurred
at a dye concentration of 2.3 wt%, balancing the increasing fluorescence due to
more dye molecules in each nanoparticle core and self-quenching. At this con-
centration, intermolecular spacing between dye molecules is 3.9 nm, just below the
calculated Forester radius of 4.1 nm. Therefore, nonquenched particles can be
achieved at dye loadings below 2.3 wt%. Holding the dye concentration constant
and varying the nanoparticle size, the fluorescence per particle scaled with the
volume of the particle suggesting that the dye is homogenously distributed
throughout the PS core. Furthermore, the fluorescence intensity per particle was
independent of nanoparticle concentration indicating that quenching between par-
ticles does not occur due to the presence of the stabilizer. The resulting nontoxic,
photostable, nonquenching particles with high dye loadings provide enhanced
brightness compared to single molecule delivery and are thus attractive for imaging
applications (Pansare et al. 2014).
Flash NanoPrecipitation can also be used to produce inorganic/polymer com-
posite nanoparticles for medical imaging. Hydrophobic inorganic materials rst
suspended in an organic solvent with the stabilizing amphiphilic block copolymer
can assemble into sterically stabilized composite nanoparticles upon mixing with an
antisolvent. Upon mixing, there is colloidal aggregation of the inorganic material
due to net attractive interactions as well as self-assembly of the amphiphilic block
copolymer. In this case, the colloidal aggregation is capped by deposition of the
hydrophobic block of the copolymer onto the surface of the colloidal clusters. As in
the case of precipitating hydrophobic solutes, the mobility of self-assembled
copolymer chains is low and the composite inorganic nanoparticles are kinetically
trapped. Coprecipitation of the inorganic materials with organic solutes can also be
achieved by manipulating the relative kinetics of nucleation and growth, colloid
aggregation and block copolymer self-assembly. The composition of the composite
particles is dictated by the relative concentrations in the homogeneously mixed
organic solvent. These nanoparticles can include an inorganic material for diag-
nostic purposes as well as an organic therapeutic and would be a versatile platform
for developing multifunctional theragnostic nanoparticles (Gindy 2008; Gindy et al.
2008a).
For example, Gindy et al. produced multicomponent nanoparticles containing
colloidal gold as an illustrative imaging agent and b-carotene as a model therapeutic
using a PCL-b-PEG for stabilization (Gindy 2008; Gindy et al. 2008a).

Hydrophobic colloidal gold (*5 nm) capped with dodecanethiol (C12-Au) was
synthesized and encapsulated by Flash NanoPrecipitation. Encapsulation within the
PCL-b-PEG did not affect the metallic properties of the colloidal gold as no
broadening or shift in the surface plasmon resonance peak at 520 nm was observed
(Gindy et al. 2008a). The dodecane capping with an estimated thickness of 12 nm
controls the separation between neighboring colloidal gold and maintains the gold
particles in an electronically independent state (Gindy et al. 2008a). At a xed block
copolymer concentration, the average nanoparticle diameter increased from
50 2 nm (0 wt% Au loading) with increasing Au loading up to 103 6 nm (23
wt% Au loading) with relatively narrow size distributions (polydispersity indices
(PDI) less than 0.25 0.02) (Gindy et al. 2008a). To control the particle size,
homopolymer PCL (3.2 kg mol1), an inert component, was added. At a xed
colloidal gold concentration (0.016 wt% in solution), particles between 75 and
275 nm could were achieved. The size increased linearly with PCL volume fraction
for volume fractions above 33 vol%. At concentrations below 33 vol%, the particle
size remained constant and was attributed to initial lling of the interstitial voids
created by the packing of the colloidal gold. The size and gold loading could be
varied independently providing considerable flexibility (Gindy et al. 2008a).
Colloidal gold could also be encapsulated with b-carotene simultaneously forming
80 nm particles containing 30.5 wt% b-carotene, 5 wt% Au, and 64.5 wt% block
copolymer. The resulting particles were stable for at least 1 month in 155 mM
saline at room temperature, although the size increased slightly from 85 to 100 nm
was observed due to Ostwald ripening (Gindy 2008). The gold nanoparticles have
also been coprecipitated with a rifampicin-vitamin E prodrug. The resulting 300 nm
particle contained 0.4 wt% rifampicin (Liu 2007).
Lanthanide-doped nanocrystals (up-converting nanophosphors, UCNPs) are
another class of inorganic nanocrystals potentially useful for bioimaging and
phototherapy. UCNPs convert two or more near-infrared (NIR) photons to one
visible light photon via sequential electronic excitation and energy transfer pro-
cesses. Advantages of UCNPs include resistance to photobleaching, low toxicity,
and minimal autofluorescence that reduces background noise, sharp excitation, and
emission spectra, high quantum yields, and long life times. Additionally, the use of
NIR excitation enables deep penetration and the emission spectra can be tuned with
different lanthanide dopants. Such materials can be synthesized by cothermolysis of
trifluoroacetate ligands in the presence of coordinating ligands such as oleic acid
and trioctylphosphine oxide. The NaYF4 nanocrystals synthesized using this
method are inherently hydrophobic and surface modication to impart stability in
physiological conditions are needed. Flash NanoPrecipitation can be used to coat
UCNPs with a dense PEG layer. In the assembly process, the amphiphilic block
copolymer adsorb to the surface irreversibly. Hexaganol prism UCNPs (aspect ratio
1) with average diameters of 140 and 70 nm were coated with PLA-b-PEG or
PLGA-b-PEG. The hydrophobic block anchors to the UCNP surface and the PEG
end sterically stabilizes the nanoparticle in aqueous media and serum. Using a 61
ratio (by weight) of block copolymer to UCNP, PEG-protected UCNPs and empty
block copolymer micelles were obtained. The micelles could be easily removed by
centrifugation. No aggregation of UPNCs was apparent using scanning electron
microscopy (SEM) or dynamic light scattering (DLS) (Budijono 2010; Budijono
et al. 2009).
Building on this work, nanoparticles containing UCNPs with photosensitizers
for photodynamic therapy, a minimally invasive cancer therapy were made using
Flash NanoPrecipitation. In photodynamic therapy, visible light activates a pho-
tosensitizer that creates cytotoxic singlet oxygen. Issues related to this method
include limited tissue penetration with UV-visible light and toxicity of soluble
photosensitizers. When the UCNP is colocalized with a photosensitizer, the UCNP
absorb NIR light and produces UV-visible light that excites the photosensitizer.
Shan et al. used Flash NanoPrecipitation to colocalize UCNPs with
meso-tetrapheynl porphine, a photosensitizer within a 100 nm particle stabilized by
PLA-b-PEG that were stable in culture media for at least 25 h at 37 C and showed
no cytotoxicity (of cervical cancer HeLa cells) without NIR. With NIR illumination
(134 W cm2, 45 min at room temperature), 75 % cell death was observed. In this
approach, the use of NIR allows for deep tissue penetration and the components are
encapsulated within a biocompatible block copolymer shell, reducing the toxicity
associated with the photosensitizer (Shan et al. 2011). Penetration depths of up to
10 mm in rats have been reported (Chatterjee et al. 2008).
3.1 In Situ Complexation
In situ complexation is another approach that can be used to achieve supersaturation

and successfully encapsulate weakly hydrophobic, ionizable materials as well as
biomolecules such as small interfering RNA (siRNA) without chemical modica-
tion. In this method, the formation of hydrophobic complex occurs prior to
nucleation and growth and sufciently rapidly to drive supersaturation resulting in
homogenous nucleation and growth and formation of stable nanoparticles (DAddio
and Prudhomme 2011; Figueroa 2014; Pinkerton et al. 2012).
Weakly hydrophobic, ionizable, small molecule active pharmaceutical ingredi-
ents (API) can be effectively encapsulated using Flash NanoPrecipitation via
complexation with a hydrophobic counter-ion to form a hydrophobic salt in situ,
i.e., during mixing. Pairing with a hydrophobic counter-ion serves to increase the
hydrophobicity that enables rapid, kinetically controlled precipitation and depresses
crystallinity. Complex (ion pair) formation requires a difference in pKa between the
acid and the base of at least 2 pH units in the solvent conditions upon mixing.
For example, Cinnarizine, a weak base used to treat motion sickness (pKa 7 in
the mixed solvent), formed macroscopic precipitates upon Flash NanoPrecipitation
with PLA-b-PEG. When pamoic acid (pKa 2.5, 3.1 in mixed solvent) at a 1:1.1
molar ratio of base to acid was included, the Cinnarizine complexed with the
pamoic acid and precipitated into 115 nm nanoparticles sterically stabilized by the
PLA-b-PEG. The resulting nanoparticles were stable over at least 2 days with 93 %
encapsulation efciency and 27 wt% Cinnarizine loading. The base to acid ratio
affected the drug loading and encapsulation efciency. Minimizing the counter-ion
concentration is advantageous for increasing the drug loading, but reduces the
encapsulation efciency as uncomplexed Cinnarizine is soluble and is removed
upon dialysis or precipitates as a macroscopic crystal. Base to acid ratios between
0.5 and 1.8 led to formation of stable nanoparticles. Outside this range, precipitation
upon dialysis was observed. At base to acid rations below 0.75, the encapsulation
efciency was essentially 100 % whereas the maximum drug loading was 24 %
using base to acid rations between 0.97 and 0.54. At a constant base to acid ratio
(1.00) and core to block copolymer ratio (0.76), the size of the nanoparticle
increased linearly from 95 and 245 nm with the solids concentration indicating
growth-dominated kinetics. Furthermore, the resulting Cinnarizine pamoate salt
was amorphous by differential scanning calorimetry (DSC) and X-ray powder
diffraction (XRPD) indicating reduced crystallinity of the Cinnarizine and improved
the stability of the API. The measured surface charge is independent of the base to
acid ratio due to steric stabilization by the PEG (Pinkerton et al. 2012).
In other examples, Clozapine could also be encapsulated (*150 nm particles)
by complexing with pamoic acid at a 1:1 base to acid ratio. The encapsulation
efciency was 74 % and the drug loading was 22 wt%. Complexes of a-Lipoic acid
and N,N-dibenzylethylene diamine, a hydrophobic base formed macroscopic
needle-like crystals which could be encapsulated with the addition of pamoic acid
to frustrate crystallization. The resulting particle size was *300 nm, with 51 %
encapsulation efciency, and 12 wt% drug loading. This method can be extended to
encapsulate a number of weakly hydrophobic, ionizable APIs, expanding the types
of APIs of molecules that can be considered for formulation with Flash
NanoPrecipitation. Another advantage of ion pairing is that the hydrophobic salt
formed via ionic interactions is not considered a new molecular entity and thus
avoids the need for full FDA reapproval (Pinkerton et al. 2012).
Electrostatic driven assembly has also been used to encapsulate siRNA within
lipid nanoparticles using Flash NanoPrecipitation. As Kumar describes, electro-
static coupling between titratable, cationic lipids and negatively charged siRNA to
produce an insoluble complex can be achieved during mixing. A cationic lipid,
neutral lipid and PEG-lipid were dissolved in ethanol and mixed with siRNA
dissolved in citrate buffer (pH 3.8). After aging for 20 h at room temperature to
maximize encapsulation efciency, dialysis was performed to remove the ethanol
which restricts the mobility of the lipids thus freezing the nanoparticle structure.
Encapsulation efciency increased upon aging due to formation of lipid lamellar
vesicles which effectively incorporate the siRNA. Aging did not affect the
nanoparticle size indicating there was no evolution to an equilibrium size. The size
of the nanoparticle decreased with increasing molar ratios of cationic lipid to
phosphate groups on the siRNA. For example, at a ratio of 1, the nanoparticles were
*625 nm whereas at a ratio of 3 the nanoparticle size was *140 nm. This trend
was attributed to the formation of a more hydrophobic cationic lipid-RNA complex
resulting in a higher degree of supersaturation. Encapsulation efciencies were
highest (92 %) using a ratio of 2 and dropped to 70 % at a ratio of 1. Increasing the
concentration of PEG-lipid reduced the resulting nanoparticle size, but also reduced
encapsulation efciently as the excess PEG-lipid sterically hindered lipid domains
and frustrated the electrostatic capture of siRNA. The size of the nanoparticle was
also affected by the ethanol concentration. Sub-100 nm particles were obtained at
ethanol concentrations below 25 % due to higher supersaturation for the
hydrophobic lipids. The ethanol concentration also affected the encapsulation
efciency. For example, at 10 % ethanol the encapsulation efciency was 65 % but
only 28 % at 25 % ethanol. The faster precipitation at higher supersaturations limits
the electrostatic capture of RNA (Kumar 2011).
A similar approach based on charge neutralization via pH shift has been used to
encapsulate a peptide with low water solubility (0.01 mg mL1) using Flash
NanoPrecipitation. Cheng et al. dissolved Peptide B, synthetic Bombesin analogue
with nine residues and three polar side chains, in 1 % acetic acid (pH 3) and mixed
with a stream of block copolymer (PS-b-PEG) in THF and 175 mM NaOH. The pH
shift from 3 to 7 neutralizes the positive charge on the histidine residue, reducing
the solubility of the peptide. Peptide B nanoparticles were initially 30 nm, but grew
to 1 micron particles within minutes. The nal nanoparticle size appears to be
determined by the surface packing of the stabilizing polymer analogous to sur-
factant systems when the micelle size is determined by the packing parameter.
Due to the relatively weak hydrophobic interactions between Peptide B and the
hydrophobic block of the stabilizing copolymer, the polymer chains become
clustered on the surface of the nanoparticle rather than forming a uniform protective
layer. Upon aggregation, the polymer surface density increases and sterically sta-
bilizes the larger particles (Chen et al. 2009). Brownian dynamic simulations
indicate that stronger hydrophobic interaction between the hydrophobic block of the
copolymer and the core materials would result in smaller, well-protected
nanoparticles (Chen et al. 2009). In practice, the hydrophobic interactions are
affected by the choice of hydrophobic block of the copolymer and hydrophobicity
of the desired core material. Thus, modifying the block copolymer to increase
hydrophobic interactions with the core material could limit particle growth, but has
not been explored experimentally.
4 Stabilizer Selection
While the choice of core material provides functionality, the selection of stabilizing
agent, typically an amphiphilic block copolymer, signicantly affects nanoparticle
assembly as well as the resulting nanoparticle stability. In a few cases, aqueous
suspensions of stable nanoparticles using Flash NanoPrecipitation without a stabi-
lizing agent have been reported. Chung et al., for example, made stable 40 nm
particles of PCL, a hydrophobic polymer, with coumarin end groups. The modied
PCL was dissolved in THF and mixed against water. Removing the THF by
evaporation yielded an aqueous suspension of coumarin-PCL nanoparticles that was
stable for at least 4 months. Particle stability was attributed to repulsion caused by
the negative surface charge on the nanoparticles due to the presence of coumarin
(zeta potential measurement of 47.1 mV comparable to citrate stabilized colloidal
gold 43 mV). The nanoparticle size could be varied from 40 nm up to 90 nm by
increasing the concentration of coumarin-PCL in THF (Chung et al. 2012).
Similarly, Flash NanoPrecipitation could also be used to produce nanoparticle
suspensions from sulfate-terminated-PS. Due to the high molecular weight, a single
PS is larger than a critical nuclei size and only growth contributes to assembly.
During mixing, nanoparticles grow until electrostatic repulsions create interaction
potential barriers to prevent further aggregation. In this example, the nanoparticle
size increased with polymer concentration in the organic solvent, polymer molecular
weight, and ionic strength of the aqueous stream (Zhang et al. 2012a). Recently,
particles of zein, a plant protein found in corn, have also been prepared using Flash
NanoPrecipitation without the addition of a stabilizer. In this case, the size of the
nanoparticle could be also be adjusted by tuning the pH (Li et al. 2014).
For most hydrophobic core materials, an amphiphilic molecule is needed to
arrest the precipitation process by adsorbing onto the hydrophobic surfaces to
stabilize the nanoparticles. Polyelectrolytes can provide steric and electrostatic
stabilization. For example, b-carotene nanoparticles without stabilizer had a slight
negative charge but were unstable. The cationic polyelectrolytes e-polylysine, poly
(ethylene imine), and chitosan were used to improve the stability of the nanopar-
ticles. When incorporated into the mixing process, adsorption of the polyelectrolyte
occurs at a rate comparable to the precipitation of the b-carotene. Higher molecular
weight polyelectrolytes provided a better stabilizing effect and branched architec-
tures provided enhanced steric stability (Zhu et al. 2010). Amphiphilic block
copolymers such as poly(ethylene glycol) (PEG)-containing triblock copolymers,
Pluronics, with a hydrophobic poly(propylene oxide) (PPO) block, PEG-b-PPO-b-
PEG have also been considered. Nanoparticles of itraconazole, a hydrophobic drug,
could be made, but the triblock copolymer did not provide long-term stability. The
triblock copolymer increases the solubility of the drug due to uptake of the drug
into the cores of polymer micelles. Given the low glass transition temperature (Tg)
of the hydrophobic block, the PPO block is sufciently mobile to adsorb and desorb
with the core material increasing the rate of solubilization (Kumar et al. 2009a).
To effectively stabilize the nanoparticle, the amphiphilic molecule must adsorb
to the hydrophobic surface forming a kinetically frozen structure. In this regard,
amphiphilic block copolymers with low critical micelle concentrations (less than
103 wt%) and high Tg hydrophobic blocks with low mobility that are not subject to
dynamic equilibrium are preferred for long-term stability. Using di-block copoly-
mers prevents bridging between two particles as the hydrophobic block is anchored
to the nanoparticle surface (Figueroa 2014; Pustulka et al. 2013).
Amphiphilic di-block copolymers that are appropriate for stabilizing nanopar-
ticles made using Flash NanoPrecipitation have a distinct hydrophobic block and
hydrophilic block. The hydrophobic block provides strong van der Waals attraction
with the precipitating nanoparticle core, leading to high adsorption. The hydrophilic
block extends away from the particle surface, providing steric stabilization and
preventing aggregation (Figueroa 2014). Poly(ethylene glycol) (PEG)-containing
block copolymers are of particular interest as the PEG minimizes protein adsorption
and prolongs nanoparticle circulation in vivo. PEG with a 5 kg mol1 molecular
weight is considered the minimum effective PEG backbone length to prevent
protein adsorption (Figueroa 2014; Pustulka et al. 2013; Pansare et al. 2014;
Pinkerton et al. 2012). Biodegradable hydrophobic blocks approved by the United
States Food and Drug Administration such as e-polycapralactone (PCL) or poly
(lactide-glycoide) (PLGA) have been used (Pustulka et al. 2013; Kumar et al.
2009b; Budijono et al. 2009; Zhu 2013). However, PCL has a tendency to crys-
tallize which can lead to particle instability, high complement activation, and low
circulation times in vivo (Budijono et al. 2009; DAddio et al. 2012). The PCL may
form dense crystal clusters on particle surfaces leaving exposed hydrophobic sur-
faces that aggregate or the lamellar structure of the PCL crystals may have exposed
hydrophobic edges that induce aggregation (Budijono et al. 2009). Other
hydrophobic blocks such as PS has also been used as a model system (Kumar et al.
2010; Pansare et al. 2014). Biodegradable poly(ester carbonate) hydrophobic blocks
have also been considered due to facile tuning of their physiochemical and bio-
logical properties (Aguirre-Chagala et al. 2013). Synthetic bioactive amphiphilic
macromolecules with a 5 kg mol1 PEG hydrophilic block and hydrophobic
domain based on galactaric acid, a polyhydroxy acid, modied with lauroyl groups
have also been explored. These copolymers were designed to mimic the
amphiphilicity and polyanionic charge distribution seen in oxidized low density
lipoprotein and are of particular interest for atherosclerosis therapies (York et al.
2012; Lewis et al. 2011). D-a-tocopheryl poly(ethylene glycol) 1000 succinate is
another PEG-based amphiphilic molecule of potential interest as it is classied as
generally recognized as safe by the United States Food and Drug Administration
(Figueroa 2014).
The hydrophobic block chemistry will affect drug loading, particle stability, as
well as water activity in the core which is particularly important if the release of the
core material is mediated by hydrolysis (Figueroa 2014; Pustulka et al. 2013; Zhu
2014a; Aguirre-Chagala et al. 2013). In terms of nanoparticle assembly and sta-
bility, several factors need to be considered when selecting the hydrophobic block.
During assembly, the hydrophobic block must adsorb to the hydrophobic surface of
the precipitating core material. Adsorption will be increased if the hydrophobic
residues of the polymer have some afnity for the precipitating core material.
Coarse-grained simulations indicate if there are no interactions between the
hydrophobic block and core material, the di-block copolymers will preferentially
form empty micelles rather than forming stable nanoparticles. Insufcient afnity
and adsorption can result in patchy surface coverage and exposed hydrophobic
surfaces that may aggregate (Figueroa 2014; Zhu 2013, 2014a, b; Spaeth 2011).
The release of an API from the core of the nanoparticle will also be affected by
interactions between the core material and hydrophobic block. The solubility
parameter or FloryHuggins interaction parameter can be a useful measure of the
afnity between the hydrophobic residues of the stabilizing polymer and core
material (Zhu 2013, 2014a; Kumar et al. 2010).
The miscibility of the hydrophobic block with the hydrophilic block must also
be considered. If the hydrophobic and hydrophilic blocks are partially miscible,
during assembly both blocks may be trapped within the nanoparticle core in a single
phase. PEG concentrations lower than expected based on the formulation have been
experimentally observed when using PLA-b-PEG suggesting that a signicant
amount of PEG was trapped within the PLA matrix during assembly (Figueroa
2014; DAddio et al. 2012). The resulting decreased PEG concentration can lead to
particle aggregation as well as potentially affect performance in vivo. Similar results
have been observed with PLGA-b-PEG (Zhu 2013). Therefore, hydrophobic and
hydrophilic blocks that are fully micro-phase-separated are preferred for particle
stability (Figueroa 2014).
The molecular weight of the block copolymer needs to be considered as it will
affect the nal conformation of the chains on the particle surface critical for particle
stability (DAddio et al. 2012). The overall chain size as well as the size of the
hydrophobic block will affect the aggregation rate of the polymer. Higher molecular
weight polymer chains have lower diffusion coefcients leading to slower
self-assembly, while higher molecular weight hydrophobic blocks reduce the crit-
ical micelle concentration of the polymer which increases the supersaturation ratio
and self-assembly rates. These factors should be balanced to match rate of
self-assembly with the precipitation kinetics of the core material as well as ensure
that the hydrophobic block is sufciently large to prevent desorption so that the
resulting nanoparticle is kinetically frozen (Figueroa 2014). The size of
hydrophobic block will also affect the density of the PEG coating which is critical
for minimizing protein adsorption. In order to create a sufciently dense PEG layer,
the area of the hydrophobic block adsorbed on the nanoparticle surface must be
closely packed so that the PEG chains extend laterally away from the core of the
nanoparticle. As the molecular weight of the hydrophobic block increases, the area
on the surface of the nanoparticle core increases thus the density of the PEG layer
decreases (DAddio et al. 2012). Generally, for hydrophobic blocks such as PCL,
PLA, and PS the molecular weight should not be considerably larger than the PEG
block (5 kg mol1) (Figueroa 2014; Pustulka et al. 2013; DAddio et al. 2012; Zhu
2010). Recently, kinetically trapped micelles from PLGA-b-PEG with a linear
PLGA and branched PEG by Flash NanoPrecipitation have been reported and may
also be considered for use as a stabilizer. Linear-dendritic amphiliphic molecules
can provide well-dened areas of functional group presentation and may also
improve particle stability. Fluorescence measurements indicate that the
linear-dendritic amphiphilic molecules can be co-assembled with linear di-block
copolymers typically used (Santos and Herrera-Alonso 2013).
Reactive processing in which the amphiphilic block copolymer is made during
mixing has also been reported. In this approach, the b-carotene was stabilized by
the amphiphilic di-block copolymer formed by reactive coupling of an
amino-terminated hydrophilic block PEG-NH2 (MW 5 or 6 kg mol1) with an acid
chloride terminated hydrophobic block (PS-COCl, MW 2.5 kg mol1 or
PCL-COCl, MW 3.6 kg mol1). The coupling reaction was performed in the
presence of triethylamie (TEA) dissolved in the aqueous stream to remove HCl
which can deactivate the PEG-NH2. Using gel permeation chromatography, the
coupling conversion was determined to by *17 % using equal concentrations
(3 mM) of PS-COCl in THF and PEG-NH2 in water, and *6 % for coupling
PCL-COCl and PEG-NH2 at the same conditions comparable to predictive models
developed by Liu and Fox (2006) and Zhu et al. (2007). The lower conversion for
PCL-b-PEG may be attributed to faster nucleation and condensation of PCL that
traps the functional groups in the core of the particle before coupling. This approach
overcomes limitations on the concentration and molecular weight of premade block
copolymer due to the critical micelle concentration and avoids kinetically trapping
the hydrophilic block of a premade block copolymer during assembly which may
adversely affect polymer stability (Zhu et al. 2007).
5 Surface Modication
In addition to being a versatile platform for encapsulation, Flash NanoPrecipitation

is a convenient platform to produce nanoparticles with tailored surface chemistry
potentially facilitating targeted delivery of a therapeutic and/or imaging agent. To
tune the surface chemistry, a functionalized block copolymer is incorporated into
the mixing/assembly process. The functionalized block copolymer can be the block
copolymer conjugated to a small molecule targeting ligand (e.g., mannose, folate).
Alternatively, the PEG end of the block copolymer can be modied with a reactive
end group to enable conjugation to a targeting ligand to the nanoparticle after it has
been assembled. These approaches are outlined in Fig. 3 and can be extended
molecules such as dyes or imaging agents. Either approach results in a random
surface distribution of the ligand of interest. Incorporating a PEG-based amphiphile
functionalized with a fluorescent dye for visualization indicated a homogenous
distribution of moieties once assembled (Santos and Herrera-Alonso 2013). Further
experiments with a PEG-based amphiphile functionalized with dithiolane to enable
labeling with gold nanoparticles after nanoparticle assembly indicate random sur-
face distribution of the functional group based on TEM visualization (Santos and
Herrera-Alonso 2013).
Nanoparticles decorated with mannose to actively target delivery to macro-
phages produced in a single Flash NanoPrecipitation step have been reported by
conjugating mannose to the PEG end of the block copolymer. DAddio et al.
synthesized a-D-mannopyranoside with azide functionality in a three reaction step
synthesis with an overall yield of 69 % and transformed a hydroxyl-terminated PS-
b-PEG block copolymer to an alkyne-terminated PS-b-PEG. The modied block
copolymer was then conjugated to the azide-functionalized mannose using copper
catalyzed click chemistry. Flash NanoPrecipitation with the PS-b-PEG conjugated
to mannose and a methoxy-terminated PS-b-PEG was performed to generated
nanoparticles from 70 to 220 nm with 075 % mannose-terminated PS-b-PEG.
Nanoparticle association with macrophage-like J774 cells was highest with a
mannose surface density of 9 %. Association also increased with nanoparticle size
Fig. 3 Approaches for nanoparticle surface functionalization
which is attributed to an increase in contact area and subsequent increase in the

number of targeting ligands than can contact a cell surface. Flash NanoPrecipitation
is a powerful tool to explore active targeting as the size and the ligand density can
be varied independently. This is a versatile approach that can be applied to range of
small molecule targeting ligands with advantage that the degree of conjugation is
dictated by the formulation (DAddio et al. 2013).
For macromolecular targeting ligands that may interfere with the assembly
process or whose function may be adversely affected by the solvent conditions used
during mixing such as antibodies, the ligand can be conjugated to the nanoparticle
after it has been assembled by incorporating a block copolymer with a specic
reactive end group into the mixing process. For example, end group modication of
a hydroxyl-terminated PCL-b-PEG to maleimide-terminated PCL-b-PEG with 70
90 % conversion has been reported. Nanoparticles with maleimide surface func-
tionalization were conjugated to L-Glutathione. The degree of conjugation was
measured indirectly by quantication of the free thiol of the unreacted glutathione,
and estimated to be 51 and 67 % for PCL-b-PEG-MAL (8.6 kg mol1-b-4.6 kg
mol1) and PCL-b-PEG-MAL (4.6 kg mol1-b-4.6 kg mol1), respectively. Thiol
conjugation with maleimides with highly efcient and expected to be quantitative,
so the relatively low degree of conjugation to the nanoparticles suggests some of the
maleimide end groups are buried within the core of the nanoparticle and/or steri-
cally hindered by other surface/interfacial phenomena that render them less reactive
(Ji et al. 2009). Using a similar approach, bovine serum albumin (BSA) was
conjugated to maleimide-functional PCL-b-PEG nanoparticles. Unreacted protein
was separated via centrifugation through a 300 kg mol1 MWCO lter membrane.
Upon conjugation to BSA, the nanoparticle size increased by approximately 15 nm
measured by DLS close to the theoretical prediction of an 20 nm increase (hydrody-
namic radius of 3.7 nm and axial ratio of 2.66 for native BSA) with no indication of
particleparticle coupling or aggregation. The highest conversion, quantied by BCA
assay, obtained was 22 % using an excess of protein, resulting in *70 BSA molecules
per 30 nm nanoparticle. In this scheme, the homogeneous maleimide-thiol occurs on
the order of minutes, thus the alignment of the protein and reactive sites on the
nanoparticle congurational docking is the rate-limiting step. Congurational
docking includes steric hindrance constraints associated with protein bound to the
nanoparticle surface, exclusion of the protein by the PEG brush, protein packing effects,
and relative orientation of unoccupied reactive chain termini and protein reactive sites.
BSA binding to the surface of a nanoparticle is rst order with respect to thiol and
inversely proportional to the maleimide concentration so the degree of ligand binding
can be precisely controlled (Gindy et al. 2008b).
In other work, Zhang et al. synthesized PS-PEG-alkyne block copolymer and
used this block copolymer to produce fluorescent particles (encapsulating hostasol
red) with alkyne functionality on the surface. The alkyne functionalized nanopar-
ticles were covalently attached to azide labeled ssDNA via click chemistry (70 C).
Each nanoparticle was functionalized with *100 ssDNA molecules based on UV
absorbance of the particles at 260 nm. The high extent of functionalization resulted
in a 20 nm increase in particle diameter (86105 nm), which may be attributed to
the intrachain electrostatic repulsion due to the negatively charged phosphate
groups of the DNA. The ssDNA modied nanoparticles were used to generate
dsDNA via polymerase chain reaction (PCR). Using UV spectroscopy, it was
estimated that there were *32 dsDNA molecules per particle which corresponds to
*75 % of the theoretical maximum surface coverage dictated by geometric con-
straints (Zhang et al. 2011b).
Similar alkyne-azide click chemistry approaches have been used to conjugate
folate, a small molecule targeting ligand, as well as a recombinant protein engi-
neered with non-natural amino acids to the surface of premade nanoparticles. In
these examples, nanoparticles with azide surface functionality were fabricated using
PLA-b-PEG-azide synthesized by ring opening polymerization of lactide from the
hydroxyl end of a heterofunctional HO-PEG-azide macroinitiator. Flash
NanoPrecipitation with functionalized PLA-b-PEG-azide yielded 70 nm particles
decorated with azide moieties. The resulting nanoparticles were conjugated to (c)-
alkyne-folate to maintain the biological activity of the folate required to facilitate
folate-receptor mediated endocytosis. Performing the conjugation reaction in water
using a 11 ratio of the alkyne to azide and Cu(I) as a catalyst (generated by in situ
reduction of Cu(II) by sodium ascorbate), 10 % of the folate was conjugated to the
nanoparticles. A recombinant protein A1 engineered with ethynyl-phenylalanine, a
non-natural amino acid, near the N-terminus to introduce a single alkyne moiety,
could also be conjugated to the azide-functionalized nanoparticles in the presence
of tris(hydroxypropyl)triazolylmethyl-amine and Cu(I) catalyst. The ratio of alkyne
to azide was 15 and unreacted protein was removed by microdialysis. Upon
conjugation to the protein, the hydrodynamic radius of the nanoparticle increased

by 16 nm, consistent with the size of the protein. Conjugation was also conrmed
using a semiquantitative spot blot method using an anti-Histidine antibody. When
conjugating protein to the nanoparticles, high extents of reaction can be obtained
without using a large excess of protein which is a major advantage click chemistry
approaches compared to the alternative maleimide-thiol approach. The stability of
the alkyne and azide moieties is another advantage when compared to the mal-
eimide:thiol or activated ester:amine chemistries which are susceptible to deacti-
vation due to thiol oxidation or ester hydrolysis, respectively (Zhang et al. 2012b).
The functionalized block copolymer with a specied reactive PEG end can allow
for site specic conjugation to a targeting ligand of interest with the appropriate
complementary functionalization. Maleimide-thiol and alkyne-azide click chemistry
are flexible platforms that can be adapted to a range of ligands of interest. Proteins
and other biomolecules are well-suited for this approach. Optimizing methods for
separating the unreacted molecules after the conjugation reaction and quantication
of the degree of conjugation to the nanoparticle are in progress. Further, these
approaches can be extended to other functional molecules of interest such as
imaging agents (e.g., gadolinium an MRI imaging agent) to further the capabilities
imparted to each nanoparticle. Overall, Flash NanoPrecipitation is uniquely suited
to developing nanomedicines for therapy and/or diagnosis. The ability to vary the
ligand, ligand density and nanoparticle size will be valuable in developing
nanoparticles that can be delivered to specic tissues of interest via active targeting.
6 Nanoparticle Stability
While the liquid nanoparticle dispersions resulting from Flash NanoPrecipitation

are generally stable for short time when stored refrigerated, Ostwald ripening
(interfacial-energy-driven dissolution and reprecipitation of solutes leading to an
increase in particle size), recrystallization, and solute degradation may affect
long-term stability (Figueroa 2014; Kumar and Prudhomme 2009). Lower organic
solvent content slows Ostwald ripening; therefore, removal of the solvent quickly
after mixing is recommended. On the laboratory scale, solvent removal is typically
achieved by dialysis against a sufciently large aqueous bath (10:1 bath volume:-
nanoparticle dispersion volume) and replenishing the bath several times (46 times
over 624 h) (Figueroa 2014).
Rapid removal of the organic solvent can also be achieved using Flash
Evaporation, especially effective for THF/water systems due to the strong
non-ideality in the THF-water vapor-liquid equilibrium (Kumar and Prudhomme
2009). Flash evaporation involves preheating for partial vaporization and a sudden
reduction in pressure. PS-b-PEG nanoparticles encapsulating b-carotene in 10 %
THF, a model system, was preheated to 57 C and fed to a vacuum chamber
operating at 2.96 kPa. The residual liquid was heated to 45 C and refed to the
chamber at an operating pressure of 2.96 kPa. The residual solvent after two steps
was calculated to be 0.21 wt% THF and empirically determined to by 0.37 wt% of
THF which is within the FDA approved limit of 0.5 wt%. After the two-stage
process, the resulting suspension was indenitely stable. The flash evaporation
approach would be especially advantageous when considering large-scale opera-
tions (Kumar and Prudhomme 2009).
Since the long-term stability of liquid dispersions can be challenging, dry dis-
persions such as powders that can be reconstituted via freeze-drying tend to be more
stable and practical. Freeze-drying involves a freezing step, primary drying to
sublime ice crystals and secondary drying to remove residual water from the sample
(Figueroa 2014). Performed immediately after Flash NanoPrecipitation,
freeze-drying can be an alternative to dialysis. For example, Kumar et al. formu-
lated nitric oxide prodrugs and anticancer lead compounds using PLA-b-PEG and
PS-b-PEG using Flash NanoPrecipitation. The initial size of the particles was 240
and 225 nm for PS-b-PEG and PLA-b-PEG, respectively. At 4 C, the particle size
increased to 440 nm over 20 h and resulted in macroscopic precipitation over 2
3 days due to Ostwald ripening. Dialysis of the dispersion to remove the organic
solvent resulted in precipitation within hours; however, freeze-drying of the sample
immediately after mixing produced a stable powder that could be reconstituted to
the initial particle size (Kumar et al. 2009b).
Generally, avoiding aggregation and maintaining particle size during
freeze-drying is a challenge. The components of the nanoparticle greatly affect the
stability of the nanoparticles during freeze-drying which facilitates particle recov-
ery. For example, the physical state of the core material impacts the recovery of
nanoparticles. Comparing nanoparticle cores of b-carotene (crystalline solid),
Vitamin E (liquid), PS (glassy solid), and poly(propylene glycol)
(PPG) (amorphous liquid), the nanoparticles with solid cores were larger after
drying than liquid core particles. This result may be due to the ability of the liquid
core particles to deform which distributes the forces associated with drying over a
greater area when compared to a solid particle that cannot deform. The higher
stresses on the solid particles may increase aggregation (Aguirre-Chagala et al.
2013; Figueroa et al. 2013). Additionally, the block copolymer that provides steric
stabilization of the nanoparticles greatly affects aggregation when freeze-drying.
When freeze-drying PEG coated nanoparticles, the phase behavior of PEG can be
benecial during freezing, but induces aggregation upon drying. Using PS-b-PEG,
and varying the molecular weight of the PEG block with a xed PS block to control
the degree of surface coverage (PEG chains/nm2), Figueroa et al. found that the
nal particle size relative to the initial particle size increased with higher surface
coverage. This trend was attributed to particleparticle bridging due to crystal-
lization of PEG during secondary drying and could be reduced by heating. Using
PLA-b-PEG or PCL-b-PEG, aggregation upon freeze-drying has been attributed to
exposed hydrophobic block surfaces resulting from lower PEG surface due to some
miscibility of the PEG with the hydrophobic block (Figueroa 2014).
The use of protectants during freeze-drying to particle growth and aggregation
has been explored. While salts such as sodium bicarbonate (Figueroa et al. 2013)
can be used as a lyoprotectant and sugars such as sucrose and trehalose can be used
as a cryo- and lyoprotectants, high concentrations of such materials are required and
contribute signicantly to osmolarity of the nal dispersion (Figueroa 2014). Since
osmolarity is a colligative property and depends on the number of molecules pre-
sent, the use polymers as protectants present an attractive alternative. With higher
molecular weights compared to sugars or salts, the osmolarity contribution is less at
an equal mass concentration. Triblock copolymers of PEG-b-PPO-b-PEG can be
used as a surfactant stabilizer during freeze-drying. For example, Pluronic F68
(PEG-b-PPO-b-PEG, MW: 3.4 kg mol1-b-1.7 kg mol1-b-3.4 kg mol1) is gen-
erally recognized as safe by the Food and Drug Administration in the United States
has been used to stabilize b-carotene encapsulated within PLA-b-PEG (Figueroa
et al. 2013), vitamin D3 within PS-b-PEG (Figueroa 2014), as well as siRNA
encapsulated within PEG-lipid during freeze-drying nanoparticles so that the size
after freeze-drying was 11.7 times the initial size (Kumar 2011). Because it is
miscible with water at 0 C, it can help protect exposed hydrophobic surfaces as
well as intercalate amongst the PEG chains in the stabilizing layers of the
nanoparticles during freezing and act as a protectant. SEM of lyophilized
progesterone-loaded nanoparticles with Pluronic F68 indicated that the primary
particles are dispersed in a polymer matrix and Pluronic F68 effectively separates
the nanoparticle. However, a mixed micelle population has been observed after
reconstitution indicating a fraction of the hydrophobic core can be stripped out of
the initial nanoparticles by Pluronic micelles (Figueroa 2014; Kumar et al. 2009a).
PEG homopolymer can also be used as an effective lyoprotectant. Since at high
molecular weights (20 kDa), phase separation from the nanoparticle can occur due
to depletion flocculation, PEG protectants should be of similar or smaller molecular
weight to the PEG used for stabilizing the nanoparticles during Flash
NanoPrecipitation. Use of PEG as a protectant resulted in improved particle sta-
bility compared to Pluronic F68, but lower API recovery. For PLA-b-PEG or PCL-
b-PEG nanoparticles with poor PEG steric stabilization, Pluronic F68 can be used
for extra steric stabilization in combination with PEG as a lyoprotectant. For
nanoparticles with dense PEG coatings such as those made with PS-b-PEG, PEG
alone serves as a robust lyoprotectant during freeze-drying (Figueroa 2014).
Freeze-thawing and freeze-drying have been compared using PEG-based
excipients. In freeze-thawing, 84 % API recovery and no increase in nanoparticle
size was observed in freeze-thawed samples with or without protectants. Therefore,
no aggregation occurs during freezing as the PEG coating provides sufcient steric
stabilization. Furthermore, examining initial cooling rates between 1.5 and *165
C min1, the effect on API recovery and nal nanoparticle size were insignicant.
This result suggests the kinetics of crystallization of the hydrated PEG layer are fast
enough to reject excess water during freezing and freezing rate is not an important
parameter in the cryopreservation of PEG-stabilized nanoparticles. In freeze-drying,
however, the API recovery was less than 26 % for samples without protectant and
85 % with protectant. By reconstituting with a lower than initial volume,
freeze-drying can result in particle concentration. Despite loss of API during
freeze-drying, the overall API concentration can still be increased by *2 fold.
Removing the organic (usually by dialysis) prior to freeze-drying improves API

recovery (Figueroa 2014).
Spray freeze-drying can also be used to convert Flash NanoPrecipitation for-
mulations into dry form. In spray freeze-drying, the liquid dispersion is sprayed
through a nozzle into a cryogenic liquid (e.g., liquid nitrogen) to freeze the sample,
and then heated under reduced pressure to sublime the ice and evaporate associated
water and residual organic solvent and leads to spherical micronsized nanopar-
ticles embedded in an excipient matrix. The resulting particles may be useful in
aerosol applications. For example, DAddio et al. produced powders of cholesterol,
PLA-b-PEG nanoparticles made by Flash NanoPrecipitation embedded within a
mannitol matrix for aerosol administration via spray freeze-drying with ultrasonic
atomization. The micron-sized mannitol particles contained up to 50 wt%
nanoparticle loading. Furthermore, with sonication the nanoparticles redispersed to
below 200 nm. The redispersability of the nanoparticles is a signicant advantage
over spray drying in which the liquid dispersion is sprayed through a nozzle to
create droplets into a hot gas that dries the droplets into micron-sized structures,
which caused irreversible aggregation of the nanoparticles. Spray freeze-drying
may be an attractive alternative to freeze-drying as it can be done as a continuous
process (Figueroa 2014; DAddio et al. 2013).
As an alternative to freeze-drying based methods, nanoparticles can also be
concentrated and dried using a hydrogen-bonding coacervate precipitation process
(DAddio et al. 2010). In this method, PEG-protected particles aggregate into a
lterable precipitate upon addition of polyacid species due to hydrogen-bonding
interactions between PEG and the polyacid. The hydrogen-bonding is pH depen-
dent thus the aggregation is reversible. When the acid is protonated, interactions are
present inducing precipitation of a hydrophobic complex. At higher pH (>7), the
complex dissociates as the ionized acrylate groups cannot hydrogen bond. The
nature of the hydrogen bond is the interaction between the oxygen and the proton
on the carboxylic acid with 1:1 stoichiometry. Using b-carotene encapsulated
within PEG-b-PLGA (5 kg mol1-b-7 kg mol1) produced by Flash
NanoPrecipitation, DAddio et al. precipitated the nanoparticles (110140 nm)
using an excess of polyacid (poly(acrylic acid), poly(aspartic acid) or citric acid),
ltered using a 1.2 micron lter, neutralized the dispersion with 0.1 N sodium
hydroxide, and resuspended the nanoparticles. Using poly(acrylic acid) or citric
acid, the nanoparticle size increased by *8 % upon redispersion and using poly
(aspartic acid) the size increased by *40 %. The size upon redispersion was
independent of polyacid concentration, and increased concentration resulted in
faster particle agglomeration. Excess acid is recoverable and recyclable while the
lter cake could be freeze-dried or vacuum dried and resuspended with dilute
sodium hydroxide. The fully dried samples redispersed to sizes below 175 nm
using a probe tip sonication and a progression of sonication at room temperature
and at 55 C. Alternatively the wet lter cake could be redispersed in a small
amount of 1 M sodium hydroxide to concentrate the nanoparticles over 80 fold.
Drying the nanoparticle dispersions to dry powders that can be reconstituted
when needed can signicantly increase the shelf life of the nanoparticle
formulation. Given appropriate lyoprotectants, e.g., PEG to prevent irreversible

nanoparticle aggregation, it is possible to produce dry powders that can be recon-
stituted in a clinical setting while maintaining nanoparticle sizes appropriate for
parenteral applications. Since freeze-drying is commonly used, industrial devel-
opment is possible.
7 Conclusion
Flash NanoPrecipitation is a simple, rapid and scalable method capable of con-

tinuously producing nanoparticles with tunable sizes between 50 and 500 nm,
narrow size distributions, high drug loading capacities, and tailored surface che-
mistries. In Flash NanoPrecipitation, an amphiphilic block copolymer is dissolved
in organic solvent with a desired core material (therapeutic and/or imaging agent).
Upon rapid mixing with a miscible antisolvent for the core material of interest and
decrease in solvent quality, self-assembly of the amphiphilic block copolymer
directs bottom-up assembly of the nanoparticle. The precipitation of the core
material is arrested by adsorption of the hydrophobic block of the block copolymer
while the hydrophilic block sterically stabilizes the nanoparticle. The choice of core
material provides desired functionality but requires suitable solubility such that high
supersaturations are achieved upon mixing. Hydrophobic materials with logP val-
ues greater than 6 are ideal. Methods to encapsulate weakly hydrophobic com-
pounds as well as biomolecules such as proteins and peptides have been explored.
Multifunctional nanoparticles encapsulating inorganic imaging agents and thera-
peutics have also been achieved. While core material selection dictates function-
ality, the choice of stabilizer is important for producing stable nanoparticles.
Amphiphilic block copolymers with low critical micelle concentrations (less than
103 wt%) and high Tg hydrophobic blocks with low mobility that are not subject to
dynamic equilibrium are preferred for long-term stability. Nanoparticles can be
further modied for active targeting as Flash NanoPrecipitation is a convenient
platform to tailor the surface chemistry of the nanoparticles. To obtain the desired
the surface chemistry, a functionalized block copolymer is incorporated into the
mixing/assembly process. The functionalized block copolymer can be the block
copolymer conjugated to a small molecule targeting ligand (e.g., mannose, folate).
Alternatively, the PEG end of the block copolymer can be modied with a reactive
end group to enable conjugation to a targeting ligand to the nanoparticle after it has
been assembled. Ligands such as folate, mannose, ssDNA, and proteins have been
attached to the nanoparticles. By leveraging the simplicity and scalability of this
process with versatile material selection, Flash NanoPrecipitation is a powerful tool
in nanomedicine uniquely suited to developing actively targeted therapeutics,
imaging agents, or theragnostic particles.
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Chapter 4
Preparation of Polymer Nanoparticles
by the Emulsication-Solvent Evaporation
Method: From Vanderhoffs Pioneer
Approach to Recent Adaptations
Nstor Mendoza-Muoz, Sergio Alcal-Alcal

and David Quintanar-Guerrero
Abstract This chapter provides an overview up to date of the emulsication-solvent

evaporation method to prepare polymer nanoparticles for pharmaceutical researchers
and formulators. It highlights the recent technological advances, assessment, and
new modalities of this method (e.g., double-emulsion and emulsication-solvent
displacement). The aim of this chapter is to review representative works and discuss
the raw materials, preparative variables, conditions, formation mechanisms, etc., in
order to make them useful for specic developments of drug nanoparticles. The
considerable progress which has been made in the Van de Hoff's method will be
reviewed with examples and applications to show its effectiveness, versatility,
advantages, and limitations. Finally, the chapter is written in such way that the reader
obtains enough criteria involved in the process to facilitate the formulation task.

Keywords Polymer nanoparticles Drug delivery systems Solvent evaporation

Single emulsion Double emulsion
1 Introduction
One of the main challenges in developing systems for the delivery of drug dosage
forms based on nanoparticles is selecting the most suitable preparation method, as
there are several physicochemical, biopharmaceutical, technological, economic,
N. Mendoza-Muoz
Laboratorio de Farmacia, Facultad de Ciencias Qumicas, Universidad de Colima, Carr.
Coquimatln-Colima km 9.5, 28400 Coquimatln, Colima, Mexico
S. Alcal-Alcal D. Quintanar-Guerrero (&)
Laboratorio de Posgrado en Tecnologa Farmacutica, Facultad de Estudios Superiores
Cuautitln, Universidad Nacional Autnoma de Mxico, Av. 1 de mayo s/n, 54745
Cuautitln Izcalli, Estado de Mxico, Mexico
e-mail: quintana@unam.mx

DOI 10.1007/978-3-319-41421-8_4
88 N. Mendoza-Muoz et al.
environmental, and other, aspects to be considered prior to adopting a robust

process that has the potential to be scaled up to an industrial level. Currently,
formulators have several techniques to choose from when producing polymer
nanoparticles. In general, those derived from preformed polymers are preferred over
methods that involve polymerization reactions. The emulsication-solvent evapo-
ration method was the rst one to obtain sub-micronic drug-charged polymer
particles. This technique was based on the pioneering work of Vanderhoff reported
in the 1970s for the preparation of pseudo-latexes. Due to its technological
advantages, this method continues to be the one most widely employed to prepare
polymer nanoparticles from preformed polymers in particular nanospheres. Since its
introduction, several modications have been reported in order to render it more
efcient and permit industrial applications; for example, the solvents dichlor-
omethane and chloroform were replaced with less toxic substances, such as ethyl
acetate; the original stabilizers were similarly replaced; the homogenization
equipment was made more efcient; and it became possible to produce batches
in-line, etc. This chapter examines the main aspects of the emulsication-solvent
evaporation method, with particular interest on outlining and explaining the features
and vital criteria involved in the process in order to facilitate the work of formu-
lators. The different methods for performing this technique are summarized in three
modalities: conventional emulsion-solvent evaporation; double emulsion-solvent
evaporation; and emulsion-solvent displacement. The advantages and disadvan-
tages of each modality are discussed critically to identify the key variables that
determine their relative success. Finally, we discuss recent developments and
possible future applications.
2 The Emulsication-Evaporation Method
2.1 Generalities
The emulsication-evaporation method (EEM) was rst described by Vanderhoff

et al. (1979) in 1979 and proposed as an alternative to the emulsication-
polymerization process for production of latexes. The major advantage of this option
is the absence of the toxic residual monomers, unreacted agents, or catalysts that
result from the emulsication-polymerization method (Quintanar-Guerrero et al.
1998; Staff et al. 2013a). As this brief description suggests, EEM was easily adapted
to develop polymer nanoparticles intended for use as drug delivery systems, and
today several patents have been granted (Nava-Arzaluz et al. 2012).
EEM is based on the emulsication of an organic phase composed of a
water-immiscible solvent in which the preformed polymer is dissolved in an
aqueous phase that contains the stabilizing agent, and high-shear agitation is used to
nally obtain an oil-in-water (o/w) emulsion. Under appropriate conditions, the
subsequent evaporation of the organic solvent induces polymer aggregation in the
4 Preparation of Polymer Nanoparticles by the Emulsication 89
Organic phase Emulsification

Polymer and drug in
immiscible solvent
Ultrasound
High Pressure
Homogenization
Aqueous phase
Stabilizer
Nanoparticles suspension Solvent evaporation
Fig. 1 Schematic representation of the steps involved in the EEM
form of nanoparticles with a diameter of a few hundred nanometers. Drugs with

lipophilic characteristics are then incorporated into the organic phase. Purication
stages can be performed by recovering polymer nanoparticles by ultracentrifugation
and consecutive washes with distilled water to remove the stabilizer and release the
drug. Additional freeze-drying removes water to increase shelf life. Then
cryo/lyoprotectans are added to ensure complete reconstitution and prevent changes
in particle size. A schematic representation of EEM is shown in Fig. 1.
Compared to other preparation methods based on preformed polymers, EEM has
specic advantages and disadvantages. Table 1 summarizes the most salient char-
acteristics of this method for polymer nanoparticle preparation from preformed
polymers.
The process of obtaining polymer nanoparticles by EEM involves two basics
steps, as follows: rst, emulsication, and, second, solvent removal. During
emulsication, droplets of the organic solvent solution containing both the polymer
and the drug are nely dispersed into the aqueous phase, and droplets of nanometric
sizecalled nanodropletsonce the solvent is removed polymer aggregates in
the form of nanoparticles are obtained, in the most frequent situation each
nanoemulsion droplet forms one nanoparticle (Quintanar-Guerrero et al. 1998;
Chernysheva et al. 2003). Thus, emulsication is the crucial step, and can be
achieved by: (a) direct emulsication of the organic solvent solution in water
(high-energy); or, (b) inversion emulsication of the organic solution by adding
water (low-energy) (Vanderhoff et al. 1979).
90
Table 1 Characteristics of the methods to prepare polymer nanoparticles from preformed polymers
Characteristic Emulsication-evaporation Solvent displacement Emulsication-diffusion Salting-out Double
emulsion-evaporation
Drug to Lipophilic Lipophilic Lipophilic Lipophilic Lipophilic and
encapsulate hydrophilic
Encapsulation High High High High Medium
efciency
Type of ICH class 2 and 3 ICH class 3 ICH class 3 ICH class 3 ICH class 2 and 3
organic
solvents
residuals Solvent and stabilizer Solvent and stabilizer Solvent and stabilizer Solvent, stabilizer Solvent and stabilizer
and salt
Scale-up Medium Low Medium Medium High
difculty
Main limitant High energy requirements Not easy to found High volumes of water Extensive washing High shear stress
for emulsication drug/polymer/solvent/nonsolvent to be eliminated from steps for salting-out required for primary
system the suspension agent removal. emulsion, low double
emulsion stability
References Chacn et al. (1996, Ueda Chacn et al. (1996, Quintanar-Guerrero Galindo-Rodrguez Zambaux et al.
and Kreuter (1997), Pin-Segundo et al. (2006), et al. (1998, 1999), et al. (2005, Zweers (1998, Lamprecht
Kreuter et al. (2011), Molpeceres et al. (1996), Thioune Galindo-Rodrguez et al. (2003), et al. (1999), Van de
Miller (2010) et al. (1997) et al. (2005) Galindo-Rodriguez Ven et al. (2011)
et al. (2004)
ICH International Conference of Harmonization
N. Mendoza-Muoz et al.
Direct emulsication is commonly preferred because, compared to the inversion

emulsion method, no additional care or control are required. To obtain droplets of
nanometric size, it is necessary to introduce large amounts of energy into the system
to force the creation of an enormous interfacial area. Energy in the form of
mechanical stress is frequently applied to generate nanoemulsions. The literature
shows that three main groups of equipment are used for this purpose: rotor/stator
devices; high-pressure homogenizers; and ultrasound generators (Anton et al.
2008). The rst assembly consists of a rotor with two or more blades and a stator
with vertical or slanted slots around the wall of the homogenizer cell. The rotor is
housed concentrically inside the stator. One of the two major forces that can reduce
the size of the dispersed droplets is mechanical impingement against the wall
caused by high fluid acceleration; the second is the shear force that occurs prin-
cipally in the gap between the rotor and the stator (Maa and Hsu 1996). Two kinds
of systems are available; the rst intended mainly for discontinuous operation
e.g., Ultraturraxand the second for continuous operation (e.g., colloid mills). In
the Ultraturrax rotor/stator homogenizer type, the turbulence is expected to be
much more intensive than in an agitated vessel or a static mixer, because emulsi-
cation in the rotor/stator homogenizer is achieved by rapid-flow circulation and
high shear force in the narrow gap between the rotor and the stator (<0.5 mm).
Factors that could influence the circulation rate and shear force are the design of the
rotor/stator assembly, the rotational speed of the rotor, total volume, and the vis-
cosity of the emulsion (Maa and Hsu 1996); while homogenization intensity
(power) and the residence time of the emulsion droplets in the shearing eld are the
main parameters that control emulsion droplet size.
The energy introduced is mostly dissipated by generating heat, or spent in
viscous friction. In colloid mills, the discontinuous phase is disrupted in an annular
conic slit between the rotor and stator. The width of the slit is in the range of a few
hundred micrometers (usually 100500 lm, but sometimes up to 3000 lm). Due to
the conic construction, the width of the annular slit can be adjusted by axial
movement of the stator. Thus, it is possible to vary both the mean residence time of
the emulsion in the colloid mill and the power density at a constant rotational speed.
A typical peripheral velocity of the rotor in colloid mills is in the range of 540 m/s,
equivalent to 20003000 min1 with rotor diameters of 50250 mm (Urban et al.
2006).
High-pressure homogenizers are the most important, continuously operated
emulsifying devices. These devices consist essentially in a high-pressure pump and
a homogenizing nozzle. The pump is used to force the crude emulsion through the
nozzle where it is depressurized and disrupts the drops. The homogenizing pressure
used is typically between 50 and 500 bar (Stang et al. 2006). As a result, shear,
impact, and cavitation forces are applied to very small volumes to generate
nanoscale emulsion droplets (Freudig et al. 2013). The homogenizing nozzle is a
critical element for the efciency of disruption when producing emulsions using
high-pressure homogenizers. The standard-size nozzle is the one most widely uti-
lized. In this device, the crude emulsion is pumped through a central inlet bore by
means of a high-pressure pump, diverted by 90, and then forced through the radial
gap between the valve seat and valve plug. Due to this specic flow path, these
nozzles are also called radial diffusers.
During preparation of nanoparticles, an ultrane emulsion in the nanometric
range should form during the emulsication step. On some occasions, high-speed
homogenizers are used to obtain a coarse emulsion, which is later passed through a
high-pressure homogenizer to obtain ne droplets. Table 2 exemplies the ideal
conditions for obtaining polymer nanoparticles using a high-speed homogenizer in
combination with a high-pressure homogenizer at laboratory scale.
In general, the mechanism for forming nanoemulsions using high-energy
methods includes: as a rst step in drop creation, the deformation and disruption of
macrometric initial droplets, followed by surfactant adsorption at their interface to
insure steric stabilization (Anton et al. 2008). The formation of such
nanometric-scale droplets is governed by directly controllable formulation param-
eters, such as the amount of energy, the amount of surfactant, and the nature of the
components.
Nanoemulsions generated by soniers are generally less energy demanding than
those produced by a rotorstator. Dispersal by ultrasound has been found to be
competitive, or even superior, in terms of droplet size and energy efciency
(Kentish et al. 2008). Ultrasonic emulsication is believed to occur through two
mechanisms: rst, the application of an acoustic eld that produces interfacial
waves which become unstable, eventually resulting in the outburst of the oil phase
into the aqueous phase in the form of macro-droplets; and, second, the application
of low-frequency ultrasound that causes acoustic cavitationi.e., the growth and
collapse of microbubblesunder the effects of an ultrasonic eld in liquids. Each
microbubble implodes on a microscopic scale in a collapsing action that causes
extreme levels of highly localized turbulence. These turbulent micro-implosions act
as a very effective method of breaking up primary droplets of dispersed oil into
droplets of sub-micron size (Kentish et al. 2008). Parameters that affect the emul-
sication process include power, ultrasound time, surfactant concentration, the
oil/water ratio, the viscosity of the continuous phase, hydrostatic pressure, gas
content, and pre-emulsication (Gaikwad and Pandit 2008; Cucheval and Chow
2008). The viscosity of the discontinuous phase also plays an important role, for it
becomes a qualitative measure of molecular interaction in a liquid; i.e., the higher
the viscosity, the higher the intensity of ultrasound for the onset of cavitation
(Behrend et al. 2000). Industrial systems for large-scale production consist of
several ultrasonic processors with from 2 to 16,000 W. When ultrasound is applied
to an identical liquid formulation with an identical processing parameter congu-
ration, the same energy per volume is required to obtain an identical result,
regardless of the scale of processing. This allows for a linear scale-up of the
optimized parameter conguration to full commercial scale (Hielscher 2005). In
addition, emulsions can be produced in continuous flows or in batches. The
ultrasonic devices utilized are low maintenance and very easy to operate and clean.
Table 3 shows the specic conditions for obtaining polymer nanoparticles using
ultrasonic emulsication at the laboratory scale.
Table 2 Conditions for the preparation of polymer nanoparticles by EMM using high pressure homogenization during the emulsication step
Nanoparticle composition Pre-emulsication Conditions Emulsication step Conditions Final Ref.
step nanoparticle
size (nm)
Polymer: PLGA 50:50, 65:35 and High-speed Share velocity: High-pressure Cycles: 4 <250 Jaiswal et al.
PEGylated PLGA homogenizer, 13,000 rpm. homogenizer, Rannie, Pressure: (2004)
Organic solvent: dichloromethane Ultra-turrax, IKA Time: 2 min Model mini-lab type 500 bar
Stabilizer: PVA (2 % w/v) volume: 8.30 H, Volume:
Drug: Cyclosporine A 130 ml 130 ml
Polymer: cCDC6 NA NA High-speed Share velocity: 141175 Lemos-Senna
Organic solvent: dichloromethane homogenizer, 20,500 rpm et al. (1998)
Stabilizer: Pluronic F68 (0.64 % Ultra-turrax T25, IKA Time: 2 min
w/v) Volume:
Drug: Progesterone 125 ml
Polymer: PLGA 75:25 High-speed Share velocity: High-pressure Cycles: n.r. 140220 Kreuter et al.
Organic solvent: dichloromethane homogenizer, 15,100 rpm. homogenizer, APV Pressure: (2011)
stabilizer: PVA (0.5 % w/v) Ultra-turrax, IKA Time: 2 min Micron Lab 40 400 bar
Drug: doxorubicin HCl Volume: 30 ml Volume: 30 ml
Polymer: ethyl cellulose High-speed Share velocity: High-pressure Cycles: n.r. 104 Miller (2010)
Organic solvent ethyl acetate homogenizer, 10,000 rpm. homogenizer, Pressure: 80 psi
stabilizer: Polytron 3100, Time: 4 min Microfluidics, Volume: 25 ml
4 Preparation of Polymer Nanoparticles by the Emulsication
d-alpha-tocopheryl-polyetilenglycol kinematica Volume: 25 ml M-1105-F12Y

1000-succinate
Drug: valdecoxib
Polymer: PLGA NA NA High-speed Share velocity: 100140 Grandls
Organic solvent: dichloromethane homogenizer, 24, 000 rpm et al. (1997)
and DMSO stabilizer: Ultra-turrax T25, IKA Time: 2 min
cholesterol-3-sulfate and pluronic Volume:
F68 755 ml
drug: somatropin
NA not apply, n.r. not reported, PLGA: poly(lactide-co-glycolide), PEG: poly(ethylene glycol), PVA: poly(vinyl alcohol), gamma CDC6: gamma
93
cyclodextrin modied with carbon chains in C6, DMSO: dimethylsulfoxide

Table 3 Conditions for the preparation of polymer nanoparticles by EMM using ultrasound for
the emulsication
Nanoparticle composition Conditions for Nanoparticle Ref.
emulsication size (nm)
Polymer: PLGA 50:50, 75:25 and Device: laboratory probe 228301 Gonsalves
PLLA (3 % w/v) Potency: 90 W et al. (2011)
Organic solvent: dichloromethane: Time: 15 min
acetone (8:2 v/v) Stabilizer: PVA Volume: 30 ml
(02.5 % w/v)
Drug: Nafcillin
Polymer: PLGA 50:50 Device: Misonix 485695 Feng (2006)
Organic solvent: dichloromethane Potency: 50 W
Stabilizer: PVA (4 % w/v) and Time: n.r.
d-alpha-tocopheryl-poly(ethylene Volume: n.r.
glycol) 1000-succinate
Drug: Paclitaxel
Polymer: PLGA 50:50 Device: Laboratory Probe 501000 Lim (2011)
Organic solvent: dichloromethane Potency:700 W
Stabilizer: PVA (4 % w/v) Frequency: 20 kHz
Drug: Magnetite and indocyanide Time: 5 min
green Volume: n.r.
Polymer: PLA and PEG-PLA Device: Laboratory 336785 Hildgen et al.
copolymer Probe, Sonic (2006)
Organic solvent: chloroform Dismembranator 550
Stabilizer: PVA (0.5 % w/v) Potency: 15 %
Drug: Rhodamine Time: 3 min
Volume: 510 ml
Polymer: PLGA Device: micro tip probe, 216220 Labhasetwar
Organic solvent: chloroform XL 2015, Misonix and Sahoo
Stabilizer: PVA (5 % w/v) Potency: 55 W (2010)
Drug: paclitaxel Time: 2 min
Volume: 15 ml
Polymer: PLApoly(trimethylen Pre-emulsication: 256 Klee and
e-carbonate) 90:10 High-speed homogenizer, Hilgers
Organic solvent: dichloromethane ultra-turrax T25, IKA. (2007)
Stabilizer: PVA (4 % w/v) 15,000 rpm, 30 s
Drug: dexomethasone Emulsication:
Device: branson sonier
450
Potency: n.r.
Time: 2 min
Volume: 55 ml
(continued)
Table 3 (continued)
Nanoparticle composition Conditions for Nanoparticle Ref.
emulsication size (nm)
Polymer: PLGA Device: sonicator 115263 Sung et al.
Organic solvent: dichloromethane VCX-750, Sonics (2007)
Stabilizer: sodium cholate (0.1 % Potency: n.r.
w/v) Time: pulse 1 s by 1 s
Drug: Paclitaxel pause during 20 min
Volume: 51 ml
n.r. not reported, PEG-PLA: poly(ethylene glycol)-poly(lactide)
As dened here, low-energy emulsication methods require only a modest

amount of applied energy. Emulsication using low-energy methods tends to
produce smaller and more uniform droplets (Calder et al. 2001) based on the phase
transitions that take place during the emulsication process and the changes in the
spontaneous curvature of the surfactant. Phase inversion is a process by which a
water-in-oil (w/o) emulsion can be inverted to an oil-in-water (o/w) emulsion, or
vice versa. Phase inversion can be classied in two types: catastrophic phase
inversion (CPI), and transitional phase inversion (TPI). In CPI, inversion is induced
by increasing the volume fraction of the dispersed phase, depending on starting type
of emulsion (o/w or w/o) addition of oil to a water surfactant mixture or addition of
water to a solution of the surfactant mixture in oil should be performed. In contrast,
in TPI methods, factors such as temperature and/or electrolyte concentration are
suitably modied to induce phase inversion, thus directly affecting the
hydrophilic-lipophilic balance (HLB) of the system. TPI can also be induced by
changing the HLB number of the surfactant at a constant temperature using different
surfactant mixtures. The best known low-energy emulsication method is called
phase inversion temperature (PIT). This concept, introduced by Shinoda and Saito
(1968) and Shinoda and Sation (1969), uses the specic ability of surfactants,
usually nonionic ones such as polyethoxylated surfactants, to modify their afnities
to water and oil as a function of temperature, and thus achieve phase inversion.
Scale-related assessments should be performed when deciding between CPI or TPI
phase inversion as the more adequate emulsion preparation method. With regards to
the PIT method, although it is easy to quickly cool a sample prepared in a test tube
by immersing it in ice, it seems to be more difcult to achieve fast cooling when a
large tank is involved. In CPI, conditions such as mixing and the addition rate
should be taken into account to assure a proper transition of phases, regardless of
the scale.
To collect useful data, the geometric ratio, or form factor, should be held con-
stant when using larger sizes. Also, it is assumed that fluid characteristics such as
density (q), diffusivities (D), and viscosity () will be held constant. Under these
conditions, if the two systems (small and large batches) are geometrically similar
they can be described by an adimensional relationship, regardless of the scale. If the
two different systems have the same Froude (Fr), Reynolds (Re) and Schmidt (Sc)
numbers, both can be described by the same adimensional differential equations

(Sol et al. 2010). Equation 1 describes the mass balance of the water (component
A) into an agitated tank in contact with a fluid B (oil and surfactants).
@CA ~ C 1 r2 C
~
vr 1
@t A
ReSc A
where CA is the concentration of water in the system, v is velocity of the agitator

and the adimensional numbers Re = qvl/ and Sc = /qDAB,.
Equation 2 allows to obtain the reduced linear velocity at any reduced time and
any point of the tank before applying a microscopic momentum balance in non-
stationary regime.
v
@~ ~~ 1 2 ~ p 1 ~g
v r
~ v r ~
v r 2
@t Re Fr g
where p is the pressure, g is the gravity force and Fr = v2/gl.

In this cases, the Schmidt number can be kept equal if the formulation are the
same, the Froude number is not considered a relevant factor since vortex is not
formed if the system has a high viscosity or could be avoided if deflectors are
installed in the tank, however in practice Reynolds numbers can only be maintained
constant if high angular rates are keep at small scales (Sol et al. 2010). In a study
by Sol et al. (2010) of a potassium oleate-oleic acid-C12E10/hexadecane/water
system, a nearly linear correspondence between scales was obtained when the
scaling variable was the linear mixing rate, indicating that it is the proper scale-up
variable to be used, together with total emulsication time, in order to assure
conservation of the concentration analogy between scales.
2.2 Mechanism of Formation
Understanding the mechanism of the formation of polymer nanoparticles by EEM is

the key to control the variables involved, including size, surface morphology, and
encapsulation efciency as the main responses. In addition, better comprehension
will lead to optimization. The mechanism of formation of polymer nanoparticles by
EEM has been studied by Desgouilles et al. (2003) and Staff et al. (2013a). At rst
sight, the formation of polymer nanoparticles by EEM is simple: a preformed
polymer is dissolved in an efcient water-immiscible solvent, then this oily phase is
emulsied in water that contains a stabilizer to form a stable o/w emulsion with
droplets of nanometric scale using one of the methods mentioned above.
Subsequent evaporation of the organic solvent leads to precipitation of the polymer
on the water-solvent interface (bad solvent); while the stabilizing agent prevents
flocculation and coagulation of the polymer nanoparticles by steric and/or elec-
trostatic repulsion.
(a) (b)
Emulsion droplet
at the end of
emulsification
Solvent concentration in the system during evaporation process
Coalescence
No Coalescence
Nanoparticle
Fig. 2 Obtaining polymer nanoparticles by EEM: scheme of the mechanism of formation, adapted
from the text in Desgouilles et al. (2003)
Desgouilles et al. (2003) and Galindo-Rodrguez et al. (2005) described two

ways to form nanoparticles by EEM (Fig. 2). In the rst (A), each droplet from the
initial emulsion produces one nanoparticle. In their study, the particle size and zeta
potential of the initial emulsion was measured using the solvent evaporation pro-
cess, and showed that the zeta potential values did not change during evaporation,
so no size increase was evident. These authors (Galindo-Rodrguez et al. 2005)
suggested that no, or only very limited, coalescence occurred. In other words, they
assumed that one nanoparticle was formed after solvent evaporation from just one,
or only a few, emulsion droplets. The emulsion droplets should be perfectly sta-
bilized during solvent evaporation. This study demonstrated that nanospheres of
poly(L-lactide acid) (PLLA) apparently follow this mechanism when ethyl acetate
is used as the solvent in the experimental protocol (since ethyl acetate is partially
soluble).
In the second sequence (B), emulsion droplets fuse during the solvent evapo-
ration step to form one nanoparticle from two or more emulsion droplets. In the
study by Desgouilles et al. (2003), contrary to PLA nanospheres, nanoparticles
formed from ethylcellulose (EC) (4849.5 % ethoxylation, and MW of 56,600 and
98,600). The size of the droplets increased after approximately 30 min because
droplet or nanoparticle nuclei fusions occurred, leading to an increase in the
diameter of the dispersed species (emulsion droplet or nanoparticle nucleus).
Recent studies by (2013a) focused on evidence of droplet coalescence during
solvent evaporation using indirect and direct measurements. Dynamic light scat-
tering (DLS) and fluorescence correlation spectroscopy (FCS) were used as indirect
methods to determine the level of droplet coalescence. By measuring the average
droplet and particle sizes using DLS it was possible to calculate the aggregation
ratio, A, dened as the average number of droplets required to form one polymer
nanoparticle in accordance with the Eq. 3:
mP=NP q
A 3
mP=Drop DDrop 3
c DNP
where, mp/NP is the mass of the polymer in the particles, mp/Drop is the mass of the
polymer in the droplets, q is the density of the polymer in the nanoparticles, c is the
concentration of the polymer in the dispersed phase, DDrop is the average diameter
of the nanodroplets, and DNP is the average diameter of the nanoparticles. If one
nanoparticle originated from just one or a few emulsion droplets, then lower values
of A should be calculated. On the other hand, FCS can provide information on the
number of fluorescent-diffusing species in a specic volume and, therefore, on the
concentration of fluorescently labeled species. In the absence of coalescence, no
change in the concentration of fluorescent species should be found. Staff et al.
(2013a) calculated the A value for poly(styrene) (PS) nanoparticles prepared with
chloroform as the solvent, and determined a value of approximately 14, which
indicates that on average 14 droplets merged to create one particle. No changes in
the concentration of fluorescent species were apparent. In a similar study, A values
were reported by Desgouilles et al. (2003) for EC nanoparticles and PLLA
nanoparticles, at 32 and 4, respectively. Despite these ndings, it is important to
note that in the information that could be obtained on the DLS mechanism, the
hydrodynamic diameter of droplet emulsions and polymer particles depended on
the properties of the solvent and dilution conditions when measurements were
taken. These considerations should also be taken into account if zeta potential
measures are used as a parameter for elucidating the behavior of emulsion droplets
in the evaporation step, since the partition of the surfactant as the adsorbed species
or molecularly dissolved species on the particles surface may not be the same for
undiluted and diluted samples. The study by Staff et al. (2013a), as mentioned
above, employed direct methods to determine the coalescence of droplets during the
evaporation process of EEM, while dual-color fluorescence cross-correlation

spectroscopy (DC-FCCS) showed that coalescence is not signicant if the droplets
are sufciently stabilized. In that study, emulsion droplets were separately labeled
with two different dyes and then mixed. Next, the solvent was evaporated to yield
nanoparticles, and the cross-correlation curves of the temporal evolution of the
fluorescence intensity of the labeled nanoparticles were calculated. The amount of
double-labeled nanoparticles was found to be below 10 %, indicating that no sig-
nicant coalescence occurred.
In conclusion, the main mechanism that governs the formation of polymer
nanoparticles by EEM assures that each emulsion droplet forms one polymer
particle when the solvent is removed, since a high correlation between the size of
the primary emulsion and that of the resulting nanoparticles has been found. In only
a very few cases (perhaps less than 10 %) does coalescence take place. Thus, these
ndings demonstrate that the preparative variables during the emulsication process
and the stabilization of the nanodroplets play important roles in achieving poly-
meric nanoparticle dispersion within a narrow size distribution.
2.3 Raw Materials and Their Importance for the Synthesis

of Polymer Nanoparticles by EEM
2.3.1 The Polymer
The polymers most frequently used to prepare nanoparticles by EEM are the same
as those utilized in other polymer nanoparticle synthesis methods. Polymers such as
PLLA, PLGA, a copolymer of PLLA and poly(glycolide) (PGA), EC, cellulose
acetate phthalate (CAP), poly(e-caprolactone) (PCL), poly(b-hydroxybutyrate) and
copolymers derived from esters of acrylic and methacrylic acid, for instance
Eudragits RL and RS, are those most frequently used. The polymer composition
(hydrophobicity, surface charge, biodegradation prole) of the nanoparticles exerts
a strong influence on drug absorption, the biodistribution pattern, and elimination
(Pinto Reis et al. 2006).
PLGA is one of the most successfully used biodegradable polymers for the
development of nanomedicines. This is because once it is inside the body it
undergoes hydrolysis to produce biodegradable metabolite monomers. The
copolymer ratio composition is the most important factor in determining
hydrophilicity and the degradation rate. In general, an increase in the percentage of
glycolic acid in the oligomers accelerates weight loss in the polymer. For example
in the case of implant devices, PLGA 50:50 (PLLA/PGA) degradation time is
approximately 12 months; 75:25 in 45 months; and 85:15 in 56 months. The
faster degradation results from the preferential degradation of the glycolic acid
portion assigned by higher hydrophilicity (Kumari et al. 2010). In the case of micro-
and nanoparticles interesting results have been found about degradation rate and the
mechanism release. Dunne et al. (2000) and Panyam et al. (2003) studied in vitro
degradation of PLGA 50:50 nanoparticles and microparticles and found that
polymer degradation behaves biphasic in both nano- and microparticles, with an
initial rapid degradation for 2030 days followed by a slower degradation phase
over 6070 days. I was observed that the particles maintained their structural
integrity during the initial degradation phase; however, this was followed by pore
formation, deformation, and fusion of particles during the slow degradation phase.
Specically, when nanoparticles of 0.1 lm were compared with 1 and 10 lm size
microparticles, the nanoparticles demonstrated relatively higher polymer degrada-
tion rate (P < 0.05) during the initial phase as compared to the larger size
microparticles (rst-order degradation rate constants of 0.028, 0.011, and
0.018 day1 for 0.1, 1 and 10 lm particles, respectively), however the degradation
rates were almost similar (0.0080.009 day1) for all size particles during the
second period despite a 10- and 100-fold greater surface area to volume ratio for
nanoparticles as compared to microparticles. Its results could be explained by the
relatively higher amounts of the stabilizer (poly(vinyl alcohol) (PVA)) found in the
smaller size nanoparticles (0.1 lm) as compared to the larger size. PLLA, a similar
polymer, is a biocompatible and biodegradable material that in the body undergoes
scission into monomeric units of lactic acid as a natural intermediate step in car-
bohydrate metabolism. PLLA is a slow-degrading polymer compared to PLG, and
has good tensile strength, low extension, and a high modulus. However, because it
is more hydrophobic than PLG, the degradation rate of PLLA is very low. It has
been reported that high molecular weight PLLA can take between 2 and 5.6 years
to achieve total resorption of implants in vivo (Nair and Laurencin 2007). On the
other hand, PCL is an easy-to-process homopolymer that is soluble in a wide range
of organic solvents, has a low melting point (5560 C) and glass transition tem-
perature (60 C), while conserving the ability to form miscible blends with a wide
range of polymers (Nair and Laurencin 2007). In physiological conditions, the
polymer undergoes a two-stage degradation process: rst, the nonenzymatic
hydrolytic cleavage of ester groups, and second, when the polymer is more highly
crystalline and of low molecular weight (less than 3000) the polymer is shown to
undergo intracellular degradation as evidenced by observation of PCL fragments
uptake in phagosomes of macrophages and giant cells and within broblasts
(Bodmeier and Chen 1990); however, the rate of degradation is rather slow (2
4 years) (depending of the starting molecular weight of the device or implant)
(Woodruff and Hutmacher 2010), no reports about the degradability of PCL from
microparticles or nanoparticles have been reported at the date.
The concentration of the polymer in the discontinuous phase has a signicant
impact on particle size. Generally, an increase in polymer concentration leads to an
increase in particle size; a fact explained by the increasing viscosity of the dispersed
phase that results in a poorer dispersability of the PLGA solution into the aqueous
phase (Mainardes and Evangelista 2005). Babak et al. (2007) studied the intrinsic
viscosity of different polymers used in preparing nanoparticles by EEM. The
intrinsic viscosity [] order was as follows: PCL > PLGA > Eudragit
RS Eudragit RL, after dilution in methylene chloride. In general, the better the
solvents thermodynamic quality for a given hydrophobic polymer, the larger the
size of the macromolecular coil, and the higher the intrinsic viscosity []. The
morphology of the nanoparticles is very closely related to the viscosity inside the
droplets (Staff et al. 2013b). As diffusion of the chains is necessary for phase
separation, one possibility for obtaining kinetically trapped morphologies consists
in increasing the viscosity by means of high molecular weight polymers. Also,
coarse primary emulsions are obtained at higher polymer concentrations, thus
contributing to the buildup of larger particles during diffusion. The polymer not
only plays an important role in the structure of the nanoparticle, when dissolved in
the dispersed phase, it prevents growth of droplet size by exerting sufcient osmotic
pressure to prevent Ostwald ripening.
On occasion, changes occur in the properties of the polymer during preparation
of nanoparticles, and such alterations may have repercussions for degradation rates
or drug release proles. Staff et al. (2012) studied changes in the crystallization of
semicrystalline polymers, including PLLA, in particles produced by EEM.
The DSC study revealed changes in the PLLA microstructure and displayed cold
crystallization at 88 C; in contrast to bulk PLLA, with which no cold crystal-
lization was found. The extent of cold crystallization proved to be size-dependent,
since it was greater for smaller particles than larger ones. This can be explained by
the strong connement of the particles due to their limited diameter. The limited
volume restricts the number of states of the polymer chains and, hence, the prob-
ability of forming a supercritical nucleus in the interior, as has been explained by
the authors.
During solvent evaporation, the discontinuous phase undergoes signicant
changes due to the loss of solvent. These changes can be divided into three stages:
(1) solution state; (2) gel state; and, (3) glassy state (Li et al. 1995) (Fig. 3). When
the polymer solution becomes concentrated due to the progressive evaporation of
the solvent, the polymer in the discontinuous phase becomes more viscous, and
eventually reaches the gelation stage. This behavior can be described as
thermo-reversible gelation. The ensuing solvent removal induces glass transition in
the polymers rich phase. In this stage, the particles are in the glassy state (i.e., the
long, flexible polymer chains tend to become entangled and attract each other by
secondary valence forces), and phase separation is complete. The solid gel sepa-
ration makes it more difcult for the solvent to pass through the solidied region. In
the gel state, the particle structure begins to stabilize, and movement of the active
agent inside the polymer aggregate is restrained. The time required to reach the gel
state may be a crucial parameter for the nal characteristics of the particles and for
their drug encapsulation efciency.
2.3.2 The Solvent and the Extraction Process
The main challenge in selecting the suitable solvent consists in nding an organic
solvent with high pressure vapor that can solubilize both the polymer and the
substance to be encapsulated. As mentioned above, the solvent must be immiscible
Sol State Gel State Glass State

Viscous Boundary Glass Boundary
(Gelation Point)
Fig. 3 Structural changes in the polymer due to the solvent evaporation during the formation the
nanoparticles based on phase separation methods
with water and its boiling point must be below that of water to ensure complete
evaporation from the nal dispersion due to the solvents potential toxicity. The
most common solvents used in EEM are dichloromethane and chloroform; the
former because it can dissolve large amounts of biodegradable polymers, shows
low solubility in water (2.0 % w/v), and has a low boiling point that allows con-
venient removal by evaporation. When the drug or polymer is not completely
soluble, a mixture is required. The most commonly used co-solvents are short-chain
alcohols, such as methanol or ethanol. Recently, hexafluoroisopropanol (HFIP) was
proposed as a suitable candidate for preparing polymer nanoparticles via the
emulsionsolvent evaporation method. In that case, HFIP was used as the dispersed
phase and several apolar solvents were tested for the continuous phase (Bohlender
et al. 2013).
Water is the number one option for the continuous phase, though it is not
suitable for all cases and has been replaced in nonaqueous emulsions by other polar
solvents, such as dimethylformamide (DMF), formic acid, formamide, or dimethyl
sulfoxide (DMSO). However, these polar solvents are difcult to remove because of
their high boiling points.
In pharmaceutical products, solvents often cannot be completely removed by
practical manufacturing techniques such as freeze-drying or high-temperature
drying under vacuum conditions, and levels that exceed regulatory limits can be
toxic for humans or the environment (Han et al. 2012). The International
Conference on Harmonization (ICH) of Technical Requirements for Registration of
Pharmaceuticals for Human Use classies regularly used residual solvents into
three groups based on their toxicity. Solvents that are known to cause unacceptable
toxicities (Class 1) should be avoided in the production of drug substances,
excipients, or drug products, unless their use is strongly justied by a risk-benet
assessment. The use of solvents with less severe toxicity (Class 2) should be limited
to protect patients from potential adverse effects. Ideally, less toxic solvents (Class
3) should be utilized wherever practical. According to ICH guidelines, the limit of
residual chloroform (Class 2) is below 60 ppm; for dichloromethane (Class 2) no

more than 600 ppm; and for ethyl acetate (Class 3) below 5000 ppm.
Forming micro- and nanoparticles from EEM involves two basic processes:
namely, mass transfer, and phase separation. Also, two main components will be
described: the dispersed (polymer dissolved in organic water, an immiscible sol-
vent), and continuous phases (water with or without a stabilizer).
Following the emulsication step and in the course of the solvent removal stage,
the organic solvent is generally eliminated from the disperse phase which occurs
as droplets. During typical solvent extraction, the continuous phase is mixed vig-
orously so that turbulence is created to promote the process. The removal process
can be divided into two steps (both of which involve mass transfer phenomena):
(1) extraction or diffusion of the solvent from droplets to the continuous phase; and,
(2) the subsequent evaporation of the solvent from the continuous phase. Mass
transfer in the continuous phase should occur rst at the interface between the
continuous and dispersed phases (droplet surface). At this point, such physico-
chemical properties as the diffusion coefcient of the organic solvent into the water
(nonsolvent) and solvent-polymer system, and the saturation solubility of the
respective solvents all play important roles (Sawalha et al. 2008). The diffusivity of
the solvent from the droplet to the continuous phase provides information on lm
resistance. This diffusivity can be estimated using various correlation equations
(Hayduk and Laudie 1974); whereas the solubility of the organic solvent in the
aqueous phase determines the initial rate of extraction. Other associated physico-
chemical properties include interaction parameters, such as those of the following
phases: organic solvent-polymer, organic solvent-non-solvent, and
non-solvent-polymer (Li et al. 1995).
Once the organic solvent diffuses into the aqueous phase and reaches saturation
solubility, a second mass transfer event occurs at the interface between the con-
tinuous phase and air. In fact, both mass transfer processes take place simultane-
ously. The driving force of solvent evaporation from the surface of the container is
the volatility of the solvent, such that higher volatility speeds up extraction from the
continuous phase. Volatility is related to the tendency of atoms or molecules to
escape from the liquid in the form of gas, and is an interpretation of the vapor
pressure, a physicochemical parameter that indicates the pressure exerted by vapor
on thermodynamic equilibrium in condensed phases. In other words, the use of
organic solvents with high vapor pressure promotes an increase in the evaporation
rate during this step of EEM. Agitation speed, flow regime, effective container area
for evaporation, and pressure, are all variables intimately related to the nal char-
acteristics of size distribution and morphology of the nanoparticles (Conti et al.
1995). Also, variables such as the ratio of the dispersed-to-continuous phase,
temperature, and composition of the dispersed phase should predict the particle
properties based on those process variables. See Fig. 4 for a schematic
representation.
Kinetically, the evaporation process is simple and comprised of two stages. At
the beginning of evaporation the rate is constant (zero order), but when the con-
centration of the organic solvent decreases below saturation, evaporation continues
Extraction Variables:
External pressure
Evaporation /Solvent difusion Aerodynamical conditions
Continuous Phase Variables:

Internal/External phase ratio
Boiling point
Mass transfer Viscosity
Temperature
Diffusion Hydrodynamic Variables:

Stirr speed
Emulsion Flow regime
Geometry of the container
droplet Geometry of the blade of
agitation
Internal Phase Variables:

Internal/External Phase Ratio Vapour Pressure
Diffusion Coefficient Boiling Point
Polymer concentration Saturation solubility
Viscosity in the continous pase
Fig. 4 Schematic representation of the mass transfer process and relevant variables involved
during the solvent evaporation process
as a rst-order process (Li et al. 1995; Sawalha et al. 2008). The full prole removal
should show that solvent evaporation is the rate-limiting step in the process, once
diffusion of the solvent from the globules into the discontinuous phase becomes fast
and is favored by the large interfacial area.
In summary, organic solvents with high diffusion coefcient values, high solu-
bility in the continuous phase, and high vapor pressure, promote quick removal of
the solvent such that the phase separation process of the polymer is achieved in less
time. This reduces to some degree the possibility that the droplets might coalesce
and produce much larger particles.
One particularly important observation is that it is possible to optimize the
solvent evaporation rate in a shorter time by applying reduced pressure. Using this
modication, smaller particles are produced in comparison to the conditions of
atmospheric pressure (Mainardes and Evangelista 2005). When the solvent is
removed by reduced pressure a higher solvent front kinetic energy is formed that
promotes the dispersion of ne droplets in the aqueous phase. This rate is also
extremely important for drug entrapment, because fast evaporation minimizes drug
diffusion into the external aqueous phase. Once organic solvent is chosen
depending on its physicochemical characteristics, type of polymer to be used and its
safety to be used in pharmaceutical products, the next stages in the obtaining of
polymer nanoparticles involve emulsication and solvent evaporation processes as

indicated above. Laboratory studies show that variables such as stir rate, type, and
amount of dispersing agent, type of polymer, viscosity of organic and aqueous
phases and temperature should be known, optimized and controlled in order to
prepare nanoparticles with an adequate and reproducible size and a narrow size
distribution. Thus, parameters of operation, like stir rate and temperature, were
dened and measured in a lab scale and using equipment that can be easily led to an
industrial scale. However there could be some limitations in scaling up to produce
large batches of nanoparticles, even in a pilot scale, because in EEM high shears
and high energy consumption are required during the rst step
(emulsication-homogenization) by using mixing devices and industrial pumps and
the evaporation stage must be enough efcient to achieve residual solvent within
specied limits (Pinto Reis et al. 2006). On the other hand, nanoparticle suspen-
sions are intended to be administered by a parenteral route which implies to have
sterilized products and the use of clean rooms. Not all sterilization methods are
suitable because they can modify nanoparticles and degrade the drug (Vauthier and
Bouchemal 2011). In contrast, a common nonsolvent used in emulsions and
therefore in evaporation solvent stage is water; the use of this solvent improves
process economics because recycling is eliminated and washing step is facilitated.
Regarding biopharmaceutical properties of nanoparticles, Ranjan et al. (2012)
found no signicant differences in drug loading, release rate, particle size, and
pharmacokinetic proles of PLGA-Curcumin C3 nanoparticles when a lab-batch
was scaled up.
2.3.3 The Stabilizer and Purication Step
As mentioned previously, EEM includes the creation of oil-in-water (O/W) emul-

sions by using high-speed homogenizers and/or probe-tip sonicators. The
nanometer-sized organic droplets must be stabilized using large amounts of sur-
factant, even greater than the quantity of polymer. The surfactant or stabilizer is
selected with an eye to obtaining optimum droplet size; however, the stabilizer also
aids in reducing aggregation of the particles when nanoprecipitates form during the
solvent evaporation process. Once the nanoparticles form, the stabilizer must be
removed completely or reduced to a secure concentration, depending on its
potential toxicity.
Stabilization of emulsions by these surfactants is basically attributed to the
latters ability to reduce the interfacial tension between the aqueous and organic
phases when the emulsion is rst created, while the capacity to reduce the aggre-
gation of the recently formed nanoparticles is based on the steric and/or electrostatic
repulsive forces created by the stabilizer adsorbed into the particle surface and
expressed when another nanoparticle comes sufciently close.
The most common surfactants used to prepare polymer nanoparticles by EEM
are poly (ethylene glycol) derivatives, such as Poloxamer and Tween, and phos-
pholipids, such as lecithin. Poly (vinyl alcohol) is also commonly reported. Less
usual are cetyltrimethylammonium chloride, sodium lauryl sulfate, and natural

polymers such as gelatin, albumin, etc.
Several studies have demonstrated the dependence of stabilizer concentration on
the size of the nanoparticles produced using emulsication methods
(Galindo-Rodriguez et al. 2004; Zambaux et al. 1998; Mainardes and Evangelista
2005; Wheatley and Lewandowski 2010). In most cases, a logarithmic relation is
found between stabilizer concentration and the reduction in the size of the
nanoparticles. Also, granulometric distribution became narrower as the amount of
stabilizer was increased. This behavior can be explained by the protective effect of
the stabilizer. These results make it easy to understand that an insufcient amount of
stabilizer would fail to be completely adsorbed on the surface of the nanoparticles
and, therefore, would tend to aggregate. However, the reduction in the size of the
nanoparticles formed could also be explained by the increase in the viscosity of the
external phase when a polymer stabilizer is used.
The stabilizer can be removed from the formulation by dialysis, crossflow l-
tration (CFF)also known as tangential flow ltrationor ultracentrifugation.
Birnbaum et al. (2000) studied the efciency of these three techniques in the
purication of PLGA nanoparticles stabilized by different agents. The surfactants
tested were poly(oxyethylene) sorbitan monooleate (Polysorbate 80), sodium
dodecyl sulfate, sodium cholate, human serum albumin, and PVA (MW 6000, 88 %
hydrolyzed). Dialysis was found to be the least effective method for purifying
nanoparticles because it required 24 h to remove approximately 90 % (mass bal-
ance) of the dodecyl sulfate using 50,000 MWCO membranes. Pore size was
determinant for the performance of this process; the larger the pore size the lower
the amount of stabilizer that remained. CFF was considered a more efcient process
than dialysis. When a 50 nm cutoff membrane was used, a high efciency in the
removal of sodium dodecyl sulfate from the formulation was found (approximately
1 h to remove >98 %, mass balance). Similar results were obtained for polysorbate
80 and sodium cholate; whereas PVA was found to be much more difcult to
remove by CFF due to its higher molecular weight (6000 Da). It took 67 h to
remove approximately 90 % of the PVA using CFF.
The method commonly used for surfactant removal is ultracentrifugation with
subsequent washings in distilled water. But the disadvantages of this technique
often include small sample volumes and extreme physical stress on the particles that
cause a still undetermined amount of damage. In addition, the particles may spend a
signicant amount of time in the aqueous environment where they will continue to
experience hydrolytic degradation. The method that we have found to be most
effective for removing unwanted surfactant is high-speed centrifugation, because it
allows centrifugation of much larger volumes than ultracentrifugation and is quite
rapid (Birnbaum et al. 2000).
The simplicity and feasibility of achieving industrial scale-up makes EEM one of
the preferred methods for preparing polymer nanoparticles intended for use as
nanomedicines. However, it is necessary to thoroughly understand the variables
involved in preparation and their influence on the nal characteristics of the
nanoparticles. Table 4 summarizes some of the possible effects derived from
Table 4 Effects of preparative variables to obtain polymer nanoparticles by the EEM

Category Preparative Possibilities Considerations Effects of
variable preparative
variable on
nanoparticle
suspension
characteristics
Formulation Stabilizer Type: Mechanism of Inadequate
Polymer or stabilization selection could
molecular (electrostatic cause globule
(ionic or repulsion or steric coalescence and
nonionic) repulsion) increase the
Temperature nanoparticle size
sensibility
Viscosity in
solution
Concentration: An increase in High
High or low viscosity affects concentrations
the diffusivity of could retard the
substance into the solvent migration
aqueous phase increasing the
Higher the extraction time
viscosity of the and the globule
external phase coalescence
higher the probability;
globule stability however, a better
stabilization is
achieved
Polymer Chemical Biodegradability High molecular
composition or pharmaceutical weight of the
acceptability polymer increase
Biodegradability viscosity in the
time organic phase
Grade of giving low
solubility en the diffusivities and
aqueous and reduce Ostwald
organic phase ripening, in
Pendant groups general low
Crystallinity nanoparticle sizes
Molecular weight is achieved.
Grade of Lower the
interaction with solubility in water
de drug lower the Ostwald
ripening, as
consequence more
stable emulsions
are created
Biodegradability
increase whit
polymers with low
molecular weights
Polymers module
the velocity of
drug release in the
nanoparticles
(continued)
Table 4 (continued)
variable on
nanoparticle
suspension
characteristics
Concentration An increase in High
High or low viscosity affects concentration of
the diffusivity of polymer result
substance (drug) sometimes in high
into the organic encapsulation
phase efcient
Final Larger
concentration of aggregation of
polymer in the particles could
nanoparticle appear when high
suspension concentration of
polymer is used
Organic phase Type: Should be use Low volatility
Chemical solvents cause larges times
composition pharmaceutically of evaporation and
accepted globule
Ratio phases coalescence could
(Solvent:Water) happened
Volatility (vapor increasing the
pressure) particle size
Miscibility with Emulsion
water properties are
Interfacial tension modied due to
the solvent ratio,
less stable
emulsion is
formed at high
solvent
proportions (large
nanoparticle size),
and additionally
viscosity is
increase
Drug Chemical Solubility Higher the
composition Crystallinity solubility in water
higher the
migration
(diffusion) to
aqueous phase as
result low
encapsulation
efciency are
frequently
(continued)
Table 4 (continued)
variable on
nanoparticle
suspension
characteristics
Process Emulsication Type: Rate In general,
Mechanical Propel type increasing the rate
Ultrasonic Time and time during
Potency the emulsication
reduce the globule
size therefore the
nanoparticle size
Evaporation Type: Pressure Partial vacuum
Atmospheric Bath temperature reduce the lapsed
pressure or at time for the
partial solvent removal
vacuum Less residual
solvent when
evaporation in
achieved at partial
pressure
changes in the preparation variables when polymer nanoparticles are prepared by

EEM. As mentioned above, removing traces of organic solvents, stabilizers, and
polymer residues is the main aim in the purication step in the manufacturing of
polymer nanoparticles and the methods described have been used to solve this
concern. For instance, evaporation method under reduced pressure is useful to
eliminate volatile solvents, nevertheless the volume of nanoparticle suspension
should be small. Thus, when a large volume of nanoparticle suspension is pre-
sented, methods of ultraltration and crossflow microltration have been reported
as an adequate alternative. However, besides these technologies, other methods
such as centrifugation, ultracentrifugation, dialysis, and gel ltration have been
employed to separate nanoparticles from the medium with good results, above all
when the traces or residues can be retained in the medium of dispersion (Vauthier
and Bouchemal 2011).
3 The Emulsication-Solvent Displacement Method
The emulsication-solvent displacement method was proposed and patented in 1997

as a new way to prepare concentrated pseudolatex using acceptable solvents
(Quintanar et al. 1999). Originally, this method was called the emulsion-diffusion
process involving direct displacement of partially water-miscible solvents by
distillation (Quintanar-Guerrero et al. 1999); so the emulsication-solvent dis-

placement method can be considered a hybrid technique that combines the tech-
nological steps of the EEM method with the formation mechanism of the
emulsion-diffusion technique. In general, it consists in emulsifying an organic
solution of a polymer and drug (water-saturated) in an aqueous solution of a sta-
bilizing agent, or mixtures (solvent-saturated) using conventional mechanical stir-
rers, followed by direct solvent displacement under fast evaporation using vacuum
and/or temperature (e.g., a rotary evaporator). The main distinction from the con-
ventional EEM is that it is not necessary to homogenize the emulsion in order to
obtain the nanoparticles. This technique relies on the rapid displacement of the
solvent from the internal into the external phase, which promotes the aggregation of
the drug and polymer. In contrast to the conventional EEM, one emulsion droplet
will form several nanoparticles. The nanoparticle formation mechanism operates
because rapid solvent diffusion produces regions of local super saturation near the
interface where nanoparticles form due to the ensuing interfacial phase transfor-
mations and polymer aggregation that occur in these interfacial domains. This
mechanism has been recognized as an explanation of the formation of nanoparticles
by the emulsion-diffusion method, where the diffusion step of the emulsion is
induced by adding water. In this case, diffusion is caused by the rapid displacement
of the solvent from the internal to the external phase, where it forms a new non-
solvent medium that induces aggregation in solid sub-micronic particles. Thus, the
selection of the partially water-soluble solvent is very importantthose most often
reported are ethyl acetate and 2-butanoneas is the mutual saturation of the water
and the partially water-soluble solvent (Quintanar-Guerrero et al. 1999;
Noriega-Pelez et al. 2011; Domnguez-Delgado et al. 2011; Rodrguez-Cruz et al.
2013). This approach has clear advantages; namely: (a) the use of pharmaceutically
acceptable organic solvents; (b) the possibility of solvent reuse; (c) adaptability to
several biodegradable or nonbiodegradable polymers (e.g., PLLA, PCL, Eudragits,
CAP, and cellulose acetate trimetilate, etc.); (d) no need for high-energy sources; and
(e) high reproducibility in laboratory batches. However, there are as yet no reports of
its application in industrial processes. Apparently, implementing an efcient system
to quickly extract the solvent is an important obstacle to achieving scale-up. Also, an
optimization step is required to assure that each polymer/solvent/stabilizer system
will produce only nanoparticles.
4 Preparation of Polymer Nanoparticles by the double

emulsion-solvent evaporation Technique
As mentioned above, the EEM, particularly in its single-emulsion form, was the
rst method developed to prepare nanoparticles from preformed polymers, and is
still the method most widely used to encapsulate drugs in solid polymer nanopar-
ticles (Vanderhoff et al. 1979; Rao and Geckeler 2011). However, it is most ade-
quate for entrapping primarily water-insoluble drugs and hydrophobic molecules,
since the drug is dissolved in an organic phase in which the polymer is also
dissolved (Kumari et al. 2010; Hirenkumar and Steven 2011; Tewes et al. 2007)
such that as nanoparticle formation proceeds, the interfaces formed during emul-
sication and evaporation of the organic solvent involve stages like adsorption and
polymer aggregation that enable entrapment of a larger amount of the drug into the
nanostructure (Anton et al. 2008). Despite the utilization and benets of the single
emulsion method, low encapsulation efciencies are reported when the aim is to
incorporate hydrophilic drugs like peptides, proteins, and vaccines into polymeric
nanoparticles (Rao and Geckeler 2011; Hans and Lowman 2002; Danhier et al.
2012; Rajeev 2000; Bala and Hariharan 2004). The main problem with attempting
to encapsulate hydrophilic drugs is the rapid diffusion of the molecule into the outer
aqueous phase during emulsication, which leads to poor drug loading and low
encapsulation efciency (Quintanar-Guerrero et al. 1998; Mao et al. 2007; Song
et al. 1997). Several groups have described water-in-oil-in-water (W1/O/W2)
emulsion methods in microparticle manufacture that successfully encapsulated
water-soluble drugs. This method, known as double, or multiple, emulsion-solvent
evaporation, is a slightly modied version of the single-emulsion method (see
Fig. 5), and is now the protocol best suited for encapsulating hydrophilic
(a) (b)
Organic phase Aqueous phase
(Drug + polymer +
solvent)
+ Aqueous phase
(Water + surfactant)
Organic phase
(Polymer + solvent) + (Drug + water +
surfactant)
Emulsification step using mechanical processes
Primary
Emulsion Aqueous phase
O/W (water + surfactant)
+ emulsion
W/O
Secondary emulsion
(W/O)/W
Solvent extraction
Nanoparticle Nanoparticle
suspension suspension
H
W Phase
o
(with Surfactant)
(a) Single
Emulsion
m
o
Emulsion O/W
O Phase g O phase
(API + Polymer) e Remotion
n
i S (Solvent
extraction / Nanoparticle
W1 Phase z First Emulsion t
evaporation) Suspension
(API + Surfactant) a W1/O i Second
Double
(b) Emulsion
O Phase
t
i W2 Phase
r
r
Emulsion
W1/O/W2
(with Polymer) o (with Surfactant) i
n n
g
Fig. 5 Differences in single (a) and double (b) emulsion-solvent evaporation techniques for
nanoparticle preparation. Adapted from Vauthier et al. (2009)
compounds such as peptides, proteins, and nucleic acids in micro- and nanocarriers
(Rao and Geckeler 2011; Danhier et al. 2012). Nanoparticle formation takes place
in a similar way to that of the single-emulsion method, in which conversion of the
emulsion into a nanoparticle suspension occurs through evaporation of the organic
solvent, which is allowed to diffuse through the continuous phase of the emulsions
(Hirenkumar and Steven 2011; Song et al. 1997; Soppimath et al. 2001). However,
because a W/O/W emulsion is a thermodynamically unstable system, this procedure
must be carried out in a short time to reduce the contact time between the drug and
the organic phase; meanwhile, the double-emulsion droplets are forced to solidify
so rapidly that the encapsulation efciency undoubtedly increases (Fan et al. 2003).
Thus, to avoid drug diffusion into the organic phase and improve encapsulation
efciency, the immediate deposit of a polymer membrane during the rst
water-in-oil emulsion is critically important. This can be accomplished by dis-
solving a high concentration of a high molecular weight polymer into the oil phase,
and/or by increasing the concentration of the stabilizer in the inner aqueous phase to
increase viscosity (Hans and Lowman 2002).
W/O/W methods can be employed to obtain polymer nanoparticles, nanospheres,
and nanocapsules by adjusting certain common processing parameters; basically, the
use of a small dispersed phase ratio (W1), a combination of organic solvents, soni-
cation, and a suitable stirring rate (Bilati et al. 2003, 2005; Mora-Huertas et al. 2010).
The double emulsion-solvent evaporation method is thus a modication of the
single-emulsion technique that adds an additional emulsion as a third step in the
process. Briefly, the rst step involves an aqueous phase of deionized water (W1) in
which a xed amount of hydrophilic drug is dissolved. After that, the drug solution is
added to a unique organic phase (O) that consists of a polymer solution in an
appropriate organic solvent, or combination of solvents. Substances like methylene
chloride (MC), chloroform (CF) and ethyl acetate (EA) are those most often used,
though MC is preferred in W/O/W methods due to its physical properties, including
the ability to dissolve large amounts of polymer, low solubility in water, and low
boiling point (39.8 C), all of which favor its later removal by evaporation. More
recently, however, EA has become the preferred solvent because of its low toxicity
(ICH, class 3) (Quintanar-Guerrero et al. 1998; Fan et al. 2003; Pinto Reis et al. 2006).
A wide variety of polymers are currently being employed to obtain nanoparticles
using this technique, including such natural molecules as chitosan, alginate, and
gelatine, as well as synthetic polymers like PCL, poly(alkylcyanoacrylates), PLLA,
poly(hydroxybutyrate), and copolymers like PLGA (Nagavarma et al. 2012). In
addition, several studies have reported using biodegradable polymers, especially
PLGA and PLLA. The application of PLGA has shown immense potential as a drug
delivery carrier because it is one of the most successful biodegradable polymers since
its hydrolysis produces endogenous metabolite monomers (lactic and glycolic acid)
that are metabolized via the Krebs cycle. This polymer has good biocompatibility and
was approved by the food and drug administration in the United States of America
(FDA) and the European medicines agency (EMA) in various drug delivery systems
for use in humans (Tewes et al. 2007; Pinto Reis et al. 2006; Vauthier and Bouchemal
2009). Once the W1 and O phases are mixed, a second stage begins in which the two
phases are subjected to vigorous stirring until a primary water-in-oil emulsion is

achieved. Commonly, nanosized polymer droplets are induced by sonication or
homogenization (Hans and Lowman 2002; Pinto Reis et al. 2006). At this point, it is
important to note that selection of the solvent and stirring speed plays a key role
because they affect two of the parameters that are taken into account to evaluate the
efcacy of the drug nanocarrier; namely, encapsulation efciency and particle size
(Hirenkumar and Steven 2011).
In the third step, the primary water-in-oil emulsion is poured into a second
aqueous phase (W2), prepared as a solution that contains a stabilizer like PVA or
poloxamer (Pluronic). This second emulsion is further emulsied under smoother
mixing and stirring conditions for a short time. In the fourth step, the organic
solvent is evaporated under the same conditions as in the single-emulsion technique
(i.e., continuous magnetic stirring at room temperature or under reduced pressure)
(Pinto Reis et al. 2006; Hirenkumar and Steven 2011; Bilati et al. 2005; Pinto Reis
et al. 2006). Finally, the nanoparticles are usually recovered by centrifugation,
washed with distilled water to eliminate or remove additives like surfactants, and
then lyophilized by adding some cryoprotectants (Danhier et al. 2012; Nagavarma
et al. 2012). Table 5 shows formulations for the preparation of solid polymer
nanoparticles, while Table 6 includes suggested formulations for obtaining poly-
meric nanocapsules using the W/O/W method. Regarding O/W surfactants, sorbitan
esters are preferred, and in the external aqueous phase, the stabilizing agents most
frequently used are PVA and polysorbates. In a typical procedure for preparing
nanocapsules by double emulsication, the primary emulsion is formed by ultra-
sound and the W/O surfactant stabilizes the interface of the W/O internal emulsion.
To contribute to nanocapsule dispersion, the same external aqueous phase com-
position is used for the dilution phase if the procedure followed includes a nal
dilution stage (Mora-Huertas et al. 2010).
Extensive research has been performed using W/O/W methods mainly in the
eld of biodegradable microparticles. For instance, Hirenkumar et al. described
how the manipulation of phases and manufacturing parameters creates changes in
Table 5 Formulations for the preparation of polymer nanoparticles by W/O/W method. Taken
and modied from Rao et al. (2011)
Polymer Organic solvent Stabilizer Particle size References
(nm)
PLGA Dichloromethane Span 40 200 Lemoine and Preat
(1998)
PLA Methylene PVA 200 Zambaux et al. (1998)
chloride
PEG-PLA Methylene Sodium 200 Quellec et al. (1999)
chloride cholate
mPEO-PLA Methylene Sucrose 268 4 Zambaux et al. (1999)
chloride
PLGA Chloroform SDS 76 Musyanovych et al.
(2008)
Table 6 Suggested composition for preparation of nanocapsules by W/O/W method. Taken and
modied from Mora-Huertas et al. (2010)
Material Suggested composition
Inner aqueous phase Variable (0.525 mg)
Active substance 0.150.5 ml
Water
Organic phase 510 % of organic phase solvent
Polymer 57 % of organic phase solvent
W/O surfactant 1.55 ml
Solvent
External aqueous phase 15 % of external aqueous phase solvent
Stabilizer agent 25 ml
Water
Dilution phase 15 % of dilution phase solvent
Stabilizer agent 50100 ml
Water
the properties of the carrier. Thus, in the case of PLGA microparticles obtained by
the W/O/W method the burst effect is slightly increased during drug release when
volume in the internal phase (W1) is increased. Meanwhile, an increment in the
volume of the continuous phase in the second emulsion (W2) provokes an increase
in surface porosity and, according to those authors, polymer and PVA concentration
modies particle size as they increase (Hirenkumar and Steven 2011). Regarding
nanoscaled carriers, Zambaux et al. studied the optimization of parameters such as
the volume of the internal aqueous phase, temperature, solvent evaporation,
washing, and the concentration of surfactants in the double-emulsion method. They
reported a slight reduction in particle size (from 203 13 to 197 26 nm) when
the organic solvent was evaporated under vacuum conditions using a rotating
evaporator, instead of gentle magnetic stirring at room temperature. In the case of
the volume of the internal phase, it has been shown that larger volumes generate
larger particles; though when the concentration of the surfactant increases, particle
size decreases due to the improved emulsication process and the high viscosity of
the medium (Hans and Lowman 2002; Zambaux et al. 1998). Bilati et al., mean-
while, examined the formulation and processing parameters of the W/O/W method
in the encapsulation of bovine serum albumin in polymer nanoparticles of PLGA.
They used sonication and vortex processes in the emulsication steps and found
high entrapment efciencies (>80 %) when sonication is used in the two emulsi-
cation steps instead of vortex (*25 %). This nding was explained by the poor
quality of the W1/O emulsion obtained with vortexing (coarser and less homoge-
nous than that obtained with sonication), which clearly promoted droplet coales-
cence during the second step, and subsequent drug leakage into the dispersing
aqueous phase. They also reported that the high amount of energy dissipated
through the sonicated sample for a longer time period favored droplet splitting,
leading to smaller nanoparticles (from 405 116 to 288 10 nm) (Bilati et al.
2003). Other parameters, such as high molecular weight, high hydrophilicity, and
the presence of free carboxylic end groups in PLGA, enhanced drug entrapment
efciency because a longer chain in the polymer generates a higher inherent vis-
cosity that affects drug entrapment, while an uncapped carboxylic end group pro-
motes better encapsulation efciency as a result of ionic interactions between the
drug and the polymer. In general, however, the double-emulsion method creates
nanoparticles of much larger size compared to the single-emulsion method. Finally,
those authors reported that a larger volume in the inner aqueous phase increases
entrapment efciency and particle size because the W1/O emulsion is coarser and
less stable, thus favoring the coalescence of suspended droplets (Hirenkumar and
Steven 2011; Bilati et al. 2003). Other studies have shown that particle size is
influenced by the type and concentrations of the stabilizer, homogenizer speed, type
of solvent, and polymer concentration. To produce a small particle size, high-speed
homogenization or ultrasonication may be employed (Nagavarma et al. 2012).
Lemoine et al. prepared PLGA nanoparticles of about 200 nm utilizing dichlor-
omethane 1.0 % (w/v) as the solvent, a small volume in the inner aqueous phase
(W1 = 1 mL), and PVA as the stabilizing agent in both emulsions (at a higher
concentration in the rst) (Lemoine and Preat 1998). Song et al., in turn, prepared
BSA-loaded nanoparticles of PLGA with a typical particle size of around 100 nm
by employing dichloromethane and acetone (8:2, v/v) as the solvent system,
Pluronic F68 at different concentrations (from 3 to 6 %) as the stabilizing agent in
the primary emulsion, and PVA as the stabilizing agent in the second emulsion
(Song et al. 1997). Table 7 presents examples of drugs that were encapsulated in
nanoparticles using the double emulsion-solvent evaporation technique. Recently,
Cohen-Sela et al. have proposed a new modication of the double-emulsion
technique in which they combined the use of a partially water-soluble organic
solvent (ethyl acetate) that results in better encapsulation, an improved yield of
hydrophilic drugs in polymer nanoparticles, smaller size, and a lower size
Table 7 Encapsulated model drugs into nanoparticles by W/O/W technique

Polymer Drug Size of nanoparticle References
(nm)
PLA Testosterone <1000 Gurny et al. (1981)
Albumin 100 or 120 Landry et al. (1996)
Tetanus 150 Tobio et al. (1998)
toxoid *300 Ueda et al. (1998)
Loperamide
PLGA DNA *100 Ueda and Kreuter (1997)
Cyclosporine *300 Prabha et al. (2002); Sanchez et al.
A *300 (1993)
Praziquantel 280 or 320 Mainardes and Evangelista (2005)
Doxorrubicin *300 Tewes et al. (2007)
Paclitaxel Mu and Feng (2003)
EC Indomethacin *200 Bodmeier and Chen (1990)
distribution, compared to the classic method. They called this new technique double
emulsion-solvent diffusion (Cohen-Sela et al. 2009).
Due to the need for high energy requirements in the homogenization process,
emulsication-solvent evaporation methods are sometimes considered adequate for
laboratory-scale operations, but for large-scale pilot production alternative methods
using low-energy emulsication are preferable to counteract this principal limitation
to scale-up. Options include the spontaneous emulsication-solvent diffusion
method, the salting-out emulsication-diffusion method, production of NPs using
supercritical fluid technology, and polymerization methods (Rajeev 2000;
Soppimath et al. 2001).
5 Conclusions and Perspectives
Over the past 35 years, great technological advances have been achieved using the
emulsication-solvent evaporation method. In this chapter, this method, its modi-
cations, adaptations, and applications in developing polymer nanoparticles were
reviewed in order to serve as an initial guide for pharmaceutical formulators. Given
that the emulsication-solvent evaporation method is the Research and
Development (R&D) departments rst option for developing a specic polymer
nanoparticle product, its three modalitiesi.e., via single emulsion, via solvent
displacement, or via double emulsionare critically evaluated. In each case, the
advantages, drawbacks, and crucial parameters are covered in order to provide
consolidated information and sufcient criteria to make decisions on their respec-
tive usefulness, and the best starting materials and operating conditions to satisfy
specic aims in polymer nanoparticle design. It is important to point out that the
implementation of this method to industrial level requires the development of new,
appropriate, and specic manufacture equipment in particular for dispersing the
system with less stress and analytical devices to follow the solvent removal in-line.
The evidence showed in this chapter conrms that the method is adequate to
encapsulate lipophilic drugs but new modalities are required to resolve the low
encapsulation of hydrophilic drugs in particular those sensitive to chemical and/or
physical stress such as peptides, proteins, DNA, RNA, genes, etc. In this sense, an
understanding of the interaction between polymer and these drugs may be useful to
provide answers to this challenge.
Although several technological aspects of the emulsion-solvent evaporation
method have been well documented, other issues require further research; for
example: (a) implementations combined with new technologies (e.g., dispersion
and homogenization equipment); (b) large-scale industrial production, especially
via solvent displacement; (c) the search for less toxic ingredients; (d) improvement
of operating conditions to incorporate green processes; and, (e) development of
multifunctional formulations with better targeting properties (f) new ways to
eliminate solvent and stabilizer residual. Finally, the emulsion-solvent evaporation
method will continue to be the principal choice for formulating polymer
nanoparticles for different drugs and pharmaceutical purposes, such as drug

delivery and drug targeting. It is anticipated that the technique will undergo
numerous modications in the near future due to the application of creative ideas.
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Chapter 5
Methods for the Preparation
of Nanoparticles by Polymerization
Christine Vauthier
Abstract This chapter describes methods of preparation of nanospheres and

nanocapsules by polymerization giving advises to carry on the polymerization
reactions to produce nanoparticles with well-dened characteristics. The number
one polymer that composes nanoparticles prepared by polymerization in the aim to
use them as drug carrier is poly(alkylcyanoacrylate) (PACA). A comprehensive
view of the polymerization reactions of the corresponding monomer is given
considering the conditions that are applied to produce the nanoparticles. It is then
explained how to prepare PACA nanospheres by emulsion polymerization and oil-
and water containing PACA nanocapsules by interfacial polymerization of these
monomers and how to play with the reaction conditions to give the nanoparticles
the desired characteristics. Besides nanoparticles made of PACA, nanocapsules
with envelopes composed of polyurethane, polyamide, and polyesters can be
synthesized by interfacial polycondensation methods carried out in very thin
emulsions. This method will be described at the end of the chapter.
Keywords Alkylcyanoacrylate
Poly(alkylcyanoacrylate)
Nanocapsules
Nanospheres
Anionic polymerization
Radical polymerization Interfacial
polymerization
Interfacial polycondensation
Emulsion
Microemulsion
Miniemulsion
Solvent diffusion
Ouzo effect
Polycondensation

Polymerization Inverse Ouzo effect
1 Introduction
Polymer nanoparticles can be made by two types of methods whether the material
used is a polymer or a monomer that is transformed into polymer at the same time
than nanoparticles are formed. Methods based on the use of a polymer are described
C. Vauthier (&)
Institut Galien Paris Sud, Faculty of Pharmacy, UMR CNRS, University of Paris-Sud,
University Paris Saclay, 5 Rue J.B. Clment, 92296 Chtenay-Malabry Cedex, France

DOI 10.1007/978-3-319-41421-8_5
124 C. Vauthier
in Chaps. 2, 3 and 4 of this book. The present chapter focuses on the methods in
which both the polymer and the nanoparticles are formed together, thanks to the
achievement of a polymerization reaction. Different modalities of polymerization
are used to produce nanospheres (matrix type nanoparticles) or nanocapsules
(reservoir type nanoparticles). All types of nanoparticles produced to be used as
nanomedicines need to fulll stringent requirements in terms of degradability and
safety. The number of polymers that suits with these requirements is low and this
number is further reduced to very few when it is synthesized at the same time than
the nanoparticles. Thus, nanoparticles obtained by polymerization methods were
almost all composed of poly(alkylcyanoacrylates) (PACA) resulting from the
polymerization of alkylcyanoacrylates (ACA), their corresponding monomers. In
vivo, the degradation of PACA is mainly due to the activity of esterases. Enzymes
cleave ester bonds included in the monomer unit structure releasing an alkylalcohol
and a poly(alkylcyanoacrylic acid) chain. While parent polymers are not soluble in
biological fluids that are aqueous media, the produced degradation products are
soluble making possible their elimination from the body by kidney ltration
(Grislain et al. 1983; Leanaert et al. 1984). Degradation of nanoparticles occurs
through a surface erosion process that takes a few hours depending on the length of
the alkyl side chain of the ACA unit composing the polymer (Gautier et al. 1992;
Muller et al. 1990). Although not demonstrating yet, PACA might also degrade
in vivo according to an unzipping depolymerization reaction that is immediately
followed by the repolymerization of the released monomer giving a polymer of
second generation of a lower molecular weight. This mechanism was described to
occur in conditions that exist in vivo where it may be initiated by amino acids and
proteins (Ryan and McCann 1996; Wu et al. 2009a). Other mechanisms of
degradation of PACA described in the literature occur at a much slower kinetic
while the required conditions are far from those found in vivo (Vauthier and
Couvreur 2002; Nicolas and Couvreur 2009; Nicolas and Vauthier 2011). Thus, it is
believed that they are not effective in vivo where the enzymatic mediated degra-
dation and/or degradation by the unzipping depolymerization process can occur at a
much faster kinetic. The safety of nanoparticles composed of PACA is well doc-
umented from the large number of works that have demonstrated their success to be
used as drug delivery devices. Above all, it is acknowledged by their evaluation in a
new treatment for the hepatocellular carcinoma in human phase II/III clinical trials
(Kattan et al. 1992; Dufour-Lamartinie et al. 2006; Soma et al. 2012).
ACA used to produce nanoparticles by polymerization methods are different
derivatives of the compounds found as constituent of surgical glues applied to repair
wounds (Bot et al. 2010; Al-Mubarak and Al-Haddab 2013) and of the well-known
instant glue used in the industry and at home. They are vinyl monomers having a
unique chemistry due to their extreme reactivity (Limouzin et al. 2003; Nicolas and
Couvreur 2009). Couvreur et al. 1979 were the rst to have described suitable
conditions of the polymerization of ACA producing nanoparticles (See the historical
perspective from Kreuter 2007). Since then, different methods of polymerization
have been developed making possible the production of both nanospheres and
nanocapsules. The physicochemical characteristics of the nanoparticles can be well
5 Methods for the Preparation of Nanoparticles by Polymerization 125
controlled from experimental conditions applied during the synthesis (Murthy and
Harivardhan 2006; Vauthier et al. 2007; Nicolas and Vauthier 2011; Nicolas and
Couvreur 2009, Vauthier 2015).
Polymerization methods of ACA are the majority of methods of polymerization
applied to produce suitable nanoparticles to be used as nanomedicines. These methods
will be presented after a brief description of the reactivity of ACA and of their
mechanisms of polymerization. Besides these methods, an interfacial polycondensa-
tion method was proposed to synthesize nanocapsules (Bouchemal et al. 2004).
Polymers form from the reaction of complemental bifunctional reagents at the surface
of droplets dispersed in the continuous phase of a very thin emulsion. Nanocapsule
envelopes are composed of polyesters, polyurethanes, or polyamides. The method of
interfacial polycondensation is presented in the second part of the chapter.
2 Obtaining Nanoparticles from the Polymerization

of Alkylcyanoacrylates
Polymerization of ACA was widely applied to produce nanoparticles by polymeriza-

tion methods for in vivo applications as drug delivery systems. Due to their chemical
structure, the chemistry of ACA monomers is unique. In this chapter, their polymer-
ization is explained while it is conducted in conditions used to produce nanoparticles. In
the next parts, strategies to design nanospheres and nanocapsules with suitable prop-
erties to be used in various drug delivery applications will be explained.
2.1 Polymerization of ACA
The general chemical structure of ACA is given in Fig. 1a together with that of the
corresponding polymer (Fig. 1b).
The structure of the molecule reveals amphiphilic properties hence the molecule
may sit on interfaces occurring in various types of dispersed systems including
non-miscible lipophilic and hydrophilic phases as illustrated in Fig. 2.
The two electron withdrawing groups (nitrile and ester groups) on the -carbon
of the double bond give this monomer an unusually high reactivity. The poly-
merization can be initiated spontaneously by traces of anions or nucleophilic group
(a) (b)
CN CN
n
COOR COOR
Fig. 1 Chemical structure of alkylcyanoacrylate monomers (a) and of the corresponding

polymers (b). R represents the alkyl chain that is n-butylcyanoacrylate (nBCA), isobutyl-
cyanoacrylate (IBCA) and isohexylcyaoacrylate in most works carried out to design nanoparticles
to be applied as nanomedicines
126 C. Vauthier
Hydrophilic (aqueous) phase
+
CN +
CN +
CN +
CN
- - - -
O O O O
O O O O
R R R R
Lipophilic (oily) phase
Fig. 2 Molecules of ACA as they can take place at the interface of lipophilic and hydrophilic
phases found in dispersed systems like emulsions, miniemulsions and microemulsions due to their
amphiphilic characteristics. R represents the alkyl chain which is lipophilic
containing compounds that are present in polymerization media. For instance, a

spontaneous anionic polymerization is initiated by bases or by hydroxyl ions
resulted from the autoprotolysis of water in conditions used to prepare nanoparticles
(Fig. 3a).
In general, polymerization media used in that purpose consist of aqueous solutions
of a quite complex composition. Besides water that can initiate spontaneously an
anionic polymerization, they contain compounds with nucleophile groups able to
initiate a zwitterionic polymerization (Fig. 3b). Both of these mechanisms of initi-
ation of the polymerization of alkylcyanoacrylates lead to the formation of a car-
banion on the -carbon of the double bond which serves as the active center for the
propagation stage of the polymerization (Fig. 3a, b). It is assumed that the poly-
merization ends by a reaction with a proton that can be provided from the dissociation
of a water molecule. In these polymerizations, the water can have two opposite roles
being possible initiator with hydroxyl ions on the one hand and terminator or inhi-
bitor of the polymerization with the proton on the other hand.
It is noteworthy that the anionic and zwitterionic polymerizations occurred
spontaneously. There is no need to provide the system with a source of energy to
allow the polymerization to start. In addition, the polymerization can be extremely
fast and is generally difcult to control. The fast speed of the anionic/zwitterionic
polymerization of alkylcyanoacrylate in polymerization conditions developed to
produce nanocapsules was the key for the obtaining of nanocapsules by interfacial
polymerization. However, to form nanospheres, the speed of polymerization carried
out in emulsions needs to be slown down to control the rate of formation of the
polymer and promote the formation of nanoparticles with well-dened character-
istics. Strong acids can be used on this purpose slowing down the initiation of the
polymerization. Polymerization media suitable for the obtaining of PACA nano-
spheres to be applied as drug delivery systems include acid aqueous phases with pH
bellow 2.5. It is noteworthy that even under very strong acid conditions (pH < 1),
inhibition of the spontaneous polymerization may be not fully effective (Wu 2013;
Iniators and iniaon Acve species of Polymer

of the polymerizaon the polymerizaon
(a) CN
HO CN CN
H
Base + - n
HO HO
Base COOR Base COOR
COOR
(b) - CN
R-OH CN CN
H
+ - n
R-NH2 R-OH R-O
R-SH COOR R-N COOR
COOR R-NH
(c) R-SH R-S
AIBN
70C
I CN CN CN
Cerium IV I I n
PEG COOR COOR COOR
Polysaccharide
Fig. 3 Mechanisms of polymerization of ACA. a Anionic polymerization initiated by hydroxyl
ions generated by the autoprotolysis of water or by bases, b zwitterionic polymerization initiated
by nucleophilic groups of chemicals added in the polymerization medium. These nucleophile
groups can be included in macromolecules like polysaccharides and poly(ethylene glycol) (PEG),
c radical polymerization initiated by an oxidationreduction reaction involving cerium and the
thermal decomposition of azobis(isobutyronitrile) (AIBN)
Chauvierre et al. 2003b, c). For instance, the use of a pure aqueous solution of nitric
acid at a concentration of 0.2 M is suitable to inhibit the polymerization of
isobutylcyanoacrylate (IBCA) over a period of 24 h. However, if a polysaccharide
including a poly(glucose) like dextran is added at a low concentration to the nitric
acid solution, the polymerization starts after a few minutes (Chauvierre et al. 2003b,
c). Although the pH of the polymerization medium is a critical parameter to control
the polymerization hence to achieve the production of nanospheres, the type and
amount of polymerization inhibitors added as preservative to the monomer can also
greatly influence the course of the polymerization (Lescure et al. 1992;
Page-Clisson et al. 1998; Cournarie et al. 2004). In general, compounds that initiate
the polymerization of alkylcyanoacrylates remain attach to the polymer that forms
(Fig. 4). This can be used to add functionalities to PACA hence to nanoparticles
(Nicolas et al. 2013). Several compounds were formally identied to serve as
initiators of the polymerization of alkylcyanoacrylate while preparing the
nanoparticles by polymerization methods. These include saccharides units found
on polysaccharides and poly(ethylene glycol) (PEG) chains that contain hydroxyl
groups able to initiate the reaction of the polymerization through a zwitterionic
mechanism (Perrachia et al. 1997, Zandanel and Vauthier 2012a). Compounds
128 C. Vauthier
Fig. 4 Composition and structures of polymers obtained from the initiation of the polymerization
of ACA by anionic/zwitterionic mechanisms. Initiations by macromers including polysaccharides
(a), poly(ethylene glycol) (PEG)-containing molecules terminated by an hydroxyl group (b, c) and
any molecules that contain a nucleophilic group (d). It is noteworthy that the initiating compounds
are included in PACA chains at the end of the polymerization. Molecules having several potential
initiator groups in their structure can initiate the formation of several PACA chains (a, c). When
they are macromolecules, copolymers are produced (ac). Their structure greatly depends on the
location of the initiating group(s) on the macromolecular chain that serves as macroinitiator of the
polymerization of ACA
bearing amino functions are other potential initiators of the polymerization of

alkylcyanoacrylate monomers (Gallardo et al. 1989; Guize et al. 1990; Kulkarni
et al. 1971; Zandanel and Vauthier 2012a).
ACA are vinyl monomers. As all vinyl monomers, their polymerization might be
initiated by radicals (Fig. 3c). However, the initiation of the polymerization of ACA
by the radical route is not straightforward because of the extremely high reactivity
of these monomers and considering the aqueous environments used to prepare
nanoparticles for biomedical applications which generally contain many com-
pounds susceptible to initiate polymerization via the anionic/zwitterionic routes.
The achievement of a radical polymerization supposes an effective inhibition of the
anionic/zwitterionic polymerizations. Only few works have reported conditions of
polymerization of ACA that were suitable to produce nanoparticles by radical
polymerizations (Chauvierre et al. 2003c; Wu et al. 2009b, c). Details about the
different mechanisms of initiation of radical polymerization of ACA are illustrated
in Fig. 5. The Fig. 5a shows the initiation triggered by a very common initiator
Fig. 5 Radical polymerization of ACA initiated by azo-bis-isobutyronitrile (AIBN) (a), poly

(ethylene glycol) (PEG) containing molecules (b) and polysaccharides including dextran (c) and
chitosan (d). The structure and composition of the resulting polymers are illustrated as well
130 C. Vauthier
used in radical polymerization of vinyl monomers. Figure 5bd illustrate mecha-

nisms of initiation of radical polymerization of ACA through oxidationreduction
reaction involving cerium IV ions together with different macromolecules.
In the approach proposed by Chauvierre et al. (2003c), the polymerization was
initiated by an oxidationreduction reaction using the couple cerium IV dextran
(Fig. 5c). While the reaction is carried out at pH 1, the polysaccharide is cleaved in
two chains and the radical created at the chain end initiates the radical polymer-
ization of ACA (Bertholon et al. 2006a). The activated polysaccharide on which the
radical appears serves as macroinitiator of the radical polymerization. At the end of
the polymerization, it remains attached to the nal polymer forming an amphiphilic
copolymer having surface active properties (Fig. 5c). A similar situation was
described by addition of Pluronic F68, a poly(ethylene glycol) (PEG) containing
surfactant, in the polymerization medium (Zandanel and Vauthier 2012a) (Fig. 5b,
d). In this case, the radical was created next to the terminal hydroxyl group of the
PEG moiety of the macromolecule by reaction with cerium IV (Fig. 5b). In both
cases, the initiation of the polymerization carried out in the presence of cerium IV
occurred faster than the spontaneous polymerization that can be initiated in the
absence of cerium IV ions or in pure acid, hence the nanoparticles were generated
by radical polymerization. In the work of Wu et al. the radical polymerization of the
monomer was initiated in droplets of a miniemulsion, thanks to the decomposition
of azo-bis-isobutyronitrile (AIBN) at 75 C (Fig. 5a) (Wu et al. 2009b, c). The
continuous aqueous phase of the miniemulsion was acidied to pH ranging from 1
to 2 to control the occurrence of the spontaneous anionic/zwittertionic polymer-
ization. It is noteworthy that, in both of these examples and despite the acidication
of the aqueous phase of the polymerization medium, the spontaneous polymer-
ization of ACA was not completely inhibited and competed with the radical
polymerization. The balance between the two types of polymerizations could be in
favor of the radical polymerization when its initiation occurred much faster than
that of the spontaneous anionic/zwitterionic polymerization. This was actually the
case with the initiation caused by the cerium IV ions (Chauvierre et al. 2003b, c).
Whatever the mechanism of polymerization used to produce the drug carrier, it
can be taken as an advantage that the polymerization is initiated by one component
added to the polymerization medium (Douglas et al. 1985; Chauvierre et al. 2003a
b, c, Bertholon et al. 2006a, Zandanel and Vauthier 2012a). This component that
will be grafted to PACA can be used to functionalize the surface of the carrier.
When the component of the polymerization medium is a polysaccharide or a
PEG-containing surfactant like Pluronic F68, a copolymer with amphiphilic
properties will form. In general, this copolymer is suitable to achieve the stability of
the small polymer particles that form without the need of the further addition of a
surfactant in the preparation medium. Indeed, the copolymer behaved like a sur-
factant (Chauvierre et al. 2004). The nanoparticles take a corecorona structure as
illustrated on the transmission electron micrograph shown in Fig. 6.
The hydrophobic moiety of the copolymer, i.e. PACA, composes the core of the
nanoparticles while the hydrophilic moiety is exposed at the nanoparticle surface
forming the corona. The architecture of the amphiphilic copolymer generated
Fig. 6 PACA nanoparticles obtained from the polymerization of ACA initiated by chitosan and
Pluronic F68 highlighting the core corona structure of by transmission electron microscopy
(image on the right side). Schemes ae represent the various structures of copolymers formed by
the assembly of lipophilic chains (in black) and hydrophilic chains (in gray or blue) and the
corresponding spatial arrangements of the hydrophilic chains in the nanoparticle corona while they
are anchored in the nanoparticle core by their lipophilic chains. Copolymers including PACA
chains are shown on the schemes (bd)
during the polymerization greatly depends on the mechanism of the initiation of the
polymerization. This step of the polymerization is critical as it is decisive in the way
the initiator covalently attaches with the nascent PACA chain(s) in the nal
copolymer (Figs. 4, 5). Taking polysaccharides as initiators of the polymerization,
comb copolymers were described to form by anionic/zwitterionic polymerization
(Fig. 4a) while formation of block copolymers was highlighted after a radical
polymerization was initiated by the reaction of cerium IV ions with the hydrophilic
polymers (Fig. 4c, d) (Bertholon et al. 2006a; Zandanel and Vauthier 2012a). The
architecture of the copolymer is important to be elucidated as it greatly influences
the spatial arrangements of the hydrophilic moieties of the copolymer in the
nanoparticle corona (Fig. 6) and in turn the type of interactions of the nanoparticles
with biological structures hence the in vivo fate of the nanoparticles. This was
revealed with dextran-coated PACA nanoparticles of almost same compositions but
132 C. Vauthier
in which the dextran chains of the nanoparticle corona were taken a different spatial
arrangement. The two types of nanoparticles achieved a completely different
biodistribution of the carried drug after intravenous administration to rats (Alhareth
et al. 2012). More generally, it was shown that spatial arrangement of the
polysaccharide chains in the corona nanoparticles influences their interactions with
cells and proteins (Perrachia et al. 1997; Labarre et al. 2005; Lira et al. 2011;
Alhareth et al. 2012). Consequently, marked differences in the biodistribution of the
nanoparticles after intravenous administration were observed (Table 1).
Understanding the polymerization of alkylcyanoacrylate occurring in conditions
used to prepare nanoparticles on a fundamental standpoint is particularly chal-
lenging due to the high reactivity of the monomers. Methods that generally apply to
investigate polymerization reactions cannot be used with alkylcyanoacrylate as the
spontaneous anionic/zwitterionic polymerizations are difcult to control because
they occur with an extremely fast kinetics. The difculty is further enhanced
knowing that depolymerization followed by an immediate repolymerization may be
trigger on a timescale of a few seconds in the presence of amines in slightly basic
conditions (Ryan and McCann 1996). In this context, only few works have
investigated the reaction of polymerization during the course of the reaction
(Limouzin et al. 2003; Behan et al. 2001; Wu et al. 2009b, c). The polymerization
was also understood from analysis of characteristics of polymers extracted from the
produced nanoparticles (Douglas et al. 1985; Gallardo et al. 1993; Page-Clisson
et al. 1998; Behan et al. 2001; Chauvierre et al. 2003a; Bertholon et al. 2006a;
Zandanel and Vauthier 2012a). Data obtained from these studies are useful to set up
conditions of reactions producing nanoparticles with well-dened specications
Table 1 Obtaining PACA nanoparticles by emulsion polymerization

Mechanism of Structure of the copolymer In vitro test of the Interaction
polymerization formed and arrangement at with the complement protein C3
the nanoparticle surface (test of the activation of the
immune system) and In vivo fate
after intravenous administration
to healthy rats
Spontaneous Comb dextran-PACA Activation of the complement
anionic/zwitterionic (Fig. 6b) protein C3
polymerization initiated by In vivo, particles were cleared by
dextran (Fig. 4a) the liver and spleen
Radical polymerization dextran-block-PACA Inert toward the immune system
initiated by dextran and (Fig. 6c) as evaluated in vitro the
cerium IV (Fig. 5c) activation of the complement
protein C3
In vivo, particles escaped
massive uptake by liver and
distributed in most major organs
Relation between mechanisms of polymerization, composition and structure of resulting polymers
that compose nanoparticles and the fate of the nanoparticles in contact with biological systems
evaluated in vitro by testing the activation of a protein of the innate immune system and in vivo
after intravenous administration to healthy rats
(Vauthier 2015). The following parts of the chapter explained how to play with the
different types and conditions of polymerization to obtain PACA nanoparticles with
various properties and drug encapsulation capability. For further details on the
polymerization of ACA, readers are encouraged to refer to the following review
articles (Vauthier et al. 2007; Nicolas and Vauthier 2011; Nicolas and Couvreur
2009; Vauthier 2015).
2.2 Application of the Emulsion Polymerization of ACA

to Produce Nanospheres with Different Properties
PACA nanospheres are generally obtained by emulsion polymerization of the

corresponding monomers, i.e., alkylcyanoacrylate. Preparation of nanospheres by
emulsion polymerization is illustrated in Fig. 7. In practice, the pure monomer,
which occurs as a nonviscous liquid, is added dropwise in the aqueous solution
prepared as the polymerization medium. Eventually, the drug to be associated with
the nanoparticles can be dissolved in the polymerization medium. The polymer-
ization takes a few hours and provides with a milky dispersion that contains the
nanoparticles. It is noteworthy that the method of emulsion polymerization used to
prepare PACA nanoparticles does not require the use of organic solvent. It is a
one-step procedure that is carried out in an aqueous environment. No specic
equipments are required for a lab scale production as illustrated in Fig. 7 which
gives examples of protocols for the preparation of nanospheres by anionic and
radical emulsion polymerization.
Step 1: Preparaon of the Step 2: Start of the Step 3: Final step: Storage
polymerizaon medium polymerizaon polymerizaon recover the dispersion
Anionic/zwierionic
polymerizaon
Hydrophilic polymer Monomer

(+ drug)
HCl, pH 2.5 Ambiant temperature

or 3-4 hours, pH 2.5 Raise pH to 7
HNO3 0.2M, pH 1 24 hours, pH 1
Strong magne c +4C
agita on
Successive addi on
Hydrophilic polymer
N2 CeIV Monomer
polymerizaon
(+ drug)
Radical
Drop temperature
N2
N2
CeIV 1.6 10-2M to +4C
N2
N2
HNO3 0.2M, pH 1
N2
N2
40C Dialysis: raise pH +
1 hour remove Cerium
Nitrogen bubling Strong magne c +4C
10 min agita on
Fig. 7 Examples of procedures to achieve the preparation of nanoparticles by emulsion

polymerization. In these syntheses, dextran 70 kDa can be used as the hydrophilic polymer
added in the polymerization medium at a concentration of 1.3 %. The monomer, isobutyl-
cyanacrylate (IBCA) for instance is added at a nal concentration of 5 %
134 C. Vauthier
The composition of the polymerization medium determines the type of poly-

merization (Fig. 3), hence the architecture of the copolymer that forms as illustrated
in Figs. 4 and 5. In the presence of cerium IV ions, a redox radical polymerization
is initiated while a spontaneous anionic or zwitterionic polymerization takes place
in their absence. During the polymerization, the hydrophilic polymer added in the
polymerization medium is generally incorporated in the polymer structure pro-
ducing a polymer with surface active properties (Chauvierre et al. 2004). This
copolymer serves as stabilizer for the nanoparticles. Thus, the hydrophilic polymer
added in the polymerization medium becomes part of the resulted copolymer during
the formation of the nanoparticles. At the end of the polymerization, it is stably
anchored at the surface of the nanoparticles composing the nanoparticle corona
(Fig. 6). The composition of the nanoparticle corona can easily be tuned by varying
the nature of the hydrophilic polymer added in the polymerization media.
Eventually, it is possible to perform the polymerization in the presence of blends of
hydrophilic polymers to obtain nanoparticles with coronas composed of two dif-
ferent species of hydrophilic polymers in different proportions (de Martimprey et al.
2010; Lira et al. 2011; Chauvierre et al. 2003a; Zandanel and Vauthier 2012a).
Characteristics of the corona can also be tuned by choosing the mechanism of
polymerization as it denes the architecture of the copolymers, hence the spatial
arrangement of the hydrophilic chains composing the nanoparticle corona (Fig. 6).
Thus, characteristics of the corona can be tuned varying both the composition and
characteristics of the hydrophilic polymer added in the polymerization medium and
the type of polymerization. Surface charge of nanoparticles is greatly influenced by
changes introduced in the composition of the nanoparticle corona. It can be
investigated by the evaluation of the zeta potential of the nanoparticles (Varenne
et al. 2015). Based on the evaluation of this parameter characterizing nanoparticle
surface charge, it could be deduced that the composition of the corona of
nanoparticles prepared in polymerization medium containing a blend of hydrophilic
polymers included both types of polymers. The zeta potential varied consistently
with the composition in hydrophilic polymers added in the polymerization medium
(Bertholon et al. 2006b; De Martimprey et al. 2010; Lira et al. 2011; Zandanel and
Vauthier 2012a). The mechanism of polymerization has almost no influence on the
nanoparticle surface charge (Bertholon et al. 2006b; Lira et al. 2011; Zandanel and
Vauthier 2012a). Size of nanoparticles can also be adjusted varying different
parameters of the polymerization procedure. It varies with the nature and concen-
tration of the hydrophilic polymer added in the polymerization medium, the
mechanism of polymerization and with the rate of agitation. When the nanoparticles
are synthesized by radical polymerization with a polysaccharide as the hydrophilic
polymer, the size of the nanoparticles also depends on the molar mass of the
polysaccharide (Bertholon et al. 2006b). It increases with the molar mass of the
polysaccharide and the effect is amplied with charged polysaccharides. A general
rule that may apply to reduce the size of nanoparticles produced by emulsion
polymerization is to increase the concentration of the hydrophilic polymer. The
molar mass can also be reduced down to a certain threshold bellow which the
polymer will occur as aggregates instead of stable colloidal nanoparticles
(Bertholon et al. 2006b). The use of a surfactant like Pluronic F68 can be rec-
ommended to achieve the preparation of nanoparticles with diameters around 50
60 nm. The concentration to use is 2.5 % preparing the nanoparticles by anionic
polymerization (Seijo et al. 1990) and 3 % preparing the nanoparticles by radical
polymerization. In this latter case, chitosan with a low molar mass (Mw = 20 kDa)
is also added in the polymerization medium at a concentration of 1.3 %. The
nanoparticle corona is then composed of both chitosan and Pluronic F68 (De
Martimprey et al. 2010; Zandanel and Vauthier 2012a, b). As it was explained
above, physicochemical characteristics of the nanoparticles including their size and
surface properties can be tuned to a large range of specications by varying the
composition of the polymerization medium. Because of the versatility of the
method, these nanoparticles were found suitable for various applications as drug
carriers as witness for the wealth of the literature (Couvreur and Vauthier 2006;
Murthy and Harivardhan 2006; Vauthier et al. 2007; Andrieux and Couvreur 2009;
Nicolas and Couvreur 2009).
The size and surface properties are important specications of nanoparticles to
achieve drug delivery applications for which they are designed for. For instance,
enhancement of adherence of nanoparticles on mucosa can be achieved adding
chitosan or poloxamer in the polymerization medium that will be incorporated into
the nanoparticle corona (Bravo-Osuna et al. 2007a, b; Bertholon et al. 2006c). For
nanoparticles designed to be administered by the intravenous route, stealth property
that allows the drug carrier to escape recognition by the immune system and be
captured by macrophages are often required. This property can be given to
nanoparticles by adding dextran (Mw 70 KDa) in the polymerization medium and
preparing the nanoparticles by radical polymerization (Alhareth et al. 2012). This
polymerization allows the formation of a dense brush of dextran chains in the
nanoparticle corona that hampers proteins involved in the immune system to
interact with the nanoparticles (Vauthier et al. 2011). It is noteworthy that
nanoparticles prepared by anionic/zwitterionic polymerization in a polymerization
medium of a very similar composition but that only differs by the absence of cerium
ions provided with nanoparticles that are recognized by the immune systems.
Although having a similar composition, the two types of nanoparticles show a very
different biodistribution after intravenous administration to rats. Differences in
characteristics of the two types of nanoparticles were their size and the confor-
mation of the chains of dextran in the nanoparticle corona. Regarding the size of the
nanoparticles, nanoparticles obtained by anionic/zwitterionic polymerization were
smaller (dH = 160 nm) than those produced by the radical polymerization
(dH = 300 nm). From the literature, it was expected that the smallest nanoparticles
would be stealth. The observed in vivo fate was the opposite but it was consistent
with the capacity of each nanoparticle to activate the protein C3 of the complement
cascade that is part of the humoral immune system and involved in the recognition
of the nanoparticles by the immune system. Additionally, it was shown that the
extent of the activation of the protein C3 triggered by the nanoparticles was con-
sistent with the capacity of their corona to hamper the adsorption of this protein on
the nanoparticle surface (Vauthier et al. 2011). The efcacy of the protection of the
136 C. Vauthier
nanoparticle surface to interact with the protein C3 was correlated with the con-
formation of the dextran chains in the corona of the nanoparticles which differed
with the type of polymerization carried out to synthesize PACA nanoparticles. The
obtaining of stealth nanoparticles requires the formation of a dense brush of
hydrophilic chains of long dextran chains at the nanoparticle surface to prevent
large proteins like the protein C3 to be activated. This condition is achieved
preparing the nanoparticles by radical polymerization with a sufcient amount of
dextran (1.3 % in weight in the polymerization medium) of molar mass above
40 kDa. Nanoparticles prepared with a looser brush of dextran chains were acti-
vating the protein C3 of the complement system. It could be determined that, in this
case, the characteristics of the corona were unsuitable to hamper an interaction
between the protein C3 and the nanoparticle surface (Vauthier et al. 2011). The
obtaining of stealth nanoparticles with a corona composed of polysaccharide seems
to obey to similar requirements as those already established considering the
obtaining of stealth nanoparticles stabilized with PEG chains.
Surface properties of nanoparticles designed by emulsion polymerization can
subsequently be modied to improve the precision of the delivery of the drug to a
specic target cell type. A general approach consists of adding a ligand on the
surface of the nanoparticles that is complemental to a specic receptor that will be
used for the recognition at the target site. Different approaches were proposed to
add ligands at the surface of nanoparticles for targeting purpose (Nicolas et al.
2013). PACA nanoparticles prepared by radical polymerization were decorated
with biotin residue using a coupling reaction with EZLink coupling reagents that
can be purchased from Pierce. The nanoparticles remained stable after the coupling
of biotin residues. A biotinylated antibody was then associated with these
biotinylated nanoparticles using streptavidin to confer the nanoparticles specicity
to recognize Ewing Sarcoma cells (Ramon et al. 2013). In this example, it was
shown that post-modications can improve the delivery of the drug to the target
tumor sites, thanks to a higher degree of recognition between the nanoparticles and
targeted cells. A low number of antibodies per particle was sufcient to improve the
specicity of the delivery as shown in the previous example. Performing surface
modications of nanoparticles post-synthesis may be critical to preserve the col-
loidal stability of the nanoparticle dispersion. Size and size distribution of the
nanoparticle dispersions need to be monitored carefully at the different steps of the
process. Evaluation of the zeta potential may be used to acknowledge the modi-
cation of the surface composition that follows the grafting of the different ligands.
At the different steps of the procedure it will also be necessary to check that the
activity of the different molecules is fully preserved. A scheme summarizing the
different steps of the procedure is proposed in Fig. 8.
A large panel of drugs can be associated with poly(alkylcyanoacrylate)
nanoparticles produced by emulsion polymerization (Table 2) (Vauthier et al. 2007).
The drug is generally added directly in the polymerization medium of the nanopar-
ticles prepared by anionic/zwitterionic polymerization. In general, the yield of
association is high but the drug loading is low around a few percent expressed as the
amount of drug per mass of nanoparticles. In general also, the activity of the drug
Biotinylated nanoparticles Biotinylated - Ligand (Ab)

Synthesis
- reactivity and specificity of

- colloidal stability of nanoparticles Avidin antibody for the target receptor
- affinity of biotin for avidin - affinity of biotin residue for avidin
Assembing
- reactivity and specificity of antibody for the target receptor

- colloidal stability of nanoparticles
Targeted nanoparticle
Fig. 8 Example of method used for the coupling of an antibody at the surface of PACA
nanoparticles to design targeted nanoparticles. Details of the method are described in Ramon et al.
(2013)
molecule is well preserved. A very few examples of molecules have lost their activity
because the molecule reacted with the monomer during the initiation stage of the
anionic/zwitterionic polymerization (Guize et al. 1990; Gallardo et al. 1989). The
addition of cyclodextrin in the polymerization medium can improve the association
of lipophilic drugs with the nanoparticles (da Silveira et al. 1998; Boudad et al. 2001).
Association of drug molecules that are polyanions including oligonucleotides can be
achieved post-synthesis on nanoparticles having positive charges on their surface.
Positively charged nanoparticles can be synthesized with chitosan or by adsorbing a
polycation at the surface of already prepared nanoparticles (Zimmer 1999; De
Martimprey et al. 2010). The association of drugs with nanoparticles prepared by
radical polymerizations was less studied. The conditions applied required that the
drug molecule must resist to the extreme acidity required to carry on this polymer-
ization and to oxidation because of the presence of cerium IV ions. For instance, these
conditions are unsuitable with doxorubicin. In this case, the drug could be associated
with the nanoparticles post-synthesis (Alhareth et al. 2011). Rhodamine, that is often
used as reporting molecule to follow nanoparticles in biological media, is another
example of molecule that cannot be added in the polymerization medium at the
138 C. Vauthier
Table 2 Type of nanoparticle produced by polymerization of alkylcyanoacrylate in different

conditions and method for the association of drug molecules
Type of Method of synthesis Method of Type of drugs associated
nanoparticle drug with nanoparticles
association
Nanospheres Anionic/zwitterionic Association Small hydrophilic
emulsion polymerization during molecule (i.e.,
polymerization doxorubicin HCl,
Adsorption Ampicillin)
after Peptides (growth
polymerization hormone)
Large hydrophilic
molecules (peptide like
growth hormone)
oligonucleotides
Nanospheres Radical polymerization Adsorption Small hydrophilic
after molecule (i.e.,
polymerization doxorubicin), large
hydrophilic molecules
(i.e., oligonucleotides)
Oil-containing Interfacial polymerization Encapsulation Large hydrophilic
nanocapsules in oil-in-water emulsion during molecules (i.e., peptide
obtained by the Ouzo formation of like insulin)
effect or miniemulsions the Small hydrophobic
nanocapsules molecules (i.e.,
indomethacin,
pilocarpine,
carbamazepine,
daredipin)
Water Interfacial polymerization Encapsulation Small water soluble
containing in water-in-oil emulsion, during molecules: nucleotides
nanocapsules miniemulsion or formation of Large water soluble
microemulsions the molecules; peptides like
nanocapsules insulin, oligonucleotides
beginning of the polymerization because it losses its fluorescence in the presence of

cerium IV. The labeling of the nanoparticles with a fluorescent probe can be achieved
by adding a rhodamine-containing monomer a few minutes after the start of the
polymerization after the cerium IV was consumed by the initiation of the polymer-
ization. Using this precaution, the rhodamine-containing monomer can be incorpo-
rated in the nanoparticle core by copolymerization with the ACA monomer
(Zandanel and Vauthier 2012b).
Methods of production of nanoparticles based on emulsion polymerization can
be scaled up. As a one-pot procedure, the polymerization medium can be prepare in
the reactor in which the monomer and eventually the cerium IV solution will be
added once the polymerization medium will be ready. The agitation can be
achieved using a motor equipped with a blade agitator (Vauthier and Bouchemal
2009; Vauthier et al. 2013). The solid content of the PACA nanoparticles disper-
sions that are yet produced vary from 1 to 5 %. This could be considered as a quite
low solid content (Landfester et al. 2003, 2007). Methods of emulsion polymer-
izations allow the production of nanoparticle dispersions with much higher solid
content but concentrations of ACA above 5 % were not considered for the
preparation of PACA nanoparticles so far. Although the production of dispersions
containing higher solid content can be considered by increasing the concentration of
monomer used during the synthesis, there are a few methods that can be applied
post-synthesis to increase the concentration in nanoparticles in dispersions. In
general, PACA nanoparticles prepared by emulsion polymerization can be freeze
dried. The dispersion can be reconstituted by dispersing the nanoparticles in a
smaller volume. Addition of cryoprotectant is recommended to recover the size
distribution of the parent dispersion (Nemati et al. 1992). The nanoparticles can also
be concentrated by evaporation under reduced pressure of part of the dispersing
medium or by centrifugation and redispersion of the pellet in a smaller volume.
Another possible method is based on dialysis against a solution containing a
polymer to apply an osmotic pressure on the dispersion that forces water to move
out of the nanoparticle dispersion. This method that is easy to perform avoids
formation of aggregates (Vauthier et al. 2008). Conditions of the dialysis can be
adjusted to obtain the desired nal concentration in nanoparticles straightforward.
This method is easy to perform and does not require sophisticated material.
2.3 Miniemulsion Polymerization of ACA Applied

to the Production of Nanospheres
Miniemulsions were suggested as alternative dispersed systems to carry out poly-

merization of ACA monomers producing nanospheres. Compared with emulsions,
they are more stable dispersions, thanks to higher stability against both coalescence
and Ostwald ripening. Their formulation includes surfactants and osmotic pressure
agents to achieve the stability of the dispersed droplets while high energy
homogenization is generally required to produce droplets with a narrow size dis-
tribution. In miniemulsions, the monomer is added in an organic solvent that is then
dispersed in small droplets in the aqueous continous phase. This is in contrast
with preparations performed in emulsions in which the monomer is roughly dis-
persed and occurred as large droplet reservoirs. Thus, mechanism of polymer
particle nucleation is very different from that occurring in emulsion polymerization.
While in emulsion polymerization, particle nucleation mostly occurs in the con-
tinuous phase, particle nucleation arises within the dispersed droplets that contain
the monomer in miniemulsion (Schork et al. 2005). Size and size distribution of the
resulting dispersions of polymer nanoparticles are then very different.
In practice, to carry on a miniemulsion polymerization, the monomer with a small
amount of organic solvent such as dodecane (Wu et al. 2009b, c) or hexadecane
140 C. Vauthier
(Weiss et al. 2007a) is rapidly dispersed using ultrasounds in a cold acid aqueous
solution containing a surfactant. The polymerization is initiated after the obtaining of
the miniemulsion by adding a base in the aqueous phase. This base initiates the
anionic/zwitterionic polymerization of the alkylcyanoacrylate contained in the dro-
plets (Fig. 9a). Alternatively, an initiator of the radical polymerization can be
Fig. 9 Preparations of nanospheres by miniemulsion polymerization of n-butylcyanoacrylate

(nBCA). a Conditions for the anionic polymerization as described in Weiss et al. (2007a, b). Acid
solutions can be prepared with hydrochloric acid and sodium dodecyl sulfate as surfactant or with
phosphoric acid and one of the following nonionic surfactant: Lutensol AT50 or Tween 20. The
solution of base can be prepared with sodium hydroxide, ammonia, TRIS base. Alternatively,
amino acids at various concentrations can also be used. b Preparation based on radical
polymerization as described in Wu et al. (2009c). The acid solution can be prepared with
hydrochloric acid. Surfactants are sodium dodecyl sulfate or poly(ethylene glycol) containing
compounds such as Brij78, Brij700, Tween80
dissolved with the monomer in the organic solvent prior to the preparation of the
miniemulsion. In general, azo-bis-isobutyronitrile is used as the initiator of the
radical polymerization (Fig. 9b). The radicals that are needed to initiate the poly-
merization are produced from thermal decomposition of the molecule at high tem-
perature (70 C) (Fig. 5a).
Miniemulsion polymerizations of ACA were not developed as much as emulsion
polymerizations. One main advantage stands on the possibility to generate dis-
persions of nanospheres with narrow size distribution due to a better control of the
stability of the dispersion that serves as the polymerization system. Typical solid
content of nanoparticle dispersions obtained by miniemulsion polymerization can
reach 10 % that is signicantly higher than that generally obtained when PACA
nanoparticles are prepared by emulsion polymerization. A potential drawback of the
method of polymerization carried out in miniemulsions is the use of organic sol-
vents and of surfactants. Post-synthesis purication of the obtained dispersions is
needed to eliminate the organic solvent and the excess of surfactant prior to use the
obtained nanospheres as drug delivery systems for in vivo applications.
A wide range of conditions of synthesis can be used to produce nanospheres
having diameters ranging from 50 to 250 nm with a narrow distribution as evaluated
by dynamic light scattering (Weiss et al. 2007a). According to Hansali et al. (2011)
radical and anionic polymerizations performed in miniemulsion using similar
experimental conditions generate particles of same size range. However, molecular
weights of PACA chains composing the nanoparticle core are different influencing
the rate of degradation of the nanoparticles. The nanoparticles prepared by anionic
polymerization formed by PACA of the lowest molecular weight degrades faster than
those obtained by radical polymerization that are composed of PACA of much higher
molecular weights (Hansali et al. 2011). As in the case of the emulsion polymer-
ization, the type of the amphiphilic molecule added in the polymerization medium
influences surface properties of the formed nanospheres as it denes the nature of the
component that will stand on the nanoparticle surface. The type of molecules added
to the polymerization medium to stabilize the emulsion droplets and the forming
nanospheres depends on the mechanism of polymerization initiated to trigger the
formation of the particles. Radical polymerizations were performed with dextran
modied with epoxide residues (Wu et al. 2009b) or PEG-based surfactants (Wu
et al. 2009c). Sodium dodecyl sulfate (SDS), Lutentol and Tween 20 were used in
examples of miniemulsion polymerization initiated by the anionic route.
Nanospheres obtained from miniemulsion stabilized with SDS are very stables. As
pointed out by Weiss et al. (2007a) steric stabilizers like dextran did not attached
covalently to PACA chains during miniemulsion polymerization of ACA. In con-
trast, amines added in the aqueous phase of the miniemulsion can initiate anionic
polymerization of ACA and attach to the growing PACA chain. This method can be
used to functionalize the nanoparticle surface as the amine containing compound is
then incorporated on the nanoparticle surface. Suitable amines include amino acids.
They can be used later as an anchor for subsequent surface chemical modication to
graft bioactive ligands at the nanoparticle surface giving the nanoparticles specicity
of recognition of target cells.
142 C. Vauthier
The main differences between methods based on emulsion polymerization and

miniemulsion polymerization of alkylcyanoacrylate are summarized in Table 3.
2.4 Preparing Nanocapsules by Polymerization of ACA
Nanocapsules are hollowed nanoparticles formed by a polymer envelope that entrap a

liquid droplet in which a drug can be encapsulated. In methods of preparation based
on polymerizations, the nascent polymer forms at the surface of tiny liquid droplets
dispersed in a liquid phase of opposite lipo/hydrophilicity. The sense of the
liquid/liquid dispersion determines the type of the produced nanocapsules, i.e. oil- or
water containing nanocapsules, and in turn the nature of molecules that can be
entrapped inside the nanocapsules (Table 2). The polymerization is named interfacial
polymerization as it occurs at the interface between the two liquids composing the
dispersion. Various types of dispersions can be used to produce nanocapsules by
interfacial polymerization including emulsions, miniemulsions, and microemulsions.
Table 3 Comparisons between emulsion and miniemulsion polymerization procedures carried

out to produce PACA nanoparticles
Emulsion Miniemulsion
Organic None Hexane (Weiss et al. 2007a, b)
solvent Dodecane (Wu et al. 2009b, c)
Anionic No initiator needed, ambient No initiator needed,
polymerization temperature
Radical Cerium IV, 40 C AIBN, 75 C
polymerization
Mechanism of Nucleation in the continuous Nucleation in the miniemulsion
nanoparticle phase and growth of particles droplets. Polymerization continues
formation until stability is reached until the monomer within the droplet
is consumed
Equipment Basic laboratory equipment Need a sonicator to achieve the
required preparation of the miniemulsion
Time of 13 h 2048 h
polymerization
Solid content Up to 5 % 10 %
of the No report considering higher
dispersion solid content
Nanoparticle 50340 nm 60240 nm
size range
Zeta potential 45 to +35 mV 50 to 80 mV
Dene by the nature of the Depend on the nature of the surfactant
hydrophilic polymer added in the added in the polymerization medium
polymerization media (Wu et al. 2009b, c) or on the nature
of the polymerization initiator (Weiss
et al. 2007a, b)
Many of the methods developed to prepare nanocapsules by interfacial polymeriza-

tion were based on the anionic polymerization of ACA. Besides polymerization of
ACA, a few methods are based on interfacial polycondensation reactions.
Nanocapsules with envelopes composed of polyester, polyurethane, or polyamide are
produced by the method presented at the end of the chapter.
2.4.1 Synthesis of Oil-Containing Nanocapsules by Interfacial

Polymerization of Alkylcyanoacrylate
Two types of methods were suggested to design oil-containing nanocapsules based

on the polymerization of ACA. In the rst presented here, oil emulsion droplets
encapsulated in the course of the interfacial polymerization of ACA are formed at the
same time then the nanocapsules (Al Khouri Fallouh et al. 1986; Gallardo et al.
1993; Chouinard et al. 1994; Thioune et al. 1997; Aboubakar et al. 1999; Cournarie
et al. 2004). In practice, an organic phase is prepared adding the required amount of
monomer and oil in a solvent that is miscible in water. Many solvents can be used
provided that they are miscible with water but acetone and ethanol were preferred to
formulate oil-containing nanocapsules applied as drug carriers intended to be used
in vivo. The emulsion is then prepared by adding the organic phase in the aqueous
phase that eventually contained a surfactant. While the two phases mix together, oil
droplets form because of the occurrence of a supersaturation of oil in the new medium
composed by the mixture of water and the miscible organic solvent (Vitale and Katz
2003; Ganachaud and Katz 2005). When supersaturation of the mixture with the oil is
reached, nucleation of oil droplets occur. This method of formation of emulsion has
the advantage to be very gentle as it does not require energetic methods of dispersion.
The phenomenon by which the droplets formed was named the Ouzo effect (Vitale
and Katz 2003). At the same time the oil droplets form, polymerization of ACA is
initiated by water molecules. The nascent polymer deposits at the surface of the oil
droplets to form the envelope of the nanocapsules (Fig. 10).
The size of the emulsion droplets and in turn the size of the nanocapsules can be
tuned by many parameters including parameters of operating conditions and the
composition of the polymerization medium (Mitri et al. 2012; Vauthier and
Bouchemal 2011). In this method, the very fast polymerization of the ACA
monomers is a key for successful formation of nanocapsules once the organic phase
is added in the aqueous phase (Aboubakar et al. 1999; Cournarie et al. 2004). The
aqueous phase at neutral pH is used to promote the rapid requested polymerization
of ACA. This is in contrast with pH conditions applied in emulsion polymerizations
recommended to produce nanospheres that need to be acid to slow down the
polymerization and prevent the formation of aggregates. Although the polymer-
ization must be fast once the organic phase is added in the aqueous phase, reaction
of the monomer with components of the organic phase should be kept under control
before the mixing of the two phases (Chouinard et al. 1994). Ethanol that is widely
used due to its safety prole when developing nanocapsules for drug delivery can
react with ACA to form ethanol-single monomer adducts. To limit the formation of
144 C. Vauthier
Mixing under
magne c s rring
Organic Aqueous
phase (a) phase
1. Ethanol 25 mL Water 50 mL
(b)
2.Miglyol 812 0.5mL Pluronic F68 0.25%
pH 2.5 with HCl
3. IBCA 0.125 mL
4. Drug
(c)
Dispersion of
Evapora on of Ethanol nanocapsules
under reduced pressure
Fig. 10 Protocol for the preparation of oil-containing nanocapsules by interfacial polymerization

of IBCA preparing the emulsion by solvent diffusion, i.e., Ouzo effect. Panels ac illustrate the
nucleation of the emulsion droplets occurring during the mixing of the two miscible oil and water
phases (a), the initiation of the polymerization of IBCA at the surface of the newly formed
emulsion droplets due to contact with water molecules (b) and the formation of the nanocapsule
envelop at the surface of the oil droplet (c). This step requires a few minutes at room temperature
and under gentle magnetic stirring
this adduct and promote the obtaining of nanocapsules, it is recommended to

acidify the organic phase but the pH of the aqueous phase should be neutral. The
use of solvents unable to react with the monomers is another option that limits the
formation of the solventmonomer adducts. For instance, ethanol can be replaced
by acetone or acetonitrile (Puglisi et al. 1995). Contaminations of the nanocapsule
dispersions by nanospheres or aggregates can also be avoided by optimizing the
oil/ethanol ratio of the organic phase (Rollot et al. 1986; Gallardo et al. 1993;
Wohlgemuth and Mayer 2003; Cournarie et al. 2004).
Originally, the method was proposed to achieve encapsulation of lipophilic
drugs. It generally allows almost quantitative encapsulation efciency (Al Khouri
Fallouh et al. 1986). Thanks to the fast polymerization hence the rapid in situ
formation of the polymer envelope around the oil droplets, this method was suitable
to encapsulate peptides such as insulin, octreotide, somatostatin and calcitonin,
although these molecules are hydrophilic. The peptides are added in the organic
phase together with the oil and the monomer. Encapsulation efciency greatly
depends on the molecular weight of the peptide. Peptides with the highest
molecular weight showed the highest encapsulation efciency while small peptides
like glutathione could not be encapsulated in PACA nanocapsules prepared by this
method (Damg et al. 1988, 1997; Lowe and Temple 1994, Vranckx et al. 1996;
Gat et al. 2001). With insulin (Mw 6000 g/mol), the encapsulation efciency was
above 90 %. It was similar to that of the corresponding method of nanoprecipitation
in which the polymer has just to precipitate (Aboubakar et al. 1999). The perfor-
mance of encapsulation of such highly water soluble molecules is closely related to
the conditions of polymerization of the monomer. These are controlled by the pH of
the solution of the peptide as shown with insulin and by the quality of the monomer
which varies by the nature and quantity of polymerization inhibitors added to insure
monomer stability during storage (Cournarie et al. 2004). Based on a work done
with insulin, it was shown that the encapsulated peptide remained chemically intact
in the nanocapsules, although the molecule contains several nucleophile groups that
may potentially initiate the polymerization of ACA (Aboubakar et al. 1999).
Polymerization in miniemulsion is the second types of methods proposed to
prepare oil-containing nanocapsules (Altinbas et al. 2006; Zhang et al. 2008). Two
approaches were proposed as illustrated in Fig. 11. In the rst option (method 1)
(Fig. 11a), the miniemulsion is prepared by dispersing an organic phase composed
of medium-chain triglycerides, Mygliol, and the monomer in acidied water
(solution of hydrochloric acid at 1 %) using ultrasounds (Altinbas et al. 2006).
During the preparation of the miniemulsion, the polymerization of alkyl-
cyanoacrylate should not start explaining why the aqueous phase must be acidied
using strong acids such as hydrochloric acid. The pH of the miniemulsion is raised
to 7 only when the system is ready for the start of the polymerization. Nanocapsules
are obtained by interfacial polymerization of alkylcyanoacrylate initiated at the
surface of the droplets of the miniemulsion. The preparation can be performed in
the absence of surfactant. By this method, nanocapsules characterized by a very
small hydrodynamic diameter, below 200 nm, can be prepared (Altinbas et al.
2006). This size range is twice lower than that obtained when the nanocapsules are
produced by interfacial polymerization of alkylcyanoacrylates carried out in an
emulsion of similar composition compared with that of Al Khouri Fallouh et al.
(1986) but prepared with a specically designed apparatus to achieve the mixing of
the two phases (Altinbas et al. 2006). In the alternative method (method 2)
(Fig. 11b), a miniemulsion is prepared by dispersing the organic phase composed
of medium-chain triglycerides, Mygliol, in an acidied aqueous phase that con-
tains mono-methoxy-PEG (Zhang et al. 2008). The molecule to be encapsulated is
added in the organic phase and the dispersion is achieved using ultrasounds. Then,
the monomer is added in this miniemulsion and dispersed with the help of ultra-
sounds. The polymerization is initiated by the hydroxyl end-group of the
146 C. Vauthier
Fig. 11 Preparation of oil-containing nanocapsules by interfacial polymerization of alkyl-

cyanoacrylate in miniemulsion. a method (1) (Altinbas et al. 2006), b method (2) (Zhang et al.
2008)
mono-methoxy-PEG resulting in the synthesis of a PACA-PEG copolymer which

structure is illustrated in Fig. 4b. The copolymer that composes the nanocapsule
envelope achieves the colloidal stability of the nanocapsules thanks to the PEG
chain included in the macromolecule during the synthesis. The size of the
nanocapsules produced by this method is generally larger (230400 nm) compared

to those produced by the method 1 (Zhang et al. 2008).
These two methods of interfacial polymerization carried out in miniemulsions
are suitable to encapsulate lipophilic drugs in the nanocapsules. Zhang et al. (2008)
has described the association of paclitaxel. The drug to be encapsulated is added in
the organic phase prior to the preparation of the miniemulsion.
2.4.2 Synthesis of Water Containing Nanocapsules by Interfacial

Polymerization of Alkylcyanoacrylate
Water containing nanocapsules were developed as drug carriers for hydrophilic

molecules. In general, methods used to prepare water containing nanocapsules are
based on the initiation of the polymerization of ACA in water-in-oil dispersed sys-
tems, thanks to the already discussed particular polymerization property of the
monomer. Suitable polymerization media includes inverse emulsions (Lambert et al.
2000; Toub et al. 2006) and microemulsions (Gasco and Trotta 1986;
Watnasirichaikul et al. 2002; Hillaireau et al. 2007). Formulation of microemulsions
generally required a large amount of surfactant but they formed in gentle conditions of
agitation (Gasco and Trotta 1986; Watnasirichaikul et al. 2002; Hillaireau et al. 2007).
The dispersion that serves as polymerization medium is prepared including the drug in
the aqueous phase but without the monomer. The monomer is added in the oily
continuous phase only once the inverse emulsion or microemulsion is obtained. The
polymerization starts spontaneously at the surface of the aqueous droplets thanks to
the presence of hydroxide ions due to autoprotolysis of water. A lm of polymer
precipitates at the surface of the droplet forming the envelope of the nanocapsule. Size
of water containing nanocapsules obtained from interfacial polymerization of ACA in
microemulsions is around 150 nm in diameter. Suitable emulsions can be prepared by
one of the following approaches. The most straightforward method consists to apply
strong energy to the system using an ultraturax or ultrasounds to force the water phase
to disperse in the oil continuous phase as droplets of very small size (Lambert et al.
2000; Toub et al. 2006). Nanocapsules with diameters around 350 nm are obtained by
this method. The other method of obtaining a suitable emulsion is based on the
application of the inverse Ouzo effect (Gross-Heitfeld et al. 2014). The emulsion
droplets form from a homogenous liquidliquid nucleation which occurs by simply
mixing the aqueous and oily phases together provided that both are miscible. This
method presents the advantage to avoid the use of high shear devices as needed with
the above described method. Water containing nanocapsules produced from emul-
sions prepared by the inverse Ouzo effect approach also display size around 350 nm
in diameter (Gross-Heifelt et al. 2014). The permeability of the nanocapsule envelope
is an important parameter that controls both the efciency of the entrapment of drugs
inside the nanocapsule cavity and the release of the drug once the nanocapsules have
reached the site of delivery in vivo. It can be nely tuned crosslinking the polymer
forming the envelope. This can be achieved adding a crosslinking monomer such as a
bis-cyanoacrylate monomer in the polymerization media together with the
148 C. Vauthier
alkylcyanoacrylate monomer usually used to produce this type of nanocapsules. The

copolymerization of the cross-linking monomer and the monomer led to the formation
of a reticulated polymer envelope which permeability can be tuned by varying the
proportion of cross-linking monomer added in the polymerization system
(Gross-Heifelt et al. 2014). The cross-linking monomer used in this approach is not
yet a marketed compound and needs to be custom synthesized.
Table 4 gives examples of conditions that can be used to produce
aqueous-containing nanocapsules from the different types of dispersions.
Nanocapsules are produced with different ranges of size from the different
systems. In general, preparation of nanocapsules from emulsions stabilized with
anionic surfactants produces nanocapsules with diameters ranging from 250 to
350 nm (Lambert et al. 2000; Toub et al. 2006). From microemulsions, nanocap-
sules of smaller diameters can be obtained (around 150 nm and even bellow)
(Watnasirichaikul et al. 2002). Water containing nanocapsules were designed to
encapsulate hydrophilic molecules. For instance, insulin was encapsulated with
80 % efciency (Watnasirichaikul et al. 2002) and encapsulation yield of siRNA
reached 97 % (Lambert et al. 2000; Toub et al. 2006). It is noteworthy that
encapsulation efciency of small molecules may be dramatically low especially
with nanocapsules having non-reticulated envelope. The molecular weight thresh-
old was not determined precisely but the methods were not suitable to encapsulate
antiviral drugs occurring as free nucleosides. Successful encapsulation of these
compounds was achieved by co-encapsulation of polycations that form ion-pairs
with nucleosides (Hillaireau et al. 2007). Examples of polycations are poly(ethylene
imine) and chitosan. These macromolecules are well encapsulated in the
nanocapsules hence the nucleosides that are linked to them by electrostatic inter-
actions. Improvement of the encapsulation may be expected applying in a more
systematic way the cross-linking approach of the nanocapsule envelope proposed
by Gross-Heifelt et al. (2014).
It is noteworthy that the produced nanocapsules are provided in an oily dis-
persion. Such dispersions are unsuitable for an intravenous injection to patient, that
is, the main route for the administration of drugs in treatment of severe diseases.
Methods were proposed to transfer the nanocapsules in an aqueous medium to
prepare nanocapsule dispersions suitable for intravenous injection. This transfer can
be achieved after the recovery of the nanocapsules from the polymerization medium
by centrifugation and using surfactants to achieve the dispersion of the pellet in the
aqueous phase. Aggregation is the main problem with this method. The obtaining of
an aqueous dispersion from the nanocapsules dispersion synthesized in an oily
phase is easier when the centrifugation is achieved over an aqueous solution of
surfactant. In this approach, the nanocapsules are transferred from the oily dis-
persion in an aqueous dispersion through the liquid interface occurring between the
two liquid phases, i.e., the aqueous receiving phase covered by the nanocapsule
dispersion in the oil continuous phase. This mode of transfer reduces aggregation
because nanocapsules are not forced to come in close contact with their neighbors
as it is the case when they are in a pellet recovered after centrifugation (Lambert
et al. 2000).
Table 4 Conditions for the preparation of aqueous-containing nanocapsules by interfacial

polymerization of ACA in emulsions and microemulsions
Experimental Emulsion Emulsiona Microemulsion
parameters
Type of Water-in-oil Water-in-oil emulsion Water-in-oil microemulsion
polymerization emulsion
system
Composition of 0.8 mL Water 1 mL Water 1 g insulin solution at
the aqueous 0.2 mL Ethanol 1 mL cosolvent (Methanol, 100 U mL1 (Humulin R)
phase 10 M Ethanol)
Oligonucleotide 0.08 g Surfactant (Tween
(20-mer) or siRNA 80)
5 mM
Composition of 8 g Miglyol 812 20 mL Cyclohexane 7.6 g oil (blend of
the oily phase 1 g Surfactant 1.6 % Span 80 medium-chain trigycerides
(Montane 80) 0.3 g ACA and medium-chain mono
(Methylcyanoacrylate), Part and diglycerides in a 3:1
of this amount of ACA can weight ratio)
be replaced by a 1.4 g surfactant (blend of
crosslinking monomer polysorbate 80 and sorbitan
mono-oleate, 3:2 weight
ratio)
Method of Ultraturrax, Inverse Ouzo effect Not specied
fabrication of 24,000 rpm, 1 min The aqueous phase is added
the +4 C in well stirred oily phase at
polymerization room temperature
system
Initiation of the Addition of the Because of the amphiphilic Addition of the monomer
polymerization monomer character of the ACA (0.10.2 g IBCA)
(0.1 mL IBCA) monomer, it tends to migrate
and accumulate at the
surface of the aqueous
droplet where the
polymerization can be
initiated by the water
Conditions and Mechanical stirring Mechanical stirring, 30 min Mechanical stirring, +4 C,
Duration of the 500 rpm, ambient 12 h
polymerization temperature, 4 h
Application to Antisens Not specied Insulin
drugs oligonucleotides,
siRNA nucleotides
applied as antiviral
agents
References Lambert et al. Gross-Heitfeld et al. (2014) Watnasirichaikul et al.
(2000), Toub et al. (2000)
(2006)
a
This example was included in the table to illustrate the use of the inverse Ouzo effect to prepare a
suitable polymerization medium to produce water containing nanocapsules. It is also interested by the
fact that it proposes the use of a crosslinker to tune the permeability of the nanocapsule
envelop. However, the use of cyclohexane, methanol and the nature of the surfactants suggested as
components of the polymerization medium is questionable to achieve the synthesis of nanocapsules that
are intended to be applied as drug carriers. The polymerization medium must be reformulated with
components that are acceptable preparing pharmaceutical formulations
150 C. Vauthier
3 Preparing Nanocapsules by Polycondensation
Interfacial polycondensation methods are alternative ways to induce the in situ

production of a nanocapsule polymer envelope during the preparation of
nanocapsules. In contrast with polymerization reactions consisting of polyaddition
of unsaturated monomers, polycondensation reactions involve molecules containing
complemental chemical groups that are able to react together. Examples of suitable
compounds are mentioned in Table 5.
Each of the monomer molecules contains at least 2 groups of the same nature
that can react with those of the second component. The obtaining of reticulated
polymer envelop can easily be achieved by introducing a certain proportion of
tri-functional compounds. This approach is interesting to consider to adjust per-
meability and biodegradability properties of the nanocapsule envelopes. During
reaction, a low molecular weight compound such as water or hydrochloric acid is
generally produced in the medium. The nature of this compound depends on the
reagents. To form nanocapsules, the polycondensation reaction should take place at
the interface between the dispersed phase occurring as small droplets and the
continuous phase of a dispersion, i.e. an emulsion for instance. In theory, the sense
of the dispersion denes the type of nanocapsule produced. So far, all methods were
developed in oil-in-water emulsions producing oil-containing nanocapsules. In
practice, each reagent is dissolved in one phase that will form the emulsion. The
Ouzo effect can be applied to obtain very thin emulsions suitable to produce the
nanocapsules (Bouchemal et al. 2004; Stumpo et al. 2013) (Fig. 12).
Table 5 Examples of reactions found in polycondensation and compounds that can be used
Monomer 1a Monomer 2a Linkage Type of
Soluble in aqueous phase Soluble in oily phase polymer
Alcohol Acid Ester Polyester
HOROH HOOCRCOOH ROCOR
Hexane diol 0.125 M ClOCRCOCl
Sebacoyl chloride
0.025 M
Amine Acid Amide Polyamide
H2NRNH2 HOOCRCOOH RNHCOR
Diethylene triamine ClOCRCOCl
0.125 M Sebacoyl chloride
0.025 M
Alcool Isocyanate Urethane Polyurethane
HOROH OCNRNCO ROCO
Short poly(ethylene Isophorone NHR
glycol) 102 M di-isocyanate 103 M
Amine Isocyanate Urea Polyurea
H2NRNH2 OCNRNCO RNHCO
NHR
a
Monomers must be at least bifunctional to obtain polymers. Specic examples of monomers and
concentrations used were taken from Bouchemal et al. (2004) and Stumpo et al. (2013)
Fig. 12 Scheme presenting the preparation of oil-containing nanocapsules by interfacial

polycondensation from emulsions prepared by the solvent diffusion technique. The method
described in Bouchemal et al. (2004) and Stumpo et al. (2013) were applied to encapsulate
alpha-tocopherol (Vitamin E) and Parsol MCX, a sun screen agent, in olive oil. Examples of
monomers and concentrations are indicated in Table 5. The formation of the nanocapsules is
almost instantaneous. After mixing the two phases the magnetic agitation was maintained for
30 min before proceeding to the evaporation of acetone
The polycondensation is initiated simultaneously to the formation of the emul-

sion droplets that forms instantaneously while mixing the organic and aqueous
phase together. The nanocapsules are produced in a single step from the two
initially prepared solutions in very gentle conditions. The polymer envelope can be
composed of polyurethane, polyesters, and polyamides depending on the reactive
species used to produce the nanocapsules (Bouchemal et al. 2004; Stumpo et al.
2013). The method can easily be scaled up. The produced nanocapsules are suitable
for the encapsulation of lipophilic drugs with controlled releasing properties
(Stumpo et al. 2013). Protocols proposed in the literature to synthesize drug-loaded
nanocapsules by interfacial polycondensation can be found in Table 5 and Fig. 12.
It could be expected that this method may also apply to the production of
water-containing nanocapsules preparing the emulsion by the inverse Ouzo
effect.
152 C. Vauthier
4 Conclusion and Perspectives
This chapter presents several polymerization methods that can be applied to pro-
duce polymer nanoparticles in the aim to use them as drug carriers. The different
methods were adapted to achieve association of drugs of various nature and
physicochemical properties including small molecules and oligo- or macro-
molecules. As it was discussed, the methods of polymerization are versatile making
possible the synthesis of nanoparticles characterized by a wide range of size and
surface properties in a controlled manner. Nanoparticle characteristics can be tuned
according to specications that are requested for a given application. While most of
the methods of preparation of the nanoparticles avoid the use of toxic organic
solvents, the majority were based on the polymerization of ACA, thanks to the very
specic reactivity of these monomers and of the biodegradability of the corre-
sponding polymer.
The emulsion polymerization of ACA was the rst method applied to prepare
biodegradable polymer nanoparticles showing a suitable prole to be developed as
drug carriers to be applied in human medicine. With time, the method has been
improved and diversied to design carriers that answered the numerous challenges
that needed to be fullled aiming to improve delivery and therapeutic efcacy of
various types of drugs. Interest in this method is still valid. It is used to synthesize
the rst polymer nanoparticles that were approved for clinical tests in human. This
demonstrates the potential of the polymerization methods to provide with polymer
nanoparticles that are suitable to be translated into medicine for patients.
Acknowledgments CV addresses her warm thanks to Patrick Couvreur who has introduced her
in the poly(alkylcyanoacrylate) nanoparticle world and for fruitful discussions that have been a
source of inspiration for her work. CV also thanks Denis Labarre for the collaboration that has
started at the drawn of the millennium and lasted up to his retirement in developing the radical
emulsion polymerization of ACA that opened huge possibilities to design poly(alkylcyanoacrylate)
nanoparticles giving them new properties needed to control their in vivo fate hence drug delivery
potential. Thanks to all Ph.D. students, Post Doc, and colleagues around the world who have
provided with new ideas of making poly(alkylcyanoacrylate) nanoparticles based on the poly-
merization of these very tricky monomers.
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Chapter 6
Shape-Controlled Nanoparticles for Drug
Delivery and Targeting Applications
Gilles Ponchel and Olivier Cauchois
Abstract Whatever nanoparticles are envisioned for, including vaccinal, imaging,

diagnostic, or drug targeting applications, their considerable interest originates from
a unique combination of a nanometric size and the possibility to considerably
modulate their physicochemical properties, including their geometry. There are
nowadays growing experimental evidences that the morphology and the shape of
nanoparticles can signicantly contribute to their pharmacokinetics in the body, by
influencing various physicochemical mechanisms such as their diffusivity, inter-
actions with biological materials, internalization by cells. We present here a review
of the present knowledge in this eld. After a brief discussion on the different
phenomena on which shape can have an influence, the different preparation
methods currently available to obtain nonspherical nanoparticles will be presented.
Their pro and cons will be discussed, regarding surface properties control, scale-up
potential, etc.
Keywords Nanoparticles
Drug delivery
Drug targeting Shape
Morphology
Self-assembling
Amphipilic copolymers Peptides
Manufacturing methods
1 Introduction
Nanomedicines have emerged as a new class of promising healthcare agents

because of continuous progresses both in nanotechnologies enabling their prepa-
ration and the emergence of powerful physicochemical characterization methods.
Nanomedicines represent nowadays considerable hopes for addressing diagnostic
and/or drug delivery challenges. In the eld of drug delivery, nanomedicines are
often envisioned for overcoming inadequate physicochemical and pharmacokinetics
G. Ponchel (&) O. Cauchois

Institut Galien Paris Sud, CNRS, Univ. of Paris-Sud, University Paris Saclay,
5 Rue J.B. Clment, 92296 Chtenay-Malabry Cedex, France
e-mail: Gilles.ponchel@u-psud.fr

DOI 10.1007/978-3-319-41421-8_6
160 G. Ponchel and O. Cauchois
properties of drug substances when conventional pharmaceutical approaches fail to

do so. For instance, they can be conceived for enhancing the passage of drugs
through epithelia lining many sites of delivery in the body (intestinal, nasal, pul-
monary, etc.). Alternatively, they can be of benet, e.g., for transitorily masking
inadequate physicochemical properties of drugs, promoting their accumulation in
desired tissues, the targeting specic cells or subcellular organites. The so-called
drug targeting strategy consists in attempting to modify the biodistribution of a drug
by the one of a carrier in which it is associated. This strategy aims not only to
reduce the amount of administered drugs, but also to improve the benet/risk ratio
for the patient by enhancing the delivery of the carrier in a specic organ, dedicated
cells, or subcellular compartments. Ideal carriers should increase specicity while
toxic effects caused by non specic delivery should be weakened. Consequently,
understanding the in vivo fate of the carriers is a critical step in their development.
A huge variety of carriers have been proposed so far, which are not only able to
encapsulate the therapeutic molecules, but are also meant to vehiculate the drug in
the body from the site of delivery to the target organ, and nally to interact ef-
ciently with target cells. Among them nanoparticles made from suitable polymers
are interesting objects because they present a unique combination of a nanometric
size and the possibility to considerably modulate their physicochemical and bio-
logical properties. A survey of the literature shows that these carriers are mostly
spherical particles and that until now the effect of the particles morphology on their
behavior in the body has been almost ignored. The reasons for it probably stems
from the difculty to prepare and to obtain micro and nano-objects with non-
spherical shapes and prepared from materials of pharmaceutical interest and also
from the lack of basic data on the role of morphology and shape on the behavior of
these objects in the body, when in contact with biological fluids and faced to
various cellular environments, for example when particles-cells interactions occur.
This situation is quite surprising, as when looking at living bodies, the notions of
shape and morphology appear so ubiquitous and having so much implications in the
machinery of life. For example, at the micron- and nano-range, it is well known that
the morphology of microorganisms has a great influence over their life (Lleo et al.
1990; Young 2006). A growing number of studies describe to which extent mor-
phology dictates the fate of nanoparticles in the body. Even if this knowledge would
have to be strengthed, there are now experimental evidences that not only the
motion of nanoparticles in blood and other fluids, but also their capacity to go
through various tissular and cellular barriers (or not to go through!), their capacity
to escape to the capture by immune system macrophages and, on the contrary, to be
efciently endocytosed by targeted cells, etc.
This chapter aims to present the available technological strategies available for
tuning the shape and the morphology of polymer nanoparticles of pharmaceutical
interest and envision the incidence of shape in drug targeting applications and more
generally in the eld of drug delivery.
6 Shape-Controlled Nanoparticles for Drug Delivery and Targeting Applications 161
2 Production of Shape-Controlled Nanoparticles

of Pharmaceutical Interest
Control of particle shape is a crucial issue in many branches of industry. For

example, in pharmaceutical technology, some llers used for tableting are made of
shape-engineered particles and are commercially available since decades because
surface and adequate particle shaping enable direct tableting and simplify techno-
logical manufacturing processes. More surprisingly, it is only recently that manu-
facture of shape-controlled nanoparticles intended for drug delivery has been
addressed, probably because production and characterization of shape-controlled
objects at nanoscale represents a real challenge. Indeed, not only the shape has to be
controlled but many critical properties have simultaneously to be maintained,
including degradability of the materials, control of the surface topological proper-
ties for targeting applications, loading capacities as well as controlled release
kinetics of active ingredients.
There are numerous methods for producing nano- (and micro-) particles, which
depend on the material to be used, metals, metal oxides, polymers, and generally
speaking, organic molecules including surfactants. However, when polymers are
used for their preparation, a literature survey shows that so far most available
techniques lead to spherical or at least round-shaped particles, either in the micro-
or nano range. Basically, the reasons for this is that for minimizing the energy of the
system, nano- and micro-objects naturally tend to minimize their surface tension by
reducing their surface thus adopting the geometry of a sphere which present the
lowest surface energy for a given amount of material. Further, very generally
polymers commonly used as building blocks to create nanoparticles adopt mainly
random coil conformations due to their flexibility. Whatever the method of
preparation, aggregates composed of a discrete number of polymer chains are
formed, in which chains relaxation and reorganization during the preparation pro-
cess and/or the storage phase lead to spherical geometries.
Therefore, to create nonspherical particles one has to nd a way to avoid iso-
tropic association of the building blocks. In the literature, two types of approaches
leading to nonspherical particles have been proposed so far. A rst strategy consists
to self-assemble specically conformed building blocks able to induce an aniso-
tropic aggregation and to form nonspherical objects. The second strategy consists in
applying external constraints on the materials forming the particle either during its
formation or after. From a practical point of view, these techniques can also be
combined, ranging from one step to two or more steps methods.
The rst approach that will be presented is the autoassembly. The particles can
be prepared directly in water by commonly used techniques, such as the nano-
precipitation technique or its variations (see Chaps. 2 and 3). The material used to
prepare them has specic properties which will impose the nal aspect of the
particles. The materials used in this approach can be polymers, peptides, or even
spherical particles, which all are prone to form nonspherical aggregates.
The second approach consists in imposing constraints to the material, which can
be achieved in placing the material in a conned space, which does not allow the
formation of spheres. The particles take the form of the space they are conned in.
An alternative approach is to prepare spherical particles in a rst stage, and then to
deform these particles to obtain a denite shape.
2.1 Self-Assemblying Methods for the Preparation

of Nonspherical Nanoparticles
Self-assembling of copolymers is widely used for producing aqueous colloidal

dispersions of nanoparticles because of its easiness. Further, their scale-up and
industrial production can be realistically envisioned for commercial applications.
However, the preparation of shape-controlled nanoparticles by self-assembling
methods necessitates the selection of carefully designed building blocks with a
molecularly dened structure that can assemble in a structured way and lead to
predened specic geometrical shapes and morphologies. Potential building blocks
include not only synthetic amphiphilic surfactants and copolymers molecules but
also bioinspired peptides or proteins. Further, hybrid structures can also be elab-
orated in which other materials (e.g., metallic nanoparticles) are hierarchically
assembled with the polymers.
2.1.1 Nanoparticles Prepared from Amphiphilic Building Blocks
Auto-assembling of small amphiphilic molecules, such as surfactants, is known for

long to lead to micelles (Williford et al. 2015). Micelles denominations such as
worms, lomicelles, nanoribbons illustrates pretty well how wide the variety of
shapes and morphologies can be. It has been described as early as in 1976 by
Israelachvili et al. (1976) that the relative importance of the hydrophilic and
hydrophobic blocks of the surfactants mastered the geometry of the assembling. In
many applications, micelles prepared from small surfactant molecules suffer of
many drawbacks for drug delivery, mostly because of their instability and dif-
culties to impart simultaneously various functionalities without totally modifying
the objects. Interestingly, amphiphilic block copolymers composed of two, three, or
even more blocks with opposite polarities can self-organize themselves and form
stable structures, due to the thermodynamic incompatibility between the different
blocks but also to block copolymers concentration, nature of the solvent used for
their preparation. Interestingly, their geometry can be modulated by adjusting
precisely the chemical structure of the building blocks. Figure 1 shows an example
of the variety of accessible structures when using a series of poly(styrene)-
block-poly(acrylic acid) diblock copolymers (PS-b-PAA) with varying molecular
weights of the blocks (Mai and Eisenberg 2012). As can be seen, accessible
structures range from platelets to rods, including spheres and hollow structures.
Fig. 1 Self-assembling of amphiphilic PSm-b-PAAn copolymers yields a variety of morphologies

when the ratio of hydrophobic to hydrophilic blocks is varied. In PSm-b-PAAn copolymers, m and
n stand for the degrees of polymerization of PS and PAA blocks, respectively. Transmission
electron microscopy (TEM) microphotographs are associated to corresponding morphological
schemes in which red represents hydrophobic PS blocks, while blue denotes hydrophilic PAA
segments. Even if spherical micelles (a) or hollow vesicles (f) can be formed, non spherical objects
can be otained such as rods (b and c), or lamellale (d and e). g Oppositely to rods, HHHs
represents hexagonally packed hollow hoops in which PAA segments are at the inner surface of
the hoops; h large compound micelles (LCMs) represents microscaled objects resulting of the
aggregation of inverse micelles and in which islands of PAA chain are surrounded by PS chains,
while the overall object is stabilized by some PAA chains located in the external corona. Note that
generally speaking the hydrophilic coronas of the different aggregates cannot be observed directly
in TEM without proper staining (for further information on nanoparticle imaging by TEM see
Chap. 8). Reproduced from Mai and Eisenberg (2012) with permission from The Royal Society of
Chemistry
The above example demonstrates that a careful engineering of weight fractions

of block copolymers is necessary for controlling the shape of the objects (Smart
et al. 2008). This aggregative behavior is now well related to the structure of such
copolymers. Depending on their packing parameter, i.e., the spatial organization of
the hydrophilic and hydrophobic blocks, Fig. 2, depicts to which extent the shape
of the aggregates can be different, ranging from spherical micelles to bilamellar
structures, by simply varying the lengths of the different blocks.
In some circumstances, nonspherical micro, and nanoparticles can be obtained
from the use of proteins or polypeptidic materials. Polypeptides, which consist in a
Fig. 2 Geometry of diblock copolymers in a selective solvent (described by the packing

parameter p) dictates the shape of supramolecular objects resulting from their aggregation.
Modied and reproduced with permission from Smart et al. (2008). The packing parameter p is
calculated from v and d, which are the volume and length of the solvent phobic block and a0, the
interfacial area between solvent phobic and solvent philic blocks. Reproduced with permission
from Smart et al. (2008)
controlled repeat of a L- or D-amino acids, may adopt -helix or -sheet confor-

mations, which can be used to obtain denite three-dimensional arrangements. An
example is given by poly(-benzyl-L-glutamate) (PBLG) (Martinez Barbosa et al.
2009). Due to the presence of the benzyl group and to their alpha-helix confor-
mation, PBLG blocks tend to form ovoid nanoparticles with an aspect ratio
( = length/width) varying from 1 (sphere) to 4 (oblate). These particles can be
quite easily prepared through a nanoprecipitation technique consisting in diluting an
organic solution of the peptide in a water miscible solvent in a water solution. For
example, Fig. 3 shows that the aspect ratio of PBLG nanoparticles depends on the
molecular weight of the alpha-helix rods which are used (Cauchois et al. 2013). It is
believed that the stiffness of the alpha-helix contributes to the formation of liquid
crystal-like objects, in which PBLG rods self-organize themselves in
nonspherical-structures. It represents clearly an example in which the molecular
structure of the building blocks masters the shape of the nanoparticles. For further
pharmaceutical applications, these lipophilic PBLG blocks can be associated to
Fig. 3 Self-assembling of poly(-benzyl-L-glutamate) (PBLG) in water. Left side PBLG chains

easily adopt an alpha-helix conformation, which self-associate together and form liquid crystal-like
aggregates in water due to their hydrophobicity (benzyl groups are exposed at the periphery of the
rods). Right side Oblate nanoparticles are formed in water and their elongation depends on PBLG
molecular weight. Scanning Electron Microscopy images of PBLG nanoparticles (a PBLG
MW = 10 kg/mol W; b PBLG MW = 30 kg/mol; c PBLG MW = 45 kg/mol; d PBLG
MW = 80 kg/mol). Down right mean aspect ratios (calculated from scanning electron microscopy
(SEM) images) increases with the length of the alpha helices rods, i.e., PBLG molecular weight.
Adaptated and reproduced with permission from Cauchois et al. (2013)
various hydrophilic moieties, including poly(ethylene glycol) (PEG) and recogni-

tion ligands, and still self-organize to form hydrophilic and functionalized corona
necessitated in drug targeting applications (de Miguel et al. 2015).
All those examples demonstrate the versatility of amphiphilic polymers and their
propensity to be used as building blocks in the engineering of nonspherical micro or
nano-objects. A wide range of shapes, sizes, composition, surfaces may be quite
accessible and they may be of interest for the preparation of interesting drug car-
riers. However, the potential toxicity of these molecules should not be forgotten.
Further, issues concerning the stability of the self-assembled objects in biological
fluids, all along their journey from their site of delivery to their target may be a real
concern, because the stability of these assemblies is often dependant on surfactant
concentration, temperature, shearing strains in the body, interactions with cell
surfaces, leading to unwanted early breakages, or failures of the particles.
Clearly these issues should deserve many further investigations, especially under
physiological conditions, to assess their potential as drug carriers.
2.1.2 Natural Nanostructures Based on Proteins and Peptides

Assemblying
Peptides and proteins are ubiquitely used as building blocks for creating
nonspherical-shape in the living world. It is well known that peptides often exist as
secondary structures such as -helices and -sheets. These structures are then folded
and lead to tertiary structures, which are a specic three-dimensional conformation
of the protein. The unique shape of the protein determines its function in vivo.
Depending on their properties, these 3D conformations can further arrange them-
selves into supramolecular assemblies, leading to various nanostructures, which are
one of the basis of life. Although they are not polymer materials, the following
examples show how natural materials can be a source of inspiration for creating
original building blocks with shape-orientating properties.
Synthesis of amelogenin by ameloblasts represents an interesting example of
naturally produced oblate-shaped nanoparticles (Fig. 4). The structural arrangement
of amelogenin nanoparticles (1214 nm in hydrodynamic radius, depending on pH)
and their hierarchical combination at the micrometer scale with hydroxyapatite
mineral crystals is crucial for effective protective properties of dental enamel.
Synthesis of amelogenin and its detailed aggregation properties have been studied
and reviewed in detail elsewhere (Ruan and Moradian-Oldak 2015; Aichmayer
et al. 2010) and not surprisingly, different attempts are made for producing sub-
stitutes for enamel reconstruction. Further, articially produced amelogenin
nanoparticles based on amelogenin intended, e.g., for gene therapy applications
(Bonde and Blow 2014) have been investigated.
Biological proteins and peptides have a propensity to self-assemble into
nanobrils or nanotubes (Reches and Gazit 2006). The understanding of such a
behavior has prompted research interest, since the exact of role of these objects is
not fully elucidated. For instance, as shown in Fig. 5 peptide nanotubes can orig-
inate from the self-assembling of various peptidic sequences, and in turn,
self-aggregate in vivo to form amylod bundled networks, which are responsible for
amylod diseases. A part of such deleterious behaviors, it shows the potential of
peptides to create nonspherical objects, likely to be used in biological applications
(Scanlon and Aggeli 2008). There is no doubt that the production of peptides
themselves can be really costly. However, the methods for preparing these
nano-objects would be generally quite easy to scale-up in order to produce large
amounts of drug carriers for various applications. For example, there is still no
control over length for the nanotubes; controlled functionalization of interior and
exterior surfaces has to be thought of to give these systems interesting targeting
properties while maintaining shape integrity.
Generally speaking and from a pharmaceutical point of view, the self-assembly
of peptide, proteins and/or their derivatives is an interesting strategy to access to
Fig. 4 Quaternary structures formed by recombinant amelogenin in vitro under different

experimental conditions; a AFM tapping mode image of amelogenin nanospheres formed at pH
8, adsorbed on mica and xed by Karnovsky xative (image width = 500 nm). b Amelogenin
nanochains detected by TEM following slow solvent evaporation in the presence of PEG.
c Amelogenin oligomers formed at pH 8 and detected in cryo-TEM, d AFM tapping mode
(width = 3 mM) of amelogenin nanoribbons formed at pH 4.5, and in the presence of calcium
phosphate. e pH dependency of amelogenin aggregation and hydrodynamic radius of the
aggregates. Adaptated and reproduced with permission from Ruan and Moradian-Oldak (2015)
very well sized- and shaped-controlled objects even though the building blocks are
not always easily obtained. We believe that such strategies, which are at the con-
fluence of synthetic biology and pharmaceutical nanotechnologies, will continue to
expand for the seek of really effective nanomedicines.
2.1.3 Hierarchical Assemblies
Hierarchical assembly of different materials offers the opportunity to manipulate

both the shape and the surface of the objets which are formed. As an illustration,
Fig. 5 Certain biological proteins and peptides have the intrinsic ability to self-assemble into
elongated solid nanobrils. Dipeptides from the diphenylalanine motif of the Alzheimers
-amyloid peptide are a simple example of self-assembling peptides. Depending on conditions,
dipeptide self-assembly gives rise to different structures. Upper line: schematic of the formation of
tubular (single or multiwalled), spherical, or brillar structures. Middle line: in a second step,
arrays of aligned peptide nanotube are formed. (1) Scanning electron micrograph of a nanoforest of
vertically aligned peptide nanotubes. (2) Cold eld-emission gun high resolution scanning electron
(CFEG-HRSEM) micrograph of the nanotube arrays. (3) High-magnication micrograph of an
individual nanotube obtained by CFEG-HRSEM. (Reproduced with permission from Reches and
Gazit (2006)
Deng et al. (2015a), have shown that hierarchical self-assembly of amphiphilic

copolymers made of (hydrophobic) poly(styrene) (PS) and (more hydrophilic) poly
(4-vinylpyridine) (P4VP) (PS-b-P4VP) could lead to Janus nanoparticles or to
patchy particles. As shown in Fig. 6, patchy nanoparticles, looking to raspberries,
bearing at their surface nanoscaled protuberances can be prepared in 3D conned
conditions. In fact, according to these authors (Deng et al. 2015b), these protu-
berances could be made by the self organization of the P4VP blocks at the external
surface of the particles in presence of poly(vinyl alcohol) (PVA) in the continuous
Fig. 6 Examples of patchy nanoparticles prepared from PS-b-P4VP copolymers. a SEM image
of the PS-b-P4VP patchy particles. b, c TEM images of the patchy particles before and after
selective staining the P4VP domains with iodine vapor. d Cross-sectional TEM image of the
patchy particles after staining with iodine vapor. e TEM image of the patchy particles after
cross-linking the P4VP domains with N,N-diisopropylamino ethylamine (DIP). Insets are SEM
image and schematic structure. f Janus NPs after disassembly of the cross-linked patchy particles
with THF. Insets are the highly magnied TEM image and schematic structure. Reprinted with
permission from Deng et al. 2015b
phase of the suspension, contributing to 3D connement (i.e., conditions in which

copolymers have only limited volume in which phase separation can occur, e.g., in
presence of an other polymer in the continuous phase constituting the dispersive
phase of the suspension).
Other examples of patchy nanoparticles based on a polystyrene core on which
smaller silica nanoparticles are anchored have been reported recently by Sabapathy
et al. (2016). Other super structured constructions prepared from (PS-b-P4VP)
copolymers have also been obtained by Deng et al. (2015) (Fig. 7), suggesting the
considerable potential of self-assembling strategies for manipulating not only the
shape, but also the surface of these objects with this kind of fractal structure.
Whatever the preparation method, nanoscale surface heterogeneities can be
expected to increase the interfacial area and influence the interaction phenomena
with biological materials.
2.2 Preparation of Nonspherical Nanoparticles Under

Constraint
As described above, shaping nanoparticles through self-assembling techniques

necessitates the use of on-demand and perfectly engineered building blocks. When
Fig. 7 TEM images and corresponding illustration of superstructures of self-assembled diblock

PS110 K-b-P4VP107 K copolymers. ad the 1D superstructure with the periodic P4VP domains
incorporated with gold nanoparticles (yellow). The scale bar in a is applied to bd. Reproduced
with permission from Deng et al. (2015)
the materials to be used lacks anisotropic self-assembling properties, an alternative

strategy for preparing nonspherical nanoparticles consists in applying orientated
constraints on a certain amount of the selected material. Three major methods can
be regrouped under this category, including electrospinning, particle replication in
non-wetting templates (PRINTTM), and micro/nanofluidics. Basically, in these
methods the particle material, generally polymer, either in a melt state or as a
solution in a solvent, is subjected to physical constraints. Therefore, it requires an
external device able to impose the form of the nanoparticles. Then the polymer must
undergo a stimulus which causes solidication or solvent evaporation and nally, it
necessitates the nanoparticles to be retrieved. Indeed, these methods are an inter-
esting opportunity to manipulate the shape of nanoparticles based on already
synthesized and characterized polymers. However, they probably share a major
drawback, because of the thermodynamic instability of the shape, due to progres-
sive chain relaxation during storage leading back to the sphere geometry. Although
important from a practical point of view, this point has almost not been addressed
yet in the literature. Shape-stability and back-to-sphere kinetics, if any, remain to be
determined, as well as, the parameters which are likely to be involved in such a
phenomenon, including molecular weight and relaxativity of polymer chains, size
of the objects.
2.2.1 Jet Spinning and Electrospinning
The fundamental idea of electrospinning dates back to 1934. Jet spinning, or

electrospinning, is a modied extrusion method that allows the formation of
nanobers, nanoribbons and nanowires, and other morphologies from an electri-
cally charged jet of melt polymer or polymer solution (Li and Xia 2004; Garg and
Bowlin 2011; Zong et al. 2002). Figure 8 depicts the principle of the method. The
basic electrospinning setup is quite simple. It comprises a feeding device (e.g., a
glass syringe in experimental setups) containing a polymer solution, an electrically
conductive metallic needle, a high-voltage power supply, and a metallic collector
on which surface the shaped polymer will be recovered. The electrospinning pro-
cess consists in applying electric charges to a polymer solution or melt via the
metallic needle. Electrical charges are induced in the polymer solution, which
causes instability within the polymer solution. The reciprocal repulsion of charges
Fig. 8 Left basic setup for electrospinning of polymers (DC current voltages are typically in the
range of 030 kV). As an illustration, the SEM microphotograph insert shows poly
(N-vinyl-2-pyrrolidone) nanobers deposited on the collector. Upper right electrospinning setup
enabling the preparation of coaxial core-shell nanobers. Down right TEM images of coaxial
core-shell structured nanobers consisting of a poly(-caprolactone) core gained by gelatin shell.
a Overview of nanobers observed on a copper grid; b, c segments of the nanobers with a sharp
boundary; and d segment of the nanobers with skewed inner component. Reproduced with
permission from Garg and Bowlin (2011), Zhang et al. (2004)
produces a force that opposes the surface tension which leads to fragmentation in
droplets. In turn these droplets are geometrically deformed and adopt a conical
shape (Taylor cone) when the electric eld is further increased. While the polymer
solution flows in the direction of the electric eld, nanobers spin off from the
conical polymer droplet and are nally deposited on the metallic collector.
Every soluble or fusible polymer is eligible for this method. Biodegradable
polymers, including poly(lactide acid) (PLA), poly(ethylene-co-vinyl acetate)
(PEVA), poly(ethylene oxide) (PEO), poly(4-vinyl-phenol) (P4VP), poly(vinyl
alcohol) (PVA), poly(lactide-co-glycolide) (PLGA) have already been successfully
jet spun and tested for biomedical or drug delivery applications (Huang et al. 2003).
The bers are typically a few tens of nanometer in diameter and do not seem to
have a maximal length. Coming to their structure, the bers can be plain, hollow or
porous. Obviously, many parameters, including conductivity, viscosity, surface
tension, solvent volatility of the solution phase can be varied for adjusting bers
length and structure, as shown for example for PLA, which can form either spheres
or nanobers depending on experimental conditions (Fig. 9). Electrospinning can
Fig. 9 Microspheres and/or nanobers resulting from electrospraying of a solution of poly

(D,L-lactide) in dimethyl formamide (DMF) under different concentrations and conditions: a 20 wt%
yielded bead; b 25 wt% yielded beads-on-a-string bers; c 30 wt% yielded bers with elongated
spindle shaped beads and d 35 wt% yielded nanobers. Reprinted with permission from Zong et al.
(2002)
be rened to form coaxial core-shell nanobers, e.g., made of an inner core of poly
(-caprolactone) and a shell of gelatin (Zhang et al. 2004).
Electrospinning does not necessitate an expensive setup and enables easy pro-
duction of nanoscaled polymer objets. Whatever, this process depends on several
processing and technical parameters that can be tuned in order to modify the
properties of the spun bers. Further, encapsulation during the formation of the
bers necessitates the drugs to be sufciently stable under the fabrication condi-
tions. Finally, when necessary, surface decoration by ligands should rather be
foreseen in a second step on the preformed suitably shaped particles.
2.2.2 Hard Templates Molding Techniques
Templates are widely used in industry for imparting specic shape and size to
various materials (Prez-Page et al. 2016). Prompted by electronics development,
very efcient lithographic methods are nowadays available for the production of
templates made of various sufciently rigid and stiff materials which can be used for
molding predened-shaped objects both at micro- and nanoscale.
Among these techniques the so-called patented PRINT technology (acronym
for Particle Replication IN non-wetting Templates) has been proposed by the group
of DeSimone et al., since 2004 (Rolland et al. 2005). As every molding technique,
the PRINT method is a multi-step molding technique based on the used of a
positive master mold. Nanoscaled templates exhibiting various shapes can be
obtained by photolithography on a silicon wafer. Then a perfluoropolyether (PFPE)
negative mold is imprinted on the master mold with nanometric features. Then a
polymer of interest is casted (or deposited in solution) in the PFPE mold under
pressure. Finally, the nanoparticles are removed by sticking them on an adhesive
polymer layer. Dissolution of the adhesive layer in water allows for the retrieval of
the nanoparticles (Fig. 10).
As for electrospinning, any solvent soluble or fusible polymer is eligible for the
PRINT process. For example PEG (Rolland et al. 2005), PLA and PLGA (Euliss
et al. 2006), as well as surface-pegylated cross-linked PEG hydrogels (Perry et al.
2012), have been already fabricated. This method offers the advantage of providing
a wide variety of shapes with dimensions as small as 20 nm. However, whatever
the shape it comprises mandatorily a flat surface because of the pressing of the
material in the nanocavities of the mold) and shapes have to be easily removable
from the mold. Furthermore, multi-step lling of the molds allows to create par-
ticles with multiple layers or anisotropic particles. Drug loading has been demon-
strated (drug, protein, fluorophore) provided that active ingredient stability
conditions can be attained under operating conditions. This process is very smooth
for the loading of a drug, because it requires no heating and no solvent out of the
dissolution of the adhesive layer. Being a multi-step process, mass production of
particles could be an issue, although production yields can be improved by
Fig. 10 Left flow chart of the particle replication in non-wetting templates (PRINT) method.
The methods comprises the following steps: (1) fabrication of a silicon master template (box,
upper left); (2) wetting of the silicon master with (green) liquid fluoropolymer, followed by curing
(top row); (3) preparation of a PFPE elastomeric mold reproducing the nanoscale features from the
master (upper right); (4) conning (red) organic liquid (melted polymer, polymer solution, or
ready to cure monomers mixtures) into cavities by applying pressure between mold and a flat
PFPE surface (middle row); (5) removal of nanoparticles from mold with adhesive layer (blue)
(bottom left); (6) dissolution of the adhesive layer, concentration and recovery of free particles
(bottom right). Bottom left examples of PEG micro and nanoparticles of complex shape prepared
by PRINT process. a 200 nm trapezoidal particles; b 200 nm 800 nm bar particles; c 500 nm
conical particles that are <50 nm at the tip; d 3 m arrow particles. Right scanning electron
micrograph images of particles fabricated using the PRINT method: a degradable 2 m cubic
particles; b 10 m magnetic hydrogel boomerangs; c 3 m hydrogel toroids; d 100 300 nm
hydrogel rods; e 200 nm cylindrical hydrogel particles; f 80 2000 nm lamentous hydrogel
particles. Reproduced with permission from Perry et al. (2011), Rolland et al. (2005)
continuous manufacturing, when using molding templates adapted on cylindrical

rolls. An other potential drawback of the method may result from the difculty to
control and to functionalize the surface of the particles during the manufacturing
process. The need for efcient washing and purication technique is also likely, due
to potential contamination of the surface of the nanoparticles by residuals of the
adhesive removal layer, which cannot be excluded too.
2.2.3 Micro- and Nanofluidics
Micro- and nanofluidics methods are based on the use of micro/nano channels to
form the particles either from a liquid polymer solution or in the melted state. By
forcing polymer drops slightly bigger than the size of the channel, it is possible to
strain each drop, one by one, in the channel. The drop is then solidied in an
enlargement of the channel and nally the nanoparticles are retrieved at the end of
the system (Dendukuri et al. 2005; Dendukuri and Doyle 2009). Figure 11 present
an example of an experimental microfluidic setup comprising the possibility to
rigidify the particles once they have been shaped. The nonspherically shaped par-
ticles can be further stabilized by post-deformation either by thermal cycling or by
photochemically induced cross-linking reactions within the particle.
Fig. 11 Formation of microspheres of complex shape through microfluidics; upper left a, A

representation of the flow focusing geometry used in the microfluidics droplet generator. The two
immiscible liquids A and B are forced into the narrow orice where the inner liquid core breaks to
release monodisperse droplet into the outlet channel, bc representations of the devices used for
producing photochemically and thermally solidied particles. The dashed rectangles mark the
flow-focusing device shown in part (a). The channels used for photochemical cross-linking were
elongated to allow longer durations or exposure of the droplets to UV light. For thermal settings
experiments, the flow focusing region was kept at a temperature exceeding the gelling (or solid
liquid phase transition) temperature (T0). The outlet channel was cooled to a temperature below T0,
and the droplets solidied as they traveled down the channel, (df) representations of the shape of
drops in the microfluidic channel. If the volume of the droplet exceeds that of the largest sphere
which could be accommodated in the channel, the droplet is deformed into a disk (e) or an
ellipsoid or a rod (f). Upper right experimental setup. Lower left exemples of SEM images of
nonspherical microspheres prepared by microfluidics a plug; b disk; c collection of plugs; and
d collection of disks. Reproduced with permission from Dendukuri et al. (2005), Dendukuri and
Doyle (2009)
Fig. 12 3D HFF microfluidic device for parallel synthesis of nanoparticles. Upper a device
visualized using red and blue food coloring dyes in the upper and lower microchannels,
respectively. PDMS was spin-coated thicker than the SU-8 post arrays (i.e., interconnecting holes
were closed) to isolate the channels. b Optical microscope image of the 3D HFF region. Here, red
and green food coloring dyes represent the PLGA-PEG polymer in acetonitrile solution and pure
acetonitrile, respectively. c Schematic illustration of one of the 8 parallel 3D HFF units in the
device. Bottom left nanoparticles sizes depending on the type of poly(lactide-co-glycolide)-
block-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles with sizes tunable in the range of 13
150 nm. Bottom right: SEM microphotograph of PLGA10 K-PEG5 K nanoparticles. Reproduced
with permission from Lim et al. (2014)
Figure 11 illustrates some of the attainable geometries via microfluidics, such as

plugs and disks in the micro-range. Even if the geometries are simple, the
microparticles can be easily and quickly mass produced in very smooth conditions.
The preparation of polymer nano-sized particles by this method has emerged only
recently, because of technological progresses in nanofluidics (Kleinstreuer et al. 2008;
Lim et al. 2014). Figure 12 depicts how a parallelized 3D hydrodynamic flow
focusing (3D HFF) device based on a multilayer microfluidic system, can be used for
producing PLA-b-PEG nanoparticles (Lim et al. 2014). Very interestingly, this
technique yields size-tunable nanoparticles within a 1245 nm range, depending on
the nature of the copolymer and experimental conditions. Despite having these
advantages, an issue of this method may arise from the need for an extreme purity of
the materials to be treated, in order not to obturate the channels during production. The
capability of shaping nanoparticles prepared by this technique may be a real issue

because of the expected fast rearrangement kinetics of polymer chains at nanoscale.
2.2.4 Film Stretching Method
The lm stretching method is a multi-steps technique which consists: (1) in preparing

(spherical) micro- or nanoparticles, (2) trapping them in a lm or a gel, (3) deforming
these particles by mechanically stretching the lm/gel before or after their liquefaction
by the action of heat or a solvent, nally (4) recovering the particles after dissolution of
the lm/gel in water, and (5) and concentration and purication of the particles water
suspension. Figure 13 shows the principle of this technique.
Fig. 13 Upper Principle of the lm stretching method introduced by Champion et al. (2007) and
used for making polymer particles with different shapes. Basically, the method implies the
following steps: a liquefaction of preformed spherical particles by using heat or a solvent (which is
also a nonsolvent of the lm forming material), b stretching the lm in one or two dimensions
under controlled conditions (deformation rate, temperature, etc.), c solidifying the particles by
extracting the solvent or cooling, and d dissolving the lm forming material, washing and
concentrating the particles in suspension. In the simplest conditions oblates are produced.
Adaptated from Champion et al. (2007) However, more complex shapes can be attained, e.g., by
double stretching or by introducing gaz (air) which could create voids around the particle. Lower
micrographs of PS micro- or nanoparticles made by single stretching with the following shapes:
(a) spheres. (b) Rectangular disks. (c) Rods. (d) Worms. (e) Oblate ellipses. (f) Elliptical disks.
(g) UFOs. (h) Circular disks. (Scale bars: 2 m.). Reproduced with permission from Champion
et al. (2007)
Despite its inherent complexity, this method is interesting when round-shaped

particles are looked for. As shown in Fig. 13 various modulations of shape are
feasible. Champion et al. (2007) have been working on developing it and have
achieved obtaining many shapes, characterized by their aspect ratio
( = length/width) from disks ( 1) to needles ( 1). They also have been
working on modifying the texture of their particles. However, they almost only used
so far polystyrene particles (non degradable), mostly at the micrometer level. The
method can be expanded to degradable polymers, including PLGA (Yoo and
Mitragotri 2010).
This method provides a wide variety of shapes. Interestingly, it offers an easy way
to prepare spherical particles, either nano- or micro-, made of the same material and
same volume, which is mandatory for the production of control particles necessitated
in experiments designed for understanding the effect of shape on pharmacokinetics,
cellular interactions, recognition phenomena, etc. However, these advantages have
to be modulated by the fact that the stretching method necessitates rather harsh
conditions (melting, solvents, and selection of a suitable polymer lm) and that
modication of the surface corona characteristics due to the difcult complete
elimination of contaminating materials during the step of particle harvesting. So far
this method has been mostly used for deforming polystyrene particles, but virtually
any polymer that can be melted could be deformed by this method. Whatever, as in
other deformation techniques, depending on its kinetics, shape-relaxation can be an
issue as shown by Yoo and Mitragotri (2010), who explored thermally induced
shape-reversion behavior of PLGA micro- or nanoparticles. Further, control of
surface properties at a molecular level (controlled grafting of ligands) could also be
an issue, due to direct contact of the particles surface with the lm forming material
during the deformation process. Indeed, the use of fragile and costly ligands could
hardly be achieved during the deformation process and therefore would have to be
undertaken in a second post-deformation step.
3 Shape Impact on Pharmaceutical Properties

and Pharmacokinetics
As described above, various manufacturing techniques are now available for

manipulating the shape of polymer nanoparticles intended for pharmaceutical
applications, each with pros and cons. Obviously, their development has been
prompted by the aim of investigating the possible effects of shape on their phar-
macokinetics following their delivery. Indeed, nonspherical shapes are expected to
considerably affect various phenomena, including: (1) motion in biological fluids,
(2) diffusion in tissues, (3) interactions with biological components including sur-
face fouling by adsorptive proteins, (4) recognition and ability to interact with cell
surfaces, (5) capacity to be entocytosed. Knowledge about the impact of shape on
these phenomena is still scarce despite a growing number of dedicated studies.
Indeed, there are various experimental difculties for assessing the role of shape at
nanoscale. Probably, one major issue is due to the difculty to design spherical
counter parts to nonspherical nanoparticles to be used as controls in experiments.
Indeed, if using the techniques discussed above makes possible to generate non-
spherical and control spherical particles made of the same material and with the
same equivalent diameters, some other characteristics of the particles are likely to
be different because of the shape and making difcult direct comparisons. For
example, the kinetics of drug release and drug degradation is likely to be consid-
erably affected when shapes modications result in considerable increases of the
specic surface of the objects. The presentation of recognition ligands, PEG chains,
etc., should be modied at the molecular scale, as well as their conguration and
dynamics.
3.1 Shape and Motion in Biological Fluids
Once they are delivered and whatever the route of delivery, nanoparticles are
dispersed in biological fluids. Still little is known about the impact of shape on their
motion. At the microscale level, platelets margination in blood is a well known and
studied phenomenon. Both different experimental (3D microfluidic channels) and
computational models have been elaborated to understand the mechanisms under-
lying the tendency of blood platelets (25 microns in diameter) to accumulate at the
endothelial walls in the vasculature. At the micron scale, few microns in size
prolates, or rods are generally suggested to have the higher propensity to migrate to
capillary walls because of their exclusion from the central regions of the blood flow,
mainly occupied by red cells (Thompson et al. 2013). Similarly, Serda et al. (2011)
suggested that hemispheric and discodal silicon microparticles had a higher ten-
dency to marginate, compared to spherical nanoparticles. Alternatively, the picture
is quite different at nanoscale, where modeling studies suggest that shapes modi-
cations (ellipsoids vs spheres) seem less efcient for ensuring the particles
margination (Mller et al. 2014). Finally, it should be noted that the scarcity of
experimental data about margination of nanoparticles in blood flow makes difcult
to give a more precise picture of the role of shape in nanoparticle motion in
biological fluids.
3.2 Shape and Interactions with Proteins and Other

Biological Macromolecules
Immediately after being in contact with biological fluids, surface fouling of

nanoparticles is known to occur. In blood, it consists in a selective adsorption of
plasmatic proteins, called opsonins, (and probably many other unstudied molecules)
at the surface of nanoparticles (Vauthier et al. 2010; Fornaguera et al. 2015). As a

result, the molecular architecture of the surface corona and its dynamics are
modied, which leads to mask or unmask the particles in front of the phago-
cytic cells of the immune system. In turn, these mechanisms are believed to dictate
their biodistribution as well as their persistence in the body (stealthiness). Even if
these phenomena have been extensively studied they are yet not fully understood
and the role of shape has emerged as an additional parameter to be evaluated in
depth. Indeed, protein adsorption phenomena at the surface of surface-anisotropic
objects (e.g., Janus nanoparticles) or multifaceted nanoparticles characterized by
sharp local curvature radii (oblates, ellipsods, needles) may be considerably dif-
ferent from what happens on plane surfaces. Recent experiments showed for
example that bovine serum albumin adsorption on carbon (single to multiwalled)
nanotubes decreased when the curvature of the tube was increased (Gu et al. 2015).
Thus, we suggest that shape could have a subtle impact on the bioimprint formed at
the surface of nonspherical particles in contact with biological fluids and that
regional modications in the topology of the (generally) hydrophilic corona sur-
rounding the nanoparticle could be expected.
3.3 Shape and Cell Adhesion
In the vast majority of targeting applications, nanoparticles are intended to adhere at

the surface of specic cells, while simultaneously not adhering and escaping to
others. Adhesion of nanoparticles necessitates a series of events, including colli-
sions with the cellular surface, creation of bonds between molecular partners and
nally, once the particle is attached, resistance to detachment when the particle is
sollicitated by shear forces in the body. Obviously, shape has a considerable effect
on these events as suggested by many biophysical models (Peng et al. 2015).
Decuzzi and Ferrari (2006) suggested that the probability of adhesion was con-
siderably raised for elongated particles compared to spheres. Further, the number of
intermolecular bonds formed between the two interactive partners is likely to be
considerably increased in the case of rods, oblates, platelets, and other elongated
particles, thus strengthening considerably the adhesive bond (not an additive
phenomenon).
3.4 Shape and Endocytosis by Cells
Once in contact with cellular membranes, nanoparticles are known to undergo

endocytosis, the intensity and the mechanisms of which have been discussed
elsewhere (Chap. 10 from Hillaireau). Indeed, shape can affect endocytosis.
Parakhonsliy et al. (2015), using anisotropic CaCO3 nanoparticles (spheres,
cubodal, elipsodal) suggested that elongated nanoparticles with the higher aspect
ratio were more easily endocytosed by HeLa cells. Huang et al. (2010) reported
similar trends using mesoporous silica nanoparticles with nanorods being much
easily endocytosed by cells of the melanoma A375 cell line. Such aspect
ratio-related differences could stem from different endocytosis mechanisms.
Spherical nanoparticles could favor the clathrin-dependent pathway while elongated
nanoparticles would prefer the caveolae-dependent pathway (Hao et al. 2016).
However, Li et al. (2015) have recently published opposite results using a
Dissipative Particle Dynamics (DPD) model for simulating the endocytosis of
differently shaped pegylated nanoparticles. The rate of endocytosis was the highest
for spherical nanoparticles and decreasing when geometry was altered as follows:
sphere < cubes < rods < disklike nanoparticles. This was attributed to the fact that
nanoparticles wrapping by cellular membranes necessitate minimal bending energy
compared to the case with higher aspect ratios.
3.5 Shape and Biodistribution
Very probably, many parameters involved in nanoparticles biodistribution, or at

least their real impact on distribution, are still unknown. This is the reason why a
comparative and mechanistic interpretation of biodistribution studies aiming to
compare differently shaped nanoparticles is difcult and may lead to contradictorily
conclusions. Nethertheless, despite the scarcity of experimental data, few studies
suggests on how much shape could strongly affect nanoparticles biodistribution. In
a pioneering study, Decuzzi et al. (2010) showed that uncoated nonspherical
silicon-based nanoparticles, including hemispheres, cylinders and discods, accu-
mulated differently depending on the shape in the organs of tumor bearing mice.
Discoidal particles accumulated more than others in most of the organs except the
liver. Later, Van de Ven et al. (2012) have evidenced that quite large platelod
nano-sized particles (1000 400 nm) could preferentially accumulate in tumors in
a melanoma mice model, probably because of a much higher propensity of platelets
to adhere to tumoral microvasculature compared to other shapes and sizes. More
recently, Kay et al. (2015), showed that elongated nanoparticles made of a
PEGylated hydrogel could considerably improve the pharmacokinetics of cisplatin,
simultaneously increasing circulation duration and favoring tumor accumulation.
These different examples suggest that nanoparticles shape modications combined
to specic surface engineering (e.g., pegylation) are likely to strongly affect their
distribution in the body, with possible interest for drug targeting applications.
However, accumulation of experimental observations would be highly desirable for
gaining a better understanding of the intricated mechanisms leading to modulations
in the biodistribution and pharmacokinetics of nanoparticles and associated drugs.
4 Conclusions and Perspectives
There are more and more evidences that shape of nanoparticles is a critical
parameter that could considerably affect their performances, including not only their
pharmacokinetics or the one of the associated drugs, but also very likely their
toxicokinetics, as well as their pharmaceutical characteristics. We have described a
broad set of methodologies enabling the production of nonspherical polymer
nanoparticles, all of which having specic advantages and drawbacks. As it has
been shown, they can be classied into two categories, mainly self-assembly of
tailored amphiphilic copolymers and preparation through constraint methods.
Clearly, self-assembling techniques necessitate the selection and the production of
suitable building blocks able to induce particle anisotropy. Although polymer
chemists are nowadays able to conceive and to precisely synthesize very sophis-
ticated polymers, we suggest that the search for bioinspired building blocks,
including peptides or polypeptides, could be an interesting alternative in the future.
Whatever, self-assemblying offers equally the opportunity of a precise tuning of the
surface of the particles at the molecular scale. At the opposite, deformation tech-
niques may suffer from a lack of control of surface properties but can be envisioned
for large varieties of commonly used polymer materials. Lithographic-based tech-
niques offers equally access to highly monodisperse specically shaped nanopar-
ticulate populations, which may represent a considerable advantage as it would be
expected to minimize variability.
Whatever, much knowledge has to be gained now on how shape of nanoparticles
behaves in the body. In this respect the recent progresses and developments in
preparation methods making possible to design specically shaped nanoparticles
provide a huge opportunity to focus on the role of shape in the performances of
particles. What is the effect of shape on toxicity, biodegradability, drug loading and
release kinetics, diffusibility in organs, intracellular trafc and possibly yet unre-
vealed phenomena? Obviously, many ndings can be still expected. To conclude,
we believe that shape will have to be fully considered as a morphological char-
acteristic as important as particle diameter and surface chemistry in the conception
and development of future nanomedicines.
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Part II
Characterization of Polymer Nanoparticles
Designed as Nanomedicines
Chapter 7
Physicochemical Characterization
of Polymer Nanoparticles: Challenges
and Present Limitations
Jeffrey D. Clogston, Rachael M. Crist and Scott E. McNeil
Abstract The biocompatibility, including aspects such as biodistribution, clear-

ance, and immunotoxicity, of a nanoparticle depends upon its physicochemical
properties. Characteristics such as size, charge, and hydrophobicity are well-known
parameters influencing the biological compatibility of in vivo administered
nanoparticles. Measurement and evaluation of these and other parameters, however,
are not always straightforward. This chapter describes six critical areas for
nanoparticle characterization, especially as pertaining to polymer nanoparticles
intended as therapeutics: starting polymer characterization, nanoparticle size,
nanoparticle surface properties, drug loading and release, nanoparticle stability, and
batch-to-batch reproducibility. The challenges and limitations of the most common
techniques used in assessment of these parameters are described.
Keywords Nanoparticles Physicochemical characterization Polymer nanopar-

ticles Polymers
1 Introduction
The importance of physicochemical characterization and the effects of nanoparticle

characteristics on biocompatibility are well established (Gatoo et al. 2014; McNeil
2009). For example, particle size is known to influence biodistribution and clearance
routes (Kobayashi and Brechbiel 2003). Particle charge is known to affect hema-
tocompatibility (Dobrovolskaia et al. 2012a, b). Surface modications (or lack
thereof) have been shown to affect biocompatibility and increase toxic side effects
(Adiseshaiah et al. 2010; Holgate 2010; Paciotti et al. 2004). Even slight changes in
any of these parameters can have a detrimental effect on the efcacy of the nano-
medicine, or worse, lethally increase toxicity. Because small changes in nanofor-
J.D. Clogston R.M. Crist S.E. McNeil (&)

Nanotechnology Characterization Laboratory, Cancer Research Technology Program,
Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research,
Frederick, MD, USA
e-mail: ncl@mail.nih.gov

DOI 10.1007/978-3-319-41421-8_7
188 J.D. Clogston et al.
mulation traits could have devastating effects, rigorous physicochemical character-

ization will be a necessity for successful clinical translation of a nanomedicine.
Furthermore, this is a required part of the regulatory process. A thorough Chemistry,
Manufacturing, and Controls (CMC) section is required for every Investigational
New Drug (IND) application with the Food and Drug Administration in the United
States of America (FDA).
Polymers and polymer nanoparticles, because of their diverse physical and chemical
properties and broad functionality, are continuing to see increased applications in a
variety of elds. The biomedical eld, for example, has experienced a tremendous
impact and growth over the last few decades from the implementation of polymer-based
formulations, including as cancer treatment options (Davis et al. 2008; Duncan 2006;
Kamaly et al. 2012; Petros and De Simone 2010; van Vlerken and Amiji 2006; Jain
2010). Polymer-protein conjugates and polymer-drug conjugates have both seen success
in the clinical phase and in receiving regulatory approval. Sigma-Taus Oncaspar, a
polymer-protein conjugate (PEG-L-asparaginase), was approved by the FDA 20 years
ago for the treatment of acute lymphoblastic leukemia, and Astellas Pharmas SMANC,
a styrene-maleic acid (SMA) copolymer and neocarzinostatin (NCS) polymer-protein
formulation, was approved by the Japanese Ministry of Labor, Health and Welfare in
1994 for the treatment of hepatocellular carcinoma (HCC) (Jain 2010; Ishii et al. 2003).
The rst controlled release polymer (poly(D,L-lactide-co-glycolide (PLGA)) copolymer)
formulation, AstraZenecas Zoladex, was approved by the FDA nearly 25 years ago
for the treatment of prostate cancer.
There has also been steady growth in the research and application of
polymer-based nanoparticles for parenteral administration (Duncan 2006). For
example, BIND Therapeutics BIND-014, a docetaxel containing poly
(lactide-co-glycolide) (PLGA)poly(ethylene glycol) (PEG) polymer-drug conju-
gate, is in Phase II clinical trials for advanced non-small cell lung cancer
(ClinicalTrials.gov Identier NCT01792479) and prostate cancer (NCT01812746).
Cerulean Pharmas CRLX101, a cyclodextrin polymer loaded with camptothecin, is
in Phase II clinical trials for recurrent small cell lung cancer (NCT01803269) and
ovarian, fallopian tube, and primary peritoneal cancer (NCT01652079). There are
also several other polymer nanoparticle conjugates at various stages of clinical
evaluation (e.g., CTI Biopharmas CT-2106 and CT-2103, and Enzons
EZN-2208), and countless others in various preclinical stages of development.
Regardless of polymer composition, synthesis route, or nanoparticle type reliable
and relevant physicochemical characterization (PCC) is essential for translational
progression of the formulation and a vital requirement for regulatory approval
(Davis et al. 2008; Duncan 2006; Kamaly et al. 2012). Years of research suggest the
key parameters affecting nanoparticle biodistribution and biocompatibility are size,
surface characteristics (i.e., charge, targeting ligands, PEGylation, etc.), and drug
release (Davis et al. 2008; Kamaly et al. 2012; Bertrand et al. 2014; Singh and
Lillard 2009). In addition, a thorough evaluation of formulation stability (i.e.,
storage stability, plasma stability, etc.) and batch-to-batch consistency are imper-
ative for long-term assessment of the formulation (Clogston and Patri 2013).
7 Physicochemical Characterization of Polymer Nanoparticles 189
This chapter will focus on the essential PCC requirements of polymer

nanoparticles, especially as pertaining to the eld of nanomedicine. As outlined in
Fig. 1, we will discuss six of the most critical characterization areaspreliminary
characterization of the polymer starting material, as well as size, surface, drug
Fig. 1 Characterization requirements for polymer nanoparticles. Physicochemical properties of a

nanoparticle can greatly influence its biological properties. Therefore, it is crucial to thoroughly
characterize the formulation. For polymer nanoparticles, characterization should begin with the
starting polymer, and include features such as purity, molecular weight, and polydispersity.
Characterization of the polymer nanoparticle should include aspects such as size, surface, drug
loading and release, stability, and batch-to-batch consistency. Some commonly used
techniques/instrumentation to assess each parameter are provided
loading and release, stability, and batch-to-batch consistency of the nanoformula-

tion. We will briefly highlight the most common analytical techniques and instru-
ments used for assessment of each of these parameters, as well as discuss the
challenges, difculties, and limitations associated with each.
2 Starting Material Characterization
Nanoparticle characterization, especially polymer nanoparticle characterization,

should begin with analysis of the starting materials. Typical analysis of a polymer
often begins with purication to remove unreacted monomers, side products, or
undesired molecular weight fractions, as well as measurements to determine
average molecular weight and polydispersity. It is important to evaluate these
properties whether the polymers are synthesized in house or purchased from a
commercial source. It is commonplace for researchers to assume a commercial
polymer is exactly as advertised, an assumption that can and should be avoided.
Polymers, depending on their synthesis, can have a broad molecular weight
distribution (and hence polydispersity) and include various impurities in the form of
lower and/or higher molecular weight species. The nal polymer can be character-
ized in a number of ways (Barman et al. 2009; Chen et al. 2003; DAvila Carvalho
Erbetta et al. 2012; Inkinen et al. 2011; Kou et al. 2009; Laguna et al. 2001; Vandijk
et al. 1983; Zhou et al. 2004). For example, characteristics like molecular weight and
molecular weight distribution (polydispersity) can be assessed using analytical
techniques such as gel permeation chromatography (GPC), dilute solution viscom-
etry, and multi-angle laser light scattering (MALLS). Composition, specically
end-group analysis, can be analyzed by nuclear magnetic resonance (NMR) and
Fourier transform infrared spectroscopy (FTIR), and impurities (e.g., metal catalysts)
can be assessed using inductively coupled plasma mass spectrometry (ICP-MS) and
GPC. Melting temperature, glass transition temperature, and thermal stability can be
evaluated by thermogravimetric analysis (TGA) differential scanning calorimetry
(DSC). Although it is not always practical to measure each of these parameters, it is
important to measure parameters that are sensitive to changes in reaction conditions,
monomer purity, and scale-up. At a minimum, this should include validation of the
molecular weight, polydispersity, and purity of the polymer.
One of the most common techniques to measure molecular weight is gel per-
meation chromatography (GPC) using a suitable molecular weight standard. This
approach is typically favored because it is based on instrumentation commonly
found in polymer laboratories and is relatively inexpensive as compared to other
techniques/instruments. Briefly, standards of various molecular weights, most
commonly polystyrene standards, are passed through a suitable column and their
retention times measured through UVVis, refractive index, viscometry, or light
scattering detection. A calibration curve is constructed using the logarithm of
molecular weights versus elution times, and the polymer sample molecular weight
is determined (Striegel et al. 2009; Trathnigg 1995; ASTM D5296). It is important
to note that the sample polymer should be run under identical conditions as the
standard polymer. The limitations of this approach, however, are assuming that the
polymer sample interacts with the column in the same manner as the standard, and
that the polymers conformation is the same as the standard. If either of these
assumptions is untrue, this approach could lead to inaccurate molecular weight
estimations.
Alternatively, GPC coupled with a multi-angle laser light scattering (MALLS)
detector can be used to determine the absolute molar mass of a polymer (Cheng
et al. 2009; Xie et al. 2002). This approach is based on rst principles, meaning no
calibration standards or polymer conformation information are needed. Typically
GPC-MALLS uses refractive index (RI) detection, and the change in refractive
index with change in concentration (dn/dc) of the polymer must be available. If
unknown, the dn/dc value can be measured, but must be measured under the same
solvent conditions and temperature as the intended sample run. The wavelength of
the laser in both the RI and MALLS detectors should also be very similar. This
approach is made even more powerful coupled with a viscometer. From the intrinsic
viscosity and MALLS data, details of polymer structure (i.e., degree of branching)
can be modeled (Mark Houwink analysis) (Gaborieau and Castignolles 2011;
Walkenhorst 2001). Detectors such as evaporative light scattering detector (ELSD)
or charged aerosol detector (CAD) can be used to increase the detection sensitivity
when RI or UVVis detection are not sensitive enough. For example, size exclusion
chromatography (SEC) coupled with CAD is used to detect the presence of PEG
dimers, wherein other detection methods are insensitive (Kou et al. 2009).
Either approach will afford information on the molar mass of the polymer and its
polydispersity. Purity as dened by the presence of unreacted monomers, reagents,
and reaction side products is better suited to reversed phase high performance liquid
chromatography (RP-HPLC) separation. RP-HPLC can also be used for the sepa-
ration of different end-group functionality polymers, whereas GPC would be
ineffective. For example, RP-HPLC coupled with ELSD was used to track the
presence of a PEG-OH impurity in the formation of the desired mPEG-OH product
(Barman et al. 2009).
Ultimately, if impurities are present, they could end up in the nal nanofor-
mulation, potentially causing unexpected adverse effects. Deviations in the
molecular weight of the polymer could affect the size of the nanoparticle and drug
loading, which in turn could affect safety and efcacy of the nanomedicine.
End-group functionality impurities may also affect drug loading capacity in
polymer-drug conjugates. As will be discussed later, batch-to-batch reproducibility
of the nanoparticle is a critical part of the development process. This is nearly
impossible to achieve without a consistent starting polymer.
3 Size
One of the most fundamental measurements in nanoparticle characterization is size.

Size measurements can be broadly categorized into two types, light scattering and
imaging. Light scattering techniques include dynamic and static light scattering and
laser diffraction (LD). Imaging techniques include scanning electron microscopy
(SEM), transmission electron microscopy (TEM), and atomic force microscopy
(AFM). Size can be dened in many ways, and the sizing technique will depend on
which information is desired. For example, dynamic light scattering
(DLS) measures the hydrodynamic size, i.e., the size as dispersed in a liquid
medium or the hydrated size. TEM measures the electron-dense portion of the
nanoparticle. Sizing analysis of a PEGylated nanoparticle, for example, will provide
different results depending on the technique used. Typically, the hydrodynamic size
by DLS will be larger than the core size by TEM.
Dynamic light scattering (DLS), also referred to as photon correlation spec-
troscopy (PCS) or quasi-elastic light scattering (QELS), is the most convenient and
fastest method for measuring size of nanoparticles (Singh and Lillard 2009; Lal Pal
et al. 2011). It should be noted that DLS actually measures the diffusion coefcient
and calculates the hydrodynamic size indirectly (Hackley and Clogston 2011).
Briefly, the nanoparticle dispersion is irradiated by a monochromatic laser and the
intensity fluctuations of the scattered light are measured as a function of time. These
fluctuations are the result of the Brownian motion of the nanoparticles which are
converted to a correlation coefcient and plotted as a function of time. This curve is
tted to an appropriate model which yields the diffusion coefcient, and the Stokes
Einstein equation is applied to calculate the hydrodynamic size. Of note, most DLS
instruments typically calculate the diffusion constant using a model valid only for a
spherical particle. If the tested nanoparticle deviates from this shape, the calcula-
tions will not provide accurate results. DLS theory is described in more detail in a
book by Berne and Pecora (2000). Details on sample preparation, sample mea-
surement, relevant standards, and data analysis are published elsewhere (Hackley
and Clogston 2011; NIST & NCL protocol).
There are two general types of measurements, batch-mode DLS, where the bulk
sample is measured as is, and flow-mode DLS, in which the DLS is used as a
detector in a separation technique. For example, polydispersed size distributions as
measured by batch-mode DLS can exist as either a single broad peak which may
cover a size range from 100 to 1000 nm or multiple peaks with different size
populations. While the volume-weighted distribution plots can be used to help
determine the major size population, batch-mode DLS cannot ascertain which size
population is responsible for any observed biological outcomes. Similarly, it offers
no information on which size population contains the drug or targeting ligand in the
case of inhomogeneous drug loading/distribution. Separation techniques, typically
SEC or asymmetric flow eld-flow fractionation (aFFFF), can be coupled with DLS
(and other detectors such as UV-Vis, RI, MALLS) to help address these areas of
concern (Clogston and Patri 2013). Using this type of system, the hydrodynamic
size, radius of gyration, purity, and molecular weight can all be determined for the
fractionated sample, and each individual size population can be subjected to a
relevant bioassay to ascertain the bioactive fraction (Messaud et al. 2009; Yohannes
et al. 2011; Sahin and Roberts 2012).
There are several practical issues associated with DLS measurements. The
intensity of scattered light is proportional to the size raised to the sixth power. Thus,
larger particles scatter more light. As a result, DLS measurements are biased toward
larger particles, and smaller particles may not be detected unless present in sig-
nicant amounts. DLS also has resolving issues; it cannot baseline resolve particles
unless they are approximately 35 times different in size. In this case, DLS provides
an average size of these populations. Sample preparation issues such as ltration,
dilution, and dispersing media can also influence measurement results. And, there
are nanoparticle-specic issues such as multiple scattering, viscosity effects, and
absorbance issues. Although these are very common in DLS, they are often not
recognized. There are also inconsistencies regarding which size measurement to
report: intensity-weighted, volume-weighted, number-weighted, or Z average
(Z-Avg). Briefly, the intensity-weighted distribution should normally be used for
the size measurement, while the volume-weighted distribution should be used to
assess relative amounts of the different size populations. A more detailed discussion
of these issues is offered by Clogston and Patri (2013).
Another aspect of concern for size measurement is assessment under biologically
relevant conditions (Treuel et al. 2014). PBS (pH 7.4, 134 mM salt concentration)
is often used to mimic physiological conditions, but this cannot adequately repre-
sent the nanoformulation in the presence of proteins. Blood cannot be used as a
dispersing media because the micron-sized red and white blood cells dominate the
DLS signal relative to the nanoparticle. Rather, plasma or serum (plasma without
clotting factors) has been used. DLS analysis of plasma or serum alone, results in a
multimodal size distribution (intensity-weighted distribution) with the majority of
the sample being <10 nm (volume-weighted distribution), making reliable and
conclusive size measurement of nanoparticles challenging (Clogston, unpublished
data). When whole plasma or serum has been used, the number-weighted distri-
bution is typically reported rather than intensity-weighted distribution. In general,
reporting number-weighted distributions should be avoided due to several
assumptions that go into derivation of the number plots (Clogston and Patri 2013;
Hackley and Clogston 2011). Most attempts at measuring nanoparticle size in
plasma and serum are in a diluted solution of plasma or serum, for example 10 %
(w/w) in PBS. While this approach circumvents the issues of plasma proteins
dominating the DLS signal, it still may not provide a true biological assessment of
the size. Another approach to making physiological measurements has been to
increase the nanoparticle concentration such that, from a light scattering perspec-
tive, it becomes the dominant species in the sample. However, this often results in
atypically high nanoparticle concentrations, and the measurement thus deviates
from the intended biologically relevant assessment.
Other light scattering techniques, static light scattering and laser diffraction, also
provide size information but are not as common as DLS. Static light scattering measures
the intensity of the scattered light as a function of scattering angle. The time scale for
static light scattering is on the order of milliseconds, compared to microseconds for
DLS. The radius of gyration (Rg) is determined based on the dependence of scattered
light intensity with scattering angle. The Rg, or root mean square (rms) radius, is the size
weighted by its mass distribution (of all point masses within nanoparticle) about its
center of mass (Buchholz and Barron 2001; Xu 2015). Laser diffraction is similar to
MALLS in that it measures the intensity of the scattered light as a function of scattering
angles, but uses Mie scattering theory to calculate the volume equivalent sphere
diameter (ISO 1332; U.S. Pharmacopeia General Chapter <429>).
As with any measurement, each of these techniques suffers from discrete limi-
tations. Static light scattering is not suitable for nanoparticles below 20 nm.
Nanoparticles smaller than 20 nm are isotropic scatterers at the millisecond time-
frame, meaning they have no angular dependence and Rg cannot be calculated.
Laser diffraction is generally used for particles greater than one micron, but requires
large sample volumes. Furthermore, laser diffraction requires knowledge of the
nanoparticles (NOT the dispersing mediums) refractive index and accurate
understanding of the imaginary (i) component.
Size analysis of nanoparticles can also be done with imaging techniques as well,
including TEM. There are a variety of electron microscopy techniques such as
cryogenics, three-dimensional tomography, and energy dispersive X-ray spec-
troscopy that can provide additional particle information such as shape, morphol-
ogy, and elemental composition. This can be an added benet over DLS, which
uses assumptions about the particle shape and only provides hydrodynamic size
information.
TEM has its own set of issues and nuances. For negative staining, the drying
process has potential to introduce artifacts such as sample agglomeration. This may
result in images and sizes of the nanoformulation that are unrepresentative in the
biological setting. Furthermore, size analysis by negative stain TEM is a
number-based measurement in which an operator counts and measures individual
particles. It is imperative to measure a sufcient number of particles such that it
accurately reflects the entire sample population, being sure to eliminate the possibility
of any operator prejudice when imaging and measuring particles. Cryo-TEM, while
touted as providing more of a natural state image of the nanoparticle, also has sample
preparation issues. Specically, many nanoformulations have added cryoprotectants
such as sucrose or trehalose, making cryo-TEM difcult or even impossible.
There are several papers that compare and contrast various methods for measuring
nanoparticle size, including techniques not described in detail here such as SEM,
AFM, analytical ultracentrifugation, and nanoparticle tracking analysis (Bootz et al.
2004; Filipe et al. 2010; Troiber et al. 2013). Because each sizing technique analyzes
the particle differently, at least two techniques should be used to measure size. This
helps cross-validate measurements and may also add insight to the polydispersity of
the sample. Separation techniques can also be employed to gain further insight into
the various populations present. A review by Gaumet et al. (2008) nicely illustrates
the need for multiple sizing techniques, highlighting the importance of adequate and
thorough PCC data for evaluation of in vivo study results.
4 Surface Characteristics
A thorough understanding (characterization) of the surface is required to ensure the

intended safety and efcacy of a nanoformulation, since surface characteristics of a
nanoparticle will dictate its biological performance, i.e., biodistribution and toxicity
(Moghimi et al. 2012). The most common attributes in dening the nanoparticle
surface are its charge (zeta potential), hydrophilic character (surface modied with
hydrophilic polymers such as PEG), and the presence of targeting ligands.
Zeta potential is dened as the potential difference between the bulk solution and
the slipping plane (boundary where nanoparticle and associated ions act as a single
entity and move under an applied electric eld). Zeta potential measurements are
based on light scattering and laser doppler velocimetry; the frequency shift of the
scattered light from the nanoparticle moving under an applied eld is used to
measure its electrophoretic mobility. This in turn is used to calculate the zeta
potential using an appropriate model (Hackley et al. 1995; Fairhurst and Lee 2011;
Fairhurst 2013). Of note, zeta potential is not a direct measurement of the surface
charge, but is used as an indication of the actual surface charge of the nanoparticle.
Zeta potential is highly dependent on measurement conditions such as conductivity
(ionic strength), pH, temperature, and solvent. Therefore, when using zeta potential
as a quality control or comparison parameter it is paramount to make measurements
under identical conditions. Details on the practical issues associated with zeta
potential are described elsewhere (Clogston and Patri 2011, 2013).
The challenges in zeta potential are similar to those described for DLS, as zeta
potential is also based on light scattering. The measured zeta potential value is an
average of all charged species in the sample, and like DLS, has limitations in
resolving power. Thus the results can be misleading if the sample is polydispersed.
Zeta potential measurements in a biological matrix (i.e., plasma or serum) are also
equally as complex. Proteins in either matrix are zwitterionic. Differentiating which
species (e.g., nanoparticle, protein, or protein-nanoparticle complex) contributes
most to the zeta potential value is very difcult in a batch-mode measurement.
Separation techniques can similarly be employed to measure the zeta potential for
different populations present in a formulation, although this is rarely described in
the current literature. Dilute sample concentrations following column purication
(dilution) often make these measurements difcult.
Surface modications typically include the addition of hydrophilic polymers and
targeting ligands. Hydrophilic polymers are used to help increase circulation time
and decrease opsonization (Singh and Lillard 2009). The most common polymer
used for this purpose is poly(ethylene glycol) (PEG), and is often used in combi-
nation with copolymers, i.e., PLGA-PEG. Targeting ligands such as antibodies and
their fragments, proteins, peptides, small molecules (e.g., folic acid), and nucleic
acids have been conjugated to polymer nanoparticles to enhance specicity and
efcacy (Kamaly et al. 2012; Bertrand et al. 2014; Chow 2013).
Surface characterization is one of the biggest challenges for nanoparticles, and
there are a plethora of challenges to consider. The density, distribution, and
presentation of the coating and targeting ligand can all be important (Adiseshaiah
et al. 2010). Yet, there are no straightforward means of quantitating these parameters.
Because of the difculty in assessing these, it is often assumed that surface coating is
uniform, or the targeting ligand is both bound to the surface and accessible for binding
to the appropriate receptor. The surface moieties may have varied densities and
distribution, resulting in heterogeneous nanoparticle populations with altered
biodistribution and biocompatibility. Furthermore, the activity and stability of a
targeting ligand may be different when bound to the nanoformulation, the active site
may become inaccessible, or the ligand may cleave prematurely before reaching the
intended site. Biological assays of nanoparticles with and without targeting ligands
are helpful in providing evidence of targeting ligand accessibility and presentation.
There are many approaches that could evaluate desired target-site binding and
protein interactions. Analytical techniques/instrumentation such as isothermal
titration calorimetry (ITC) (Freyer and Lewis 2008; Ghai et al. 2012), Archimedes
(Burg et al. 2007; Godin et al. 2007), quartz crystal microbalance with dissipation
monitoring (QCM-D) (Delcroix et al. 2014; Dixon 2008), Nanopore-based detec-
tion through tunable resistive pulse sensing (TRPS) (Anderson et al. 2013; Kozak
et al. 2012; Roberts et al. 2010; Willmott et al. 2010), dual polarization interfer-
ometry (DPI) (Hu et al. 2014; Swann et al. 2004), and surface plasmon resonance
(SPR) (Daghestani and Day 2010; Petryayeva and Krull 2011; Sonesson et al.
2007) all use different principles for assessing protein/ligand binding. However
robust and broadly applicable methods based on these and others are still lacking
for nanoparticles.
Although techniques to address issues such as heterogeneity and presentation of
surface moieties are limited, quantication of surface conjugates has been slightly
more fruitful. Quantication of the total amount of PEG (or any other polymer for
that matter) and the targeting ligand can be achieved using RP-HPLC. Using
appropriate conditions, the polymer nanoparticle conjugate is disrupted such that
the individual components can be detected and quantied. UVVis or fluorescence
is routinely used for detection and quantication of targeting ligands, depending on
the spectral properties. Mass spectrometry can also be employed for better sensi-
tivity. Detection and quantication of many polymers is better suited with ELSD or
CAD when UVVis detection is not sensitive enough. This is especially true for
PEG which contains neither a chromophore nor a fluorophore. Thorough reviews of
additional techniques that can be applied to PEG/polymer characterization are
offered by Rabanel et al. (2014) and Baer et al. (2013).
5 Drug Loading and Drug Release
One of the most widely explored applications for nanotechnology is for drug delivery.
Drug loading in polymer nanoparticles can be achieved two ways: (1) encapsulation
of the drug (non-covalent) within the polymer nanoparticle, or (2) attached to the
polymer backbone (covalent, with cleavable linkers). Encapsulation during
formation is the most common method employed for hydrophobic drugs. The drug
can be encapsulated during nanoparticle formation or loaded post-formation (i.e.,
adsorption) (Kamaly et al. 2012; Singh and Lillard 2009).
Regardless of the method used for loading, the total drug is typically quantied
by RP-HPLC. Similar to the approach for quantifying surface ligands, the
nanoparticles are disrupted under appropriate RP-HPLC conditions, the drug sep-
arated from the other polymer components, and detected using an appropriate
detector based on its spectral properties. UVVis or fluorescence detectors can be
used in most cases, although mass spectrometry, ELSD, and CAD can also be
employed to enhance sensitivity.
Drug loading and drug release (as well as other properties) have also been
measured using florescent tags. However, if the fluorescent tag is not a part of the
nal nanoformulation, this approach is strongly discouraged. Methods should
always be developed for characterization of the intended nal clinical formulation.
Characterization using a fluorescently labeled formulation would not be accepted as
part of the required regulatory CMC, due to overarching concerns the tag (or some
fraction thereof) may be non-covalently associated which would readily dissociate
upon administration/testing, may cleave upon administration, or may change the
formulations biological properties.
In addition to drug loading, drug release should also be examined, optimally under
physiological conditions, considering factors such as media, pH, temperature, sam-
pling time and volume. Initial measurements in PBS at 37 C can be made during the
optimization process, but release proles in plasma are highly recommended and
preferred. Using plasma not only assures physiological ionic strength and pH, but
also assesses how the nanoparticle formulation interacts with plasma proteins.
There are several techniques that can be used to monitor drug release. The
method of choice depends on each individual formulation and how easily the
released drug can be separated from the nanoparticle. Equilibrium dialysis, ultra-
centrifugation, ultraltration, and diffusion cells are among the most common
methods employed for drugs that are encapsulated (Singh and Lillard 2009).
Dialysis tends to be the most common approach as solubility issues (most drugs are
hydrophobic) can be resolved by performing release experiments under innite sink
conditions. Free drug concentration (i.e., drug release) can then be determined using
the same or a similar method as employed for total drug concentration. An addi-
tional level of complexity is added when using plasma as the media source. The
released drug can bind to plasma proteins resulting in several drug fractions (e.g.,
encapsulated drug, unencapsulated drug, unencapsulated and protein-bound drug)
and complicating the analysis. Bekersky et al. (2002) have developed an ultral-
tration method to assess the amount of total drug released in free form (nonprotein
bound) and protein-bound form. The difculty with this method is that
nonprotein-bound drug quantities will be very low in the ltrate. Most drugs will be
protein bound and remain in the retentate. Low drug quantities could lead to drug
detection sensitivity issues and ultimately erroneous concentrations. A good review
on the different methods used for evaluating drug release in plasma, including their
advantages and disadvantages is offered by Ambardekar and Stern (2015).
For drugs conjugated to the polymer backbone, measuring release in plasma is

crucial. It is important to mimic biological conditions to monitor enzymatic or
pH-mediated cleavage of the drug from the backbone and measure hydrolysis rates.
The site of cleavage as well as the activity of the cleaved drug must also be
conrmed. Measurement of the cleaved drug as a function of time by RP-HPLC is
typical. One challenge to assessing drug release in plasma is that longer incubation
times may result in degradation of the drug product (e.g., enzymatic hydrolysis of
ester linkages) resulting inaccurate drug concentrations. A possible approach to
circumvent these artifacts is to measure depletion of the intact polymer-drug con-
jugate over time, as opposed to measuring accumulation of the cleaved drug
product. An intact polymer-drug complex will elute differently than the cleaved or
compromised drug product using RP-HPLC. Preparation-induced artifacts intro-
duced during the in vitro sample handling process, as well as some possible work
around methods are described in a manuscript by Stern et al. This manuscript also
describes a pharmacokinetic modeling approach to estimating drug concentrations
that cannot be directly measured (Stern et al. 2013).
6 Stability
In addition to the quantitative measurements of the formulation components, sta-

bility and batch-to-batch consistency of the sample are also invaluable assessments
of the materials potential clinical viability. Stability, short term and long term,
dictates the shelf life of the polymer formulation. Storage conditions such as sol-
vent, temperature, pH, and form (e.g., in solution, lyophilized powder, frozen)
should be examined. For example, freezing and/or lyophilization of the sample may
compromise the integrity of the nanoparticle. Certain solvents or buffers may cause
nanoparticle precipitation or premature release of the drug. Sample handling pro-
cedures such as freeze/thaw cycles, lyophilization, centrifugation, and ltration
should be tested for their effects on nanoparticle integrity as well. Especially as it
relates to clinical translation, the stability of the sample in plasma or serum is
critical and must be assessed prior to in vivo examination of the nanoparticle. If the
nanoparticle aggregates in biological media, in vivo use can cause fatal embolisms.
Furthermore, specialized reconstitution procedures and shelf life after reconstitution
are important considerations.
Measurement of any number of parameters can be used to assess stability and the
effects of storage conditions or sample handling procedures. For example, size
measurements can be used to study the effects of ltration or lyophilization, but
would be insensitive to drug loading/release. A more thorough analysis should
include both size and drug loading before and after the ltration or lyophilization
process. While size, drug loading, and drug release are all important and recom-
mended measurement parameters to assess short and long-term stability (i.e., shelf
life), ideally one should also include parameters that have a direct effect on a
biological outcome. For example, nanoparticle zeta potential may be monitored to
avoid possible complement activation in vivo. Alternatively, an in vitro biological

assay could also be used to predict the likelihood of in vivo complement activation.
Several other examples are described in a perspective by Crist et al. (2013).
7 Batch-to-Batch Consistency
Batch-to-batch consistency is achieved by obtaining consistent results and mea-

surements in each of the characterization areas described above. Again, choosing
the relevant assessment parameters (i.e., lot release criteria) that relate to a desired
in vivo outcome is critical. This could simply be the size, zeta potential, and total
drug loading of the nanoparticle formulation, and/or include an in vitro biological
assay. If the drug is conjugated to the polymer backbone, release/cleavage rates
may also be needed to ensure batch-to-batch consistency. This is especially
important when using a previously untested batch of the polymer-drug conjugate.
Once dened, these parameters can be used as lot-to-lot release specications.
Batch-to-batch consistency can be an even bigger challenge for polymer
nanoparticles as compared to other nanoformulations. The complexity of the
polymerization process and polydispersity of polymer starting materials can mean
extra time and effort to rst ensure batch-to-batch consistency of the starting
material. This can be further complicated anytime a synthetic process is altered or
the reaction is scaled-up. Results obtained from a small scale reaction may be
different from the large scale reaction; this can be true of either the starting material
polymerization process or the subsequent nanoformulation process. Having estab-
lished lot release specications beforehand is key to ensuring the integrity and
consistent reproducibility of the nanoformulation.
8 Summary
The key parameters relating to polymer nanoparticle characterization include size,

surface characteristics, drug loading and release, stability, and batch-to-batch
consistency. Additionally, starting material characterization is equally important in
controlling nanoformulation consistency, especially as pertaining to polymer
nanoformulations. The importance of preliminary characterization of the starting
materials cannot be stressed enough. Without quality control measures in place to
assure reproducible and consistent starting materials, the lot release specications
for the nal nanoparticle may be unachievable.
Other PCC parameters not discussed here may also be an important and required
part of a nanoparticles characterization. Every nanoformulation is unique. It stands
to reason that characterization of every formulation will be unique as well, and will
pose its own distinct challenges in characterization. Measuring parameters using
multiple orthogonal techniques will afford the most comprehensive understanding
of the nanoformulations physicochemical characteristics. Furthermore, knowing

the limitations of each technique and what information can be reliably gained from
each measurement will strengthen the data analysis and overall understanding of the
formulation.
Acknowledgments This project has been funded in whole or in part with Federal funds from the
Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract
HHSN261200800001E. The content of this publication does not necessarily reflect the views or
policies of the Department of Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by the US Government.
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Chapter 8
Imaging Polymer Nanoparticles by Means
of Transmission and Scanning Electron
Microscopy Techniques
Nicolas Tsapis
Abstract This chapter describes methods of imaging polymer nanoparticles by

electron microscopy. Protocols for the preparation of nanoparticles for observations
by transmission electron microscopy, scanning electron microscopy, and
cryo-transmission electron microscopy are proposed. Possible artifacts are shown.
Keywords Electron microscopy Polymer nanoparticles Preparation methods

Negative staining Artifacts
1 Introduction
Nanomedicines consist of biodegradable or biocompatible colloidal particles with

size below the micron encapsulating a drug (Fattal and Tsapis 2014). After the early
work in the 1970s, nanomedicine technology has emerged as an attractive eld and a
tremendous number of nanometric novel delivery systems have been proposed
(Fattal and Tsapis 2014). Advances in chemistry and engineering have yielded a large
panel of biocompatible and biodegradable material nanomedicines can be built of. If
the very rst works focused on lipid-based nanomedicines (Gregoriadis and Ryman
1971; Gabizon et al. 1994; Joondeph et al. 1988; Lopez-Berestein et al. 1983), soon
polymers started to attract some attention. However, the polymer nanoparticles rst
developed in the mid-1970 by Birrenbach and Speiser were not degradable
(Birrenbach and Speiser 1976). As biodegradable polymers appeared, they were
quickly used to design nanometric drug delivery systems suitable for human appli-
cations (Couvreur et al. 1979). Polymer nanoparticles were of interest as they could
be more active than liposomes due to their better stability, leading to the encapsu-
lation of many drugs (e.g., antibiotics, cytostatics, nucleic acids) (Fattal et al. 1991).
Many different polymers were synthesized and formulated to yield nanomedicines
N. Tsapis (&)
Institut Galien Paris Sud, Faculty of Pharmacy, UMR CNRS 8612, Univ. Paris-Sud,
Universit Paris Saclay, 5 Rue J.B. Clment, 92296 Chtenay-Malabry, France
e-mail: nicolas.tsapis@u-psud.fr

DOI 10.1007/978-3-319-41421-8_8
206 N. Tsapis
among which are polyesters such as poly-DL-lactide, poly(lactide-co-glycolide) or

poly(-caprolactone), poly-alkylcyanoacrylates) and even polysaccharides.
Biodegradable polymers can be formulated into nanospheres or nanocapsules
according to different preparation processes: anionic emulsion polymerization
(Couvreur et al. 1979) (see Chap. 5 by Vauthier from Xu and Khan), solvent
emulsion-evaporation (Vanderhoff et al. 1979; Losa et al. 1993; Pisani et al. 2006)
(see Chap. 4 by Mandoza-Muoz et al.) or nanoprecipitation (Fessi et al. 1992) (see
Chap. 2 by Miladi et al. and Chap. 3 by Tang and Prudhomme from Xu and Khan).
Nanospheres are matrix systems in which the drug is dispersed throughout the whole
matrix, whereas nanocapsules are vesicular systems in which the drug is conned to a
cavity surrounded by a unique polymer membrane (Fig. 1). Polymer micelles were
also proposed, consisting of the self-assembly in aqueous phase of amphiphilic
copolymers leading to rather spherical aggregates of a few nanometers in diameter
(Jones and Leroux 1999) (Fig. 1). Amphiphilic copolymers can also form poly-
mersomes, the polymer particles equivalent of liposomes: a bilayer of amphiphilic
copolymer encloses an internal aqueous compartment (Lee and Feijen 2012) (Fig. 1).
Once polymers are formulated into nanoparticles or nanomedicines, researchers
need to characterize them in terms of size distribution and morphology. Since their
size distribution is on the order of the wavelength of light, nanoparticles are
Fig. 1 Schematic representation of polymer nanoparticles

8 Imaging Polymer Nanoparticles by Means of Transmission 207
submitted to Brownian motion, and light scattering techniques (dynamic or static)

can be used to characterize their size distribution and shape. This set of methods
provides a good statistical evaluation of the suspension as it analyzes several
millions of nanoparticles simultaneously (Moore and Cerasoli 2010). However, the
detail of single nanoparticle size and morphology cannot be determined with this
technique. Optical microscopy resolution is not sufcient to resolve the details of
single particles as it suffers from the diffraction limit (Betzig et al. 2006). Recently,
super-resolution optical microscopy techniques such as stimulated emission
depletion (STED) (Westphal et al. 2008), photo-activated localization microscopy
(PALM) (Betzig et al. 2006) or stochastic optical reconstruction microscopy
(STORM) (Rust et al. 2006) have allowed to decrease the resolution down to 20
50 nm. Betzig Moerner and Hell have received the Nobel Prize of Chemistry for
their work on super-resolution microscopy techniques in 2014. Despite these recent
achievements, these techniques have been optimized for the imaging of biological
samples and their resolution remains far above the 1 nm resolution one can achieve
with classical electron microscopy (EM) techniques. Therefore, for imaging ther-
apeutic nanosystems in general and polymer nanoparticles in particular, EM still
remains the method of choice for researchers.
Among EM techniques, one can cite scanning electron microscopy (SEM),
transmission electron microscopy (TEM) and cryo-associated techniques. All these
EM techniques are based on the interaction of an electron beam with the material on
which it is focused. We will review briefly the principles of the technique and will
try to give a short overview of the different ways to image polymer nanoparticles
focusing on practical application of these techniques.
2 Transmission Electron Microscopy (TEM)
2.1 Principle of the Technique
The original form of electron microscopy, Transmission electron microscopy

(TEM) involves a high-voltage electron beam between 80 and 300 kilovolts
(kV) emitted by an electron gun usually consisting of a tungsten lament or a
tungsten needle cathode (Klang et al. 2013). The electron beam is accelerated by an
anode and focused onto the specimen to be observed by electrostatic/electromagnetic
lenses. As electrons reach the specimen, some of them are scattered while the others
are transmitted. Electron microscope sample chambers are usually being maintained
under high vacuum although recent advances allow working at a few percents of
atmospheric pressure (Klang et al. 2013). The transmitted electrons carry informa-
tion about the structure of the specimen and the contrast results from amplitude
contrast and phase contrast of the transmitted electrons. The spatial variation of
transmitted electrons forming the image is then magnied by magnetic lenses until it
is recorded by hitting a fluorescent screen, a photographic plate, or a light sensitive
208 N. Tsapis
sensor such as a CCD (charge-coupled device) camera. Transmission electron

microscopes produce two-dimensional, black and white images and the current
resolution is commonly around 0.2 nm (Prez-Arantegui and Mulvey 2005).
2.2 Sample Preparation
For TEM observations, sample preparation is crucial. Samples should be in the

form of suspensions of nanoparticles. Ideally, the surfactants or stabilizers used for
nanoparticle preparation should be removed prior to preparation of the TEM grid
either by ultracentrifugation or dialysis. Indeed their presence could give rise to
several artifacts as one would observe them along with the nanoparticles. If
surfactants/stabilizers removal is too difcult or lead to nanoparticle aggregation,
one should always image surfactant/stabilizer solution as a control.
A typical protocol for sample preparation is presented below
1. A droplet of the suspension is deposited on a copper grid covered with a lm of
formwar (Fig. 2) and nanoparticles are let for a certain time to interact with the
formwar lm.
The formwar-coated grid should have been previously glow-discharged to
increase its hydrophilicity. Ideally glow-discharge should be performed less than
a few hours before depositing nanoparticle suspension. Otherwise,
hydrophilicity of the formwar lm is lost over time. Typically the time of
suspension deposition can be varied between 10 s to a few minutes depending
on the concentration of nanoparticles and their interaction with formwar. As an
example, as described by Grabowski et al., PLGA nanoparticle suspensions at a
polymer concentration of 1 mg/mL were deposited for 2 min on a 400 mesh
copper grid before negative staining (Grabowski et al. 2015). The interest of
negative staining is presented below.
2. To increase the contrast between the nanoparticles and the formwar lm, negative
staining can be performed. This staining step can be optional but it really eases the
observation step as polymer nanoparticles possess more or less the same elec-
tronic contrast as the lm. The idea is to use electrostatic interactions between the
nanoparticle surface and the stain to increase surface opaqueness to the electron
Fig. 2 Schematic top-view of a typical TEM grid. The number of lines can typically vary from 50
lines/inch (e.g. 50 mesh) to 600 lines/inch (600 mesh). Alternatively the grid pattern can be
hexagonal
beam. For negative staining, a droplet of the stain is deposited on the grid and let to
interact with nanoparticles for several seconds typically from 10 s to 1 min. To
increase electron opaqueness, stains should be selected with atomic numbers
higher than those of carbon, oxygen, and nitrogen that compose organic polymer
nanoparticles. The typical stains used are salt solutions of heavy metals that
absorb electrons such as ammonium molybdate, uranyl acetate, uranyl formate,
phosphotungstic acid, osmium tetroxide, osmium ferricyanide, and auroglu-
cothionate. Their concentration is between 1 and 3 % by weight. Depending on
the stain used, the interaction with the particles can be electrostatic or due to
hydrogen bounding. While uranyl salts have been used extensively, the tendency
is to progressively reduce their use to avoid workers exposure as they are
radioactive. Grids should then be loaded into the sample holder of the electron
microscope and imaged. Usually it is better to prepare the grid the very same day
of observation. The typical process for negative staining is illustrated in Fig. 3.
Alternatively, the sample and the staining solution can be deposited separately on
a paralm piece and the grid can be put sequentially in contact by flotation with
the different droplets before removing the excess liquid (blotting).
Negative staining leads to very high contrast helping to differentiate regions of
different electronic densities. In addition, the radiation damage from the absorbed
electrons can be neglected as it does not affect stain salts at the same rate as the
Fig. 3 Schematic illustrations of the negative staining methods for preparing TEM grids of
nanoparticles. Top direct drop deposition, Bottom sequential grid dipping
210 N. Tsapis
Fig. 4 PLGA-based nanoparticles observed by transmission electron microscopy (TEM):

stabilizer-free PLGA (a), poly(vinyl alcohol) stabilized PLGA (b), chitosan stabilized PLGA
(c) and pluronic F68 stabilized PLGA (d) (scale bars 500 nm) (Grabowski et al. 2015). Source:
Elsevier
organic molecules of the sample. Finally, the method is rather easy to implement
and provides quickly, the images of nanoparticles.
As an example, Fig. 4 shows images of poly(lactide-co-glycolide) (PLGA)
nanoparticles uncoated or coated with different stabilizing agents. The nanoparticle
concentration was 1 mg/mL and the suspension was let 2 min on the grid before a
30 s staining was performed using either phosphotungstic acid 1 % (w/v) (a, b, and
d) or uranyl acetate (c) at 0.5 % (w/v). These conditions yielded nanoparticles
homogeneously distributed on the grid without too many aggregates (Fig. 4)
(Grabowski et al. 2015).
There are however drawbacks to this technique. Depending on their mechanical
properties, the nanoparticles can be distorted during the drying process as it was
observed for polymersomes by Johnston et al. (2010) (Fig. 5).
Artifacts may also appear if the stain does not deposit homogeneously or does
not interact with the nanoparticle surface (Fig. 6). Negative staining may also favor
particle aggregation by creating liquid bridges between particles. The resolution is
Fig. 5 Example of distorted polymersomes observed by TEM after negative staining (Johnston
et al. 2010)
Fig. 6 Top Examples of poor interaction of the stain with nanoparticle surface. The stain that
appears dark does not wet properly the nanoparticle surface. Bottom the stain interacts better
making it easier to distinguish nanoparticles. The image on the bottom-right is the type of image
one wants to obtain
limited by the size of the salt grain around 2 nm if everything else is optimal such
as microscope alignment.
TEM provides images to assess the mean size and the polydispersity of
nanoparticle suspensions remembering that mean sizes measured on TEM images
are always smaller than those determined by dynamic light scattering where the
nanoparticle hydration layer is taken into account. TEM images can also provide
information on nanoparticle morphology. As an example, alkylcyanoacrylate
nanoparticles obtained by anionic polymerization exhibit a rough surface that was
also observed on SEM images (Fig. 7).
Fig. 7 TEM (left) and SEM (right) images of poly(alkylcyanoacrylate) nanoparticles exhibiting a
rough surface most probably arising from the polymerization method used to obtain them. By
contrast, PLGA nanoparticles possess a rather smooth surface as it can be observed on Fig. 6
(bottom, right)
212 N. Tsapis
Fig. 8 TEM images of nanoparticles prepared by nanoprecipitation using poly(-benzyl-L-

glutamate) with various molecular weights a 28 kDa, b 45 kDa, c 70 kDa and d 85 kDa (scale
bars 100 nm). The higher the molecular weight of the polymer, the more elongated the
nanoparticles (Cauchois et al. 2013). Source: Elsevier
While most polymer nanoparticles are spherical, research has started to focus on
nonspherical nanoparticles. Indeed, nanoparticle shape for drug targeting applica-
tions seems an interesting parameter to modulate their fate after in vivo adminis-
tration including their distribution in organs, interactions with cells, and even
subcellular trafcking (Mitragotri 2009; Cauchois et al. 2013). TEM imaging allows
for example to measure the aspect ratio of poly(-benzyl-L-glutamate) nanoparticles
as the polymer molecular weight varies as shown in Fig. 8 (Cauchois et al. 2013).
3 Scanning Electron Microscopy (SEM)
3.1 Principle of the Technique
In scanning electron microscopy, a focused beam of electrons is sent onto the

sample. The primary electrons interact with atoms in the sample, producing various
signals that can be detected and that contain information about the samples surface
topography and composition. The electron beam is generally scanned in a raster
scan pattern, and the beams position is combined with the detected signal to
produce an image. Unlike TEM, where the electrons in the primary beam are
transmitted through the sample, SEM produces images by detecting secondary
electrons which are emitted from the surface due to excitation by the primary
electron beam followed by elastic scattering processes. Two main types of contrast
are available with SEM. On one hand, the topographic contrast can be obtained by
detecting the secondary electrons with energy below 50 eV. On the other hand,
material contrast or atomic number contrast can also be observed by detecting
backscattered electrons of high energy (between 50 eV and the initial primary
electron energy). This contrast is not very frequently used for polymer nanoparti-
cles. Indeed, unless a very specic atom is present on their surface, the composition
cannot be determined precisely as polymers are mainly composed of carbon,
oxygen, and nitrogen. SEM can theoretically achieve resolution better than 1 nm if
all conditions are perfect. Specimens can be observed in high vacuum, in low
vacuum, or in slightly humid conditions such as those found in environmental
SEM. SEM can operate at rather low voltages (110 kV) compared to TEM, which
might be convenient for beam-sensitive materials.
3.2 Sample Preparation
Samples are usually deposited onto SEM stubs. If nanoparticles are available as
powders either obtained by spray-drying or freeze-drying, they can be sprinkled
directly onto double-sided carbon-conductive tape covering the SEM stub. Then, it is
recommended to dust gently the stub with a dry and clean gas spray to remove the
excess of powder and prevent electron column contamination if the powder is loosely
attached to the stub. As gas spray, dry computer cleaning sprays are convenient for
this step but any inert dry gas will do the job. If the sample is in the liquid state, a
droplet of nanoparticle suspension can be deposited directly on the SEM stub covered
with either a double-sided carbon-conductive tape or a clean surface such as mica,
glass, or silicon. An optical microscope coverslip can be convenient as it is cheap and
can be cleaned with an appropriate solvent before specimen deposition. Of course, as
it is the case for TEM sample preparation, the nanoparticle suspension must be clear
of all stabilizers, otherwise as water evaporates, stabilizers can create a matrix in
which nanoparticles are embedded preventing their correct observation. As polymer
nanoparticles are mostly nonconductive, a metal layer should be deposited onto
specimens. Metal sputter coating is usually used. It consists in a very thin coating of
electrically conducting metalsuch as gold (Au), gold/palladium (Au/Pd), platinum
(Pt), platinum/palladium (Pt/Pd), silver (Ag), chromium (Cr) or iridium (Ir) onto the
sample. Sputter coating prevents charging, which would occur because of the
accumulation of static electric elds. It also increases the amount of secondary
electrons that can be detected from the surface of the specimen and therefore increases
the signal to noise ratio. The typical thickness of the coating is in the range of 2
20 nm. The thicker the coating, the easier is the observation. However the thickness
should be small enough not to modify nanoparticle morphology. The different steps
of sample preparation for SEM observations are summarized in Fig. 9.
SEM of polymer nanoparticles can be very difcult due to polymer sensitivity to
the electron beam. Indeed, particles may melt under the beam. To solve this issue,
one can reduce the electron beam voltage or coat the sample with a thicker metal
layer, provided the thickness remains smaller than the nanoparticle diameter. In
some cases, the same sample can withstand better the electron beam after
freeze-drying than directly deposited as a suspension.
Experimentally, it has been observed that it is more difcult to image PEGylated
nanoparticles by SEM than their unPEGylated counterparts. Indeed, the PEG layer
as the suspension dries may create a soft coating that slightly blurs the image
(Fig. 10). In this case, one might prefer to image PEGylated nanoparticles using
TEM.
214 N. Tsapis
Fig. 9 Schematic representation of the different steps of sample preparation for SEM observations
Fig. 10 SEM image of PEGylated PLGA nanoparticles: the concentration and the PEG coating
reduce resolution and lead to particle aggregation during drying or even fusion/lmication in
certain regions
Many artifacts can be observed as nanoparticle suspensions are dried before

sputter coating. The rst artifact is nanoparticle segregation according to their size
depending where observation is performed. Indeed, as a droplet of colloidal sus-
pension is deposited on a surface and let to dry, colloids do not deposit homoge-
neously but form concentric circles of material: this is the so-called coffee ring
effect (Deegan et al. 1997). Due to pinning of the contact line and to convective
phenomena occurring during the drying process, nanoparticles deposit mostly at the
contact line. In addition, the larger the particles, the closer to the initial contact they
deposit, whereas smaller nanoparticles will deposit further. If the suspension is not
perfectly monodisperse, which is the case of most polymer nanoparticle suspen-
sions, if SEM is performed close to the initial contact line, one will observe mainly
the bigger nanoparticles. As observation is performed closer to the center of the
initial droplet, smaller nanoparticles will be observed. SEM observation should be
performed at several positions to yield a qualitative idea of nanoparticle size
Fig. 11 Schematic representation of the coffee ring effect for monodisperse or polydisperse
nanoparticles
distribution (Fig. 11). The drying process is very dependent of the exact compo-
sition of the suspension and nanoparticle shape, charge, and stabilizing forces and
deposition may vary from one suspension to another (Anyfantakis and Baigl 2015;
Eral et al. 2011). The take-home message here is to image the deposit at several
places to ensure there is no bias in the nanoparticle distribution.
Another artifact that can occur is nanoparticle fusion due to capillary interactions
and van der Waals forces. If the suspension is very concentrated, it can even lead to
the formation of a fused lm of nanoparticles and it will therefore be very difcult
to image individual nanoparticles (Fig. 10). This phenomenon depends on the
elastic properties of the nanoparticles, their surface state (charged, not charged,
polymer coating etc), their concentration, and the speed of drying. If fusion
occurs, usually it can be avoided by decreasing the nanoparticle concentration.
4 Cryo-electron Microscopy (Cryo-EM)
In addition to TEM and SEM, cryo-EM can also be considered if one wants to
investigate the inner structure of nanoparticles. It is a technique of choice when the
drying step of TEM or SEM leads to deformations of the initial shape of the
nanoparticles (polymersomes) or when nanoparticles possess an inner organization
(nanocapsules).
Cryo-EM was rst developed for imaging biological systems such as viruses while
preserving their microstructure (Adrian et al. 1984). After deposition of the
nanoparticle suspension on a carbon-coated EM grid containing holes (Fig. 12), the
excess solution is blotted and freezing is performed as fast as possible. Freezing can
be achieved either by plunge-freezing (Adrian et al. 1984) or by high-pressure
freezing (Costello 2006). The idea is to freeze the sample very fast to promote the
vitrication of ice instead of crystallization, since ice crystals may deform samples as
they grow. It has been shown that water can be vitried if freezing rates are above
216 N. Tsapis
Fig. 12 Schematic representation of the EM grid used for cryo-TEM observation of nanoparticle
suspensions
104 C/s (Klang et al. 2013). In the case of nanoparticle suspensions, plunge-freezing
is preferred as sample thickness is below 510 m. Plunge-freezing is achieved in
liquid ethane or propane, maintained at low temperature using liquid nitrogen
(Fig. 12). Then the grid is transferred to the TEM using a cryo-holder maintained in
liquid nitrogen as well, and imaged. As for TEM, grids should be glow-discharged
before depositing the suspension to favor hydrophilicity. Automated vitrication
instruments are commercially available, with humidity and temperature-controlled
chambers to reproducibly prepare cryo-TEM grids. These instruments also allow to
adjust the blotting time of the grid. As a rule of thumb, the concentration of
nanoparticle optimized for TEM can be multiplied by a factor 10 for cryo-TEM.
In Fig. 13, several examples of nanoparticles are presented. On one hand, the
plain polymer nanoparticles do not exhibit any internal structure. On the other hand,
nanocapsules of perfluorocarbons present a darker core (Diou et al. 2014) and
polymersome bilayer is clearly visible on cryo-TEM images (Scott et al. 2012).
Some artifacts can be observed by cryo-TEM such as liquid ethane/propane
contamination (Fig. 14, left) or ice crystals if the suspension lm thickness is too
important due to insufcient blotting or to water condensation (Fig. 14, right).
High-pressure freezing is not used for nanoparticle suspensions, unless freeze
fracture is performed afterwards. Freeze-fracture consists in fracturing the sample
and creating a replica of the surface that is then imaged by TEM (Hope et al. 1989).
We will not get into details about this technique as it is rather complicated and now
people prefer to perform cryo-TEM instead.
Fig. 13 Examples of cryo-TEM images of nanoparticles (left), nanocapsules (middle) and

polymersomes (Scott et al. 2012) (right)
Fig. 14 Examples of artifacts. On the left liquid ethane contamination, on the right hexagonal ice
5 Conclusion
This chapter presented the main electronic microscopy techniques used for imaging
polymer nanoparticles. While TEM and SEM are relatively easy to use and do not
require complicated equipment except the electronic microscope, cryo-TEM is
more tedious and necessitates specic cryo-equipement for the preparation and
conservation of the grids. TEM and SEM already allow to give an idea of size and
morphological characteristics of particles in the sample suspension while cryo-TEM
can provide interesting information on the inner organization of the particles and
allows to image them without any distortion and or artifact during sample drying.
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Chapter 9
Evaluating the Interactions Between
Proteins and Components of the Immune
System with Polymer Nanoparticles
Silvia Lorenzo-Abalde, Rosana Simn-Vzquez, Mercedes Peleteiro

Olmedo, Tamara Lozano-Fernndez, Olivia Estvez-Martnez,
Andrea Fernndez-Carrera and frica Gonzlez-Fernndez
Abstract The use of polymer nanoparticles in biomedicine has increased in recent

years because of their potential to improve a wide range of biomedical applications,
particularly as drug-delivery systems. However, the use of these nanoparticles in
biomedicine has been accompanied by signicant concern regarding their bio-
compatibility. The success of the use of nanoparticles in biomedical applications
will depend to some extent on their interactions with cells and other components of
the immune system. The main focus of this chapter is the way in which the
interactions between complement factors, antibodies and cells with nanoparticles
can be studied. The main guidelines, protocols, and key issues to be considered in
these assays will be discussed. Moreover, the potential immunogenicity induced by
nanoparticles will be addressed. Immunostimulation can be benecial for vaccine
purposes as nanoparticles could activate the complement system, improve the
antigenicity of weak antigens by serving as adjuvants, enhance antigen uptake, and
stimulate antigen-presenting cells. In contrast, unwanted immune activation can
lead to undesirable reactions in the hosts body, such as inflammation, allergic, or
pseudoallergic reactions and autoimmune disorders.
Silvia Lorenzo-Abalde and Rosana Simn-Vzquez shared rst-authorships.
S. Lorenzo-Abalde R. Simn-Vzquez M. Peleteiro Olmedo T. Lozano-Fernndez

O. Estvez-Martnez A. Fernndez-Carrera . Gonzlez-Fernndez (&)
Immunology, Biomedical Research Center (CINBIO), Institute of Biomedical
Research of Vigo (IBIV), Universidad de Vigo, Campus Lagoas Marcosende,
36310 Vigo, Spain
e-mail: africa@uvigo.es
R. Simn-Vzquez
Institut Galien Paris Sud, Faculty of Pharmacy, CNRS, Univ. Paris-Sud,
Universit Paris Saclay, 5 rue J.B. Clment, 92296 Chtenay-Malabry, France

DOI 10.1007/978-3-319-41421-8_9
222 S. Lorenzo-Abalde et al.

Keywords Toxicity Immunogenicity Biocompatibility Immune response

Complement Endocytic routes Nanotoxicology Nanomedicine Nanovaccines
1 Introduction
Nanoparticles (NPs) can interact with the immune system (either with cells and/or
humoral factors) with benecial or detrimental effects. In order to understand the
potential impact of these interactions, this chapter will try to give a broad overview
on several aspects: (I) First, a general view of the immune system (including the
innate and the adaptive immune components; (II) the potential consequences of the
interaction between nanoparticles and the immune system (phagocytosis, comple-
ment activation, cell toxicity, inflammation, etc.); (III) the potential use of some
NPs on vaccines; (IV, V) the interaction of NPs with other blood components and
plasma proteins; and (VI) other important issues related with immunotoxicity.
The chapter also includes the functional analysis and the techniques most fre-
quently used for the evaluation of the interaction between the NPs with the immune
components. Altogether, they will help us to know the potential benets that
polymer NPs can offer on several biomedical applications but also the risk/safety
issues.
2 Brief Description of the Immune System
Immunity can be divided into innate and adaptive immunities. Both types include
cellular and humoral components (Table 1). As far as cells are concerned, there is a
long list of innate immune cells: macrophages, dendritic cells (DCs), mast cells,
neutrophils, basophils, eosinophils, natural killer (NK) cells (Janeway and
Medzhitov 2002) and a new variety of innate cells that was recently described, such
as the nuocytes (Neill et al. 2010). On the other hand, the specic immune cells are
T and B lymphocytes.
The main difference between the innate and the adaptive immune responses is that
while innate immune cells bear germ-line recognition receptors for foreign and self
(apoptotic-damaged cells) elements, adaptive immune cells are responsible for
antigen-specic responses and their receptors are coded by rearranged gene segments.
2.1 Cellular and Humoral Innate Immunity
The innate immune system, despite the fact that it is not antigen-specic, can
discriminate between self and nonself molecules by three main strategies (Parkin
and Cohen 2001; Medzhitov and Janeway 2002; Hoebe et al. 2004). The rst
9 Evaluating the Interactions Between Proteins and Components 223
Table 1 Immune system components

Immune system
Cells Humoral factors
Innate immune cells Innate humoral components
Phagocytic cells Complement factors
Neutrophils Anaphylotoxins: C3a, C4a, C5a
Classical cascade: C1,C4,C2,C3, C5, MAC*
Dendritic cells Alternative cascade: C3, B, D, Properdin, MAC*
Macrophages Lectin cascade: MASP, C3, C5, MAC
Non-phagocytic cells Cytokines
Basophils Pro-inflammatory: TNF, IL-6, IL-1, IL-12, IL-17
Eosinophils Anti-inflammatory: IL-10
Mast cells Chemokines: IL-8, etc.
Natural Killer cells Colony-stimulating factors**: GM-CSF, G-CSF, M-CSF
Nuocytes and other innate cells Interferons , ,
Interleukins: IL-2, IL-3, IL-4, IL-5
Adaptive specic immune cells Specic humoral components
Lymphocytes Antibodies
B cells IgG
T helper (Th) CD4+ IgM and IgD
T cytotoxic (Tc) CD8+ IgA
T regulatory (Treg) foxP3+ CD4+ IgE
* MAC membrane attack complex including C5b, C6, C7, C8 and C9 complement factors
** G granulocyte, M monocyte, CSF colony stimulating factor
*** IL interleukin
strategy is microbial non-self recognition and this is based on the identication of

pathogen-associated molecular patterns (PAMPs), which are conserved products or
molecules that are unique to microorganisms recognized by the pattern recognition
receptors (PRRs) present on the surface of many immune cells. These receptors can
also recognize endogenous molecules released from damaged cells, which are
termed damage-associated molecular patterns (DAMPs). Examples of PRRs include
Toll-like Receptors (TLRs), C-type lectin receptors (CLRs), Retinoic acid-inducible
gene (RIG)-I-like receptors (RLRs), and NOD-like receptors (NLRs). Activation of
the PRRs leads to the expression of co-stimulatory molecules on the surface of
antigen-presenting cells (APCs) such as macrophages, B and dendritic cells.
Another type of receptor, Scavenger Receptors (SR), is involved in innate
immune defense (Areschoug and Gordon 2009). SRs can bind modied
low-density lipoproteins (LDL) and different microbial structures. SRs are also
involved in the recognition of dying cells. As the apoptotic process evolves,
membrane phospholipids are redistributed and oxidized and they become a target
for macrophages. These modied phospholipids (mainly phosphatidylserine) are
recognized by scavenger receptors in the macrophage surface (Fadok et al. 2001).
The second strategy is the recognition of the missing self. In a normal cell,
specialized markers of self are expressed in a constitutive way; they are recog-
nized by specic receptors in the surface of the immune cells giving an inhibitory
signal. The absence or down-regulation of these markers in the surface of a cell
makes the cell appear to be nonself and this triggers its elimination by NK cells.
The third strategy, the detection of abnormal self, is based on the fact that
markers of abnormal self are induced upon infection (mostly viral) or cellular
transformation. This phenomenon leads NK cells to eliminate infected or tumoral
cells (Medzhitov and Janeway 2002).
One of the rst events of innate immunity is the recruitment of neutrophils
towards the site of infection, where they phagocyte the pathogen and liberate
pro-inflammatory mediators, including chemokines, to attract other innate cells
(Goncalves et al. 2011) such as macrophages and dendritic cells. These cells will
also phagocyte the pathogen and, once the antigen is processed, they will present
antigen peptides to T lymphocytes through the major histocompatibility complex
molecules (MHC). There are two types of MHC molecules: MHC class I, which are
present in all body nuclear cells, and MHC class II, which are basally expressed on
APCs (B cells, macrophages and DCs) and on some cortical thymic cells. MHC
class I molecules are in charge of presenting intracellular antigens, such as virus or
tumor peptides, whereas MHC class II molecules present endocyted exogenous
antigens (Parkin and Cohen 2001).
2.1.1 Innate Humoral Components
In addition to cells, the innate response has a humoral component that includes the
complement system and cytokines (Tables 1 and 2).
Table 2 Cytokines produced by several types of immune cells

Cell types Subtypes Cytokines Prole
Macrophages M1 IL-1, IL-1, IL-6, Inflammatory
IL-12,TNF-
M2 TGF-, IL-10 Anti-Inflammatory
NK cells TNF-, IFN- Inflammatory
Dendritic cells IL-12, IFN- Inflammatory
T helper cells (CD4+) TH1 IFN, IL-2, TNF- Inflammatory
TH2 IL-4, IL-5, IL-6, IL-9, Anti-inflammatory
IL-10, IL-13
TH17 IL-17, IL-21, IL-22 Inflammatory
T regulatory cells TGF, IL-10 Anti-inflammatory
(Foxp3 + CD4+ CD25+)
B cells Primed by IFN, IL-12 Inflammatory
TH1
Primed by IL-2, IL-13 and IL-4 Anti-inflammatory
TH2
IL interleukin, TNF tumor necrosis factor, TGF tumor growth factor, IFN interferon
The Complement System
The proteins and cofactors of the complement system are, together with those of the
coagulation pathway, the main plasma proteins involved in the opsonization of the
NPs. The complement system plays a major role in the recognition, opsonization,
and elimination of foreign materials. This system includes a group of around 35
plasma proteins that are sequentially activated in a cascade, which leads to the
opsonization and further lysis of the pathogen (Fig. 1). Deciencies in one of the
components of the complement system can cause severe and recurrent infections or
systemic autoimmune disease.
The complement system can be activated by three different pathways: classical,
lectin, and alternative pathways. These three pathways use different recognition
molecules to sense a foreign particle (Fig. 1), but they all converge at the major
complement protein C3 (Salvador-Morales and Sim 2013; Harboe et al. 2011;
Gorbet and Sefton 2004).
The protease C3 convertase then cleaves C3 into C3a (an anaphylotoxin) and
C3b. C3b plays an important role in antigen opsonization. Clusters of hundreds of
C3b molecules can bind to the antigen surface, thus making it visible to other
immune components. C3 cleavage is followed by the formation of C5 convertase,
which then cleaves C5 into C5a and C5b. C5a is a potent anaphylotoxin and C5b
induces assembly of the components C6, C7, C8, and C9 to form a large protein
complex called the membrane attack complex (MAC), which can insert itself into
Fig. 1 Complement activation pathways (a) and a representative example of a Western blot to
detect the ability of different nanostructures to activate the complement cascade by measuring the
cleavage of the C3 factor (b)
lipid bilayers and make holes in the cell membrane (Fig. 1), leading to cell death
(Salvador-Morales and Sim 2013; Harboe et al. 2011). Although the capacity of the
MAC to attach and open holes on liposomes in a similar way to that described for
cells is known since many years ago (Lachmann et al. 1970; Humphries and
McConnell 1974), the capacity of the MAC to attach and open holes on polymer
nanoparticles has not been addressed yet.
In addition to the MAC, the secretion of anaphylotoxins C3a, C4a, and C5a has a
pro-inflammatory effect and chemoattractant activity for other elements of the
immune system (Parkin and Cohen 2001).
Cytokines
A group of proteins secreted by immune cells, named cytokines, synchronize

immune mechanisms (Gamucci et al. 2014). Cytokines are low molecular weight
messengers that are secreted by immune cells to alter their and other cells behavior.
There are many different types of cytokines, such as interleukins (IL-1, 2, 3, 4, 5, 6
etc.), and they have a wide variety of functions; chemokines, with chemoattractant
activity; colony-stimulating factors, which cause stimulation and proliferation of
stem cells; interferons, which interfere with viral replication and activate immune
cells, tumor necrosis factor, and many others (Table 2).
2.2 Cellular and Humoral Adaptive Immunity
Adaptive immunity also has cellular (T and B lymphocytes) and humoral (anti-
bodies) factors. These factors work in close collaboration with the innate system but
they begin when pathogens are not fully eliminated by the innate immune system.
The adaptive immune responses are also called acquired or specic responses and
they induce immune memory. An adaptive immune response against a pathogen is
much faster and stronger after a second encounter than after the rst one. This is the
basis of the vaccination strategy.
The cellular arms of the adaptive immunity are the lymphocytes. Lymphocytes
are leukocytes with antigen-specic receptors that are able to recognize a wide
range of foreign elements (Parkin and Cohen 2001). There are two main types of
lymphocytes, namely B and T lymphocytes, and both proceed from a common bone
marrow lymphoid precursor.
2.2.1 B Cells
Once B cells have maturated in the bone marrow, they travel through the blood and
lymph to the lymphoid tissues and they can directly recognize the antigen through
the clonal B cell receptor (BCR) expressed on the surface. The antigen-BCR
complex is internalized and processed, and antigen peptides bound to MHC class II
molecules are presented on the B cell surface. When this happens, B cells act as
APCs and promote T cell activation (Parkin and Cohen 2001; Yuseff et al. 2013).
The union of a B and a T cell is dependent on the presence of co-receptor molecules
on the surface of the B cell, such as CD40, which binds to CD154 (CD40-ligand)
on the T cell surface. This union promotes not only the T cell activation and
cytokine release, but also the B cell activation. When B cells are activated they can
either become antibody-secreting plasma cells or memory B cells.
2.2.2 T Cells
T lymphocytes also express clonal receptors called T cell receptors (TCR).

Unlike B cells, T cells can only recognize foreign peptides when these peptides are
bound to the MHC molecules on the cell membrane (Parkin and Cohen 2001).
There are three main populations of T cells (Table 1): Helper (Th or CD4+),
Cytotoxic (Tc or CD8+) and regulatory (Treg or CD4+ Foxp3+) cells. Th and Treg
cells are activated by antigens presented by MHC class II molecules. Depending on
the pattern of cytokine release, several major subtypes of Th cells can be dened:
Th1, Th2, Th17, Tfh, and Th22.
Th1 cells produce IFN and help in cellular responses, such as those aimed at
eliminating intracellular bacterial and viral infection. Th2 cells secrete IL4, IL5,
IL13 and promote humoral responses, such as those against extracellular bacterial
infection. Th17 cells release IL17, IL21, and IL22 and participate in autoimmunity.
Follicular Th cells (Tfh) and Th22 mainly secrete IL21 (OShea and Paul 2010) and
IL22, respectively. In contrast to the helper function of the CD4+ T cells, CD8+ T
cells have cytotoxic effects, although a similar subdivision to helper cells has been
suggested depending on the pattern of cytokines produced by these cells. Finally,
naive regulatory T cells (CD4+ CD25+ Foxp3+) induce immunosuppression through
the production of IL10 and TGF.
3 Nanoparticle Interactions with the Immune System
The physical and chemical properties of the NPs, such as size, shape, surface
charge, hydrophobicity/hydrophilicity and the steric effects of the particle coating,
can regulate the interaction with the immune system (Gamucci et al. 2014; Zolnik
et al. 2010; Naahidi et al. 2013), especially with the components of the innate
system. When NPs enter the bloodstream, the immune cascade is initiated with the
adsorption of opsonins to their surface, thus making them more visible to phago-
cytic cells (Naahidi et al. 2013). Several plasma proteins can act as opsonins, with
immunoglobulins and complement proteins being the most potent ones (Owens and
Peppas 2006). This protein corona modies the size, shape, and surface prop-
erties of the NPs, and not only enhances the nanoparticle recognition by the
immune cells, but also increases the hydrodynamic diameter (HD) and modies the
NP biodistribution and circulation time (Gamucci et al. 2014). Manipulation of NP
size and charge has proven to increase either particle delivery to the immune system
or avoid opsonization and subsequent nonspecic uptake by phagocytic cells
(Dobrovolskaia et al. 2008a). For instance, hydrophilic polymers, like poly(ethy-
lene glycol) (PEG), can be conjugated or adsorbed onto the surface of NPs to
provide steric stabilization, which prevents protein adsorption, avoids recognition
by the immune cells and thus prolongs particle circulation in the blood
(Dobrovolskaia et al. 2008a; Alexis et al. 2008). In other cases, such as vaccine
delivery systems, it has been demonstrated that surface functionalization with
chitosan or mannose delivers NPs to macrophages and dendritic cells via dened
phagocytic pathways, thus enhancing immune responses to nanoparticle-bound
antigens (Dobrovolskaia et al. 2008a).
Understanding bio-kinetics of NPs is important to anticipate their accumulation
in the body organs and therefore, their toxicity. Once NPs reach the blood circu-
lation and/or the lymphatic system, either after intravenous administration or after
their absorption by the other routes, they can be distributed to various organs,
tissues, and cells (Landsiedel et al. 2012). In the bloodstream, the transport of NPs
into tissues will be affected by their size and also by the type of blood vessel
endothelium (Almeida et al. 2011). The pore size of endothelial walls represents
another barrier and at the same time allows selective accumulation. For this reason,
the major uptake of NPs in many cases is into the liver followed by the spleen, and
bone marrow, because they have discontinuous endothelia with pores of 50
100 nm (Landsiedel et al. 2012). Moreover, all of these organs contain high levels
of macrophages (Almeida et al. 2011). The route of administration of the NP into
the body also affects its biodistribution. NPs will interact with different types of
cells depending on the chosen route and, therefore, they could induce different
immune reactions (Zolnik et al. 2010; Dobrovolskaia and McNeil 2007). A scheme
summarizing how nanoparticles may interact with the immune system is reflected in
Fig. 2.
3.1 Interaction of Complement Factors with Nanoparticles
The properties of polymer NPs that affect complement activity are related to the
hydrodynamic size, morphology, composition, surface properties, chain length,
architecture, the presence of functional groups, and hydrophobic/hydrophilic
effects.
Studies on different NPs have shown that size affects complement activation. For
instance, Pedersen et al. (2010) proved that 250 nm dextran-coated iron NPs with
Fig. 2 The physicochemical properties of NPs determine their interaction with the immune
system and blood components. Proteins rapidly attach to NP surface, forming the protein corona,
which may cause conformational changes on these proteins and/or modify the properties of NPs.
These interactions can affect tissue biodistribution and cellular uptake. NPs can affect cell viability
and activation and may trigger inflammatory responses. They can activate the complement
cascade, interact with erythrocytes promoting hemolysis, induce oxidative stress, suffer
aggregation, etc. While recognition and induction of cell activation is appropriate in the case of
a nanovaccine, or a toxic effect is desirable on tumor cell therapy, in other cases the main goal
could be to avoid the detection by the immune system to reach the target cell. This can be
frequently addressed by modulating the physicochemical properties of NPs due to its high
versatility. Note Servier Medical Art was used for this composition
IgM bound on their surface can activate complement whereas 600 nm NPs produce
less complement activation when corrected for surface area.
It has also been demonstrated that the curvature affects complement activation.
NPs with sizes similar to the cross-sectional diameter of IgM (around 40 nm)
produce a more signicant increase in complement activation because IgM C2
domains attach to C1q and this triggers the classical pathway complement activa-
tion (Pedersen et al. 2010).
Surface chemistry, such as the density of functional groups or the presence of
hydrophobic/hydrophilic domains, strongly influences complement activation. For
instance, NPs with a high surface density of amino and hydroxyl fractions can
induce nucleophilic attack on C3b, thus potentiating the alternative pathway.
Lipid-polymer hybrid NP also triggers this alternative pathway. In contrast, other
types of NPs can activate the classical cascade because C1q usually binds to
negatively charged surfaces. NPs such as anionic liposomes can activate comple-
ment either through classical, alternative or both pathways (Moghimi et al. 2011).
Surface modication of NPs with poly(ethylene glycol) (PEG) is an effective way to
prevent protein adsorption and complement activation. However, the success of this
strategy depends on the density of the PEG coating, the chain length, and the
chemical conguration of this component (Salvador-Morales and Sim 2013).
3.1.1 Methods of Analysis
The interaction between NPs and the components of the complement system may
alter its normal function and either increase or decrease its activation. Several tests
can be performed that focus on the evaluation of either the overall functional
activity or the presence of individual components.
Functional Analysis
Hemolytic Assays
Hemolytic assays are the most commonly used methods to evaluate the classical
pathway activation of complement. In the classical CH50 test, serial dilutions of the
sample (i.e., a serum pre-incubated with NPs) in a buffer containing Ca2+ and Mg2+
ions are tested to ascertain the dilution required to achieve 50 % lysis of
antibody-coated sheep erythrocytes. If Ca2+ ions are removed by a chelating agent,
the classical and lectin pathways are completely inhibited and only the alternative
pathway can be measured. In this respect, the AH50 test is similar to the CH50, but
in the former case a Ca2+ chelating agent is used along with rabbit or guinea pig
erythrocytes. In both experiments, the proportion of lysed cells is linearly correlated
to the amount of complement present in the sample (Salvador-Morales and Sim
2013; Harboe et al. 2011; Mollnes et al. 2007; Kirschnk and Mollnes 2003).
Another variant of the hemolytic assay is the hemolysis-in-gel test, in which the
same erythrocytes and buffer used in the CH50 and the AH50 tests are incorporated
in agarose gel (Mollnes et al. 2007).
Hemolytic assays have been used to evaluate how different nanomaterials, such
as liposomes (Chonn et al. 1991), interfere with the activation of the complement
system and to compare the reduction in functional activity after complement acti-
vation by NPs with different surface properties (Socha et al. 2009; Meerasa et al.
2011).
Methods Based on Enzyme Immunoassays
The enzyme-linked immunosorbent assay (ELISA) is performed using plates coated
with either IgM or lipopolysaccharide to activate the classical or the alternative
pathway, respectively. To analyze the lectin pathway, the plaques are coated with
mannan and activation of the classical pathway has to be blocked by an anti-C1q
monoclonal antibody. Then, after incubation with the sample, a monoclonal anti-
body against the C9 neoepitope is used to detect complement activation (Harboe
et al. 2011; Mollnes et al. 2007).
Individual Component Analysis
In this case, individual factors of the complement pathways can be measured, with
the C3 and C4 components being the most frequently assessed. Numerous methods
can be used to analyze the presence of individual components of the complement

cascade and these are described below.
Immunochemical Assays
These assays are based on the formation of immune complexes and include
nephelometry, radial immunodiffusion, electroimmunoassay, turbidimetry, Western
Blot, ELISA, and time-resolved immunofluorometric assay (TRIFMA). In all of
these methods it is crucial to select an appropriate antibody to detect the target
immune complex.
Western blot to detect the C3 factor and its breakdown products would be the
rst method of choice, because degradation of C3 occurs in all complement acti-
vation pathways. There are commercial polyclonal antibodies that are directed
against different epitopes on the protein molecule. For instance, antibodies against
C3 and C4 components can also react with their major soluble fragments such as
C3c and C4c, which are formed during the complement activation cascade (Harboe
et al. 2011; Mollnes et al. 2007).
Phage Libraries
The design of microarrays through the generation of phage display libraries of
numerous scFv (single-chain fragment variable) antibodies specic for selected
proteins can be used to detect different complement factors. Serum samples are
incubated on the array, stained, and scanned to give a semiquantitative measure of
concentration. The advantage of this method is that several complement proteins
can be screened simultaneously (Mollnes et al. 2007).
Immunoelectrophoresis Method
This technique characterizes proteins based on electrophoresis migration and
reaction with antibodies. C3 and its breakdown products C3b, iC3b, and C3c are
separated by electrophoresis of serum on agarose, and these proteins interact with
anti-C3 antibodies. When the proteins react with the specic antibodies they form a
visible precipitate over an area that is proportional to the protein concentration
(Salvador-Morales and Sim 2013).
3.2 Effect of NPs on Innate Cells
When pathogens cross the epithelial and mucosal barriers, the innate immune
system immediately acts by trying to eliminate them. Monocytes-macrophages,
along with DCs and neutrophils, are professional phagocytes (Table 1) and they
play a major role in the elimination of foreign elements.
Monocytes travel on blood and lymph towards different tissues, where they
differentiate to mature macrophages, which are now long-living cells distributed
throughout the body (Gonzlez et al. 2006; Beutler 2004). Their most important
functions are related with the detection and elimination of pathogens and other
nonself elements, together with the clearance of apoptotic or damaged cells. They
engulf and kill the pathogen leading to secretion of chemotactic and

pro-inflammatory cytokines for the recruitment of other myeloid cells, principally
neutrophils. Together with DCs and B lymphocytes, the other two professional
APCs, they internalize, process, and present the antigen to T lymphocytes in order
to initiate an adaptive immune response (Beutler 2004).
DCs are considered to be one of the most specialized APCs due to their cognate
interaction with T cells. DCs are present in many tissues and they capture and
process antigens to present them to T cells (Bousso 2008; Hubbell et al. 2009). In
their immature state, DCs mediate the maintenance of self-tolerance. As part of their
surveillance routine, DCs recognize, internalize, and process self-peptides. T cells
are tolerant to these peptides in the absence of co-stimulatory molecules. Once
immature DCs encounter a pathogenic antigen, they are induced to maturate by
PAMPs, inflammatory cytokines and prostaglandins present in the environment
(Ballestrero et al. 2008). Now, as mature DCs, they express co-stimulatory mole-
cules on their surfaces and present antigen peptides on their MHC molecules. They
acquire migratory capacity and move to lymph nodes, where they promote the
activation of T cells (Hubbell et al. 2009; Fesenkova 2013).
Due to the important role of DCs for the presentation of antigens to T cells, the
use of loaded NPs containing antigens (stimulatory compounds or immunosup-
pressant agents) for targeted delivery to DCs is a very promising approach to
modulate the immune response (Fesenkova 2013; Look et al. 2014). Such NPs can
be used for the design of vaccines against pathogens or for therapeutic vaccines
against tumoral cells (Danhier et al. 2012; Nestle et al. 2005).
The use of antigen-loaded NPs has stimulated the study of the potential effects
that these NPs could have on their target cells, mostly DCs (Klippstein and Pozo
2010). Uto et al. have shown that polymer NPs directed to mouse spleen DCs can
affect their maturation. Nanoparticles made of poly(-gluconic acid) (-PGA-NPs)
are able to enhance in DCs the expression of co-stimulatory molecules, including
CD40, CD80, or CD86 and MHC class I, in a dose-dependent way. This nding,
along with the secretion of inflammatory cytokines and chemokines and the acti-
vation of signaling pathways (NF-B and MAPK pathways), suggests the induction
of DC maturation (Uto et al. 2009). However, NPs can be used to modulate DCs in
addition to activating them (Shen et al. 2006). The inhibition of DCs could be very
useful for some inflammatory and autoimmune disorders, decreasing inflammation
and cell activation.
Neutrophils are the third type of specialized phagocytic cells, but they are not
APCs to T lymphocytes. Their recruitment to the site of injury is very fast after
receiving the signal of infection, and their activity is closely related to the signals
coming from macrophages and other components of the innate system. Despite
being a necessary event for host defense, excessive neutrophil inltration and
activation can lead to an undesirable inflammation, which in turn causes tissue
damage (Parkin and Cohen 2001; Wang et al. 2014a).
Other types of non-phagocytic innate cells are the eosinophils, mast cells and
basophils (which participate in allergic inflammation, response to parasites and
containment of infection), and the Natural Killer (NK) cells.
NK cells are a subclass of lymphocytes that contain cytolytic cytoplasmatic

granules. They eliminate cells infected by viruses or tumoral cells without prior
antigenic stimulation. Their surface phenotype is characterized by the presence of
CD16 (a low afnity Ig Fc receptor), which enables them to detect antibody-coated
cells (Vivier et al. 2008), and CD56 (Neural cell adhesion molecule, NCAM).
However, they lack T cell receptor (TCR) and CD3, typical markers of T cells. NK
cells use inhibitory and activator receptors to recognize cells with a reduced MHC
class I expression, which is a common feature in tumor and stressed cells (Vivier
et al. 2008; Rh ov 2002; Lanier and Phillips 1992). NK can also recognize
infectious nonself ligands expressed on infected cells by means of special receptors
in their surface. These events induce cytotoxicity and proliferation, migration to
infected tissues and cytokine release. NK cells can secrete interferon gamma
(IFN), which induces macrophage activation, and also tumor necrosis factor alfa
(TNF), a pro-inflammatory cytokine. NK proliferation and activation can also be
stimulated by the presence of IL-2 and IFN (Rhov 2002), thus producing the
so-called LAK (lymphokine-activated killer) cells.
The interaction of NPs with the non-phagocytic innate cells could affect to their
functional activity, but no major attention has been given by researchers
(Lozano-Fernndez et al. 2014).
3.3 Nanoparticle Uptake by Innate Immune Cells
Innate phagocytic immune cells work in a combined way to perform rapidly the
uptake and elimination of a foreign element that enters the body. This requires an
endocytosis process not only for antigen elimination but also, as indicated before, to
trigger cell activation and antigen presentation.
Endocytosis includes several internalization pathways such as phagocytosis,
pinocytosis, and macropinocytosis (Table 3).
Table 3 Endocytic mechanisms

Endocytic mechanisms Receptors/proteins involved
Phagocytosis Complement receptor (CR)
Fc receptor (FcR)
Scavenger receptors
Clathrin-mediated Clathrin/dynamin
Caveolar type Caveolin
CLIC/GEEC* Actin, many others
Macropinocytosis Actin, many others
Others: IL-2Rb pathway, Arf6, Flotilin, circular dorsal Dynamin, actin, contactin and
ruffles, Entosis many others
*CLIC/GEEC clathrin-independent carrier/GPI-AP-enriched early endosomal compartment
(GEEC) pathway
Phagocytosis is actin-dependent and is restricted to professional phagocytes

(macrophages, neutrophils, monocytes, and DCs). This process can be mediated by
different receptors, such as complement receptor (CR), FC receptor (FCR), and
scavenger receptors (SR).
The pinocytosis pathway involves absorption of biological fluids from the
external environment of the cell. This process can be performed by all cell types
and is divided into clathrin-mediated, caveolin-mediated, and clathrin/caveolin-
independent endocytosis, whereas the macropinocytosis pathway is a nonspecic
process to internalize fluids and particles into the cell (Sahay et al. 2010; Oh and
Park 2014).
Endocytosis can also be classied based on the way in which the cell interacts
with the engulfed material (receptor-mediated, adsorptive, or fluid phase) (Sahay
et al. 2010; Caron et al. 2013).
Different NPs can enter the innate cells by different pathways and this process
depends mainly on their physicochemical features. Of all the mechanisms men-
tioned above, phagocytosis and clathrin- and caveolae-mediated endocytosis are the
most important pathways for the internalization of the NPs into cells (Oh and Park
2014; Zhao et al. 2011a).
When NPs are internalized by clathrin-mediated endocytosis, they are entrapped
in intracellular vesicles, such as endosomes, which then fuse with acidic lysosomes
resulting in the degradation of the endosome content. This degradation can be a
disadvantage if the NP is used as an antigen, drug, or small interfering RNA (siRNA)
carrier. If this is the case, it is of interest to nd an alternative route for endocytosis,
such as macropinocytosis and caveolar-mediated pathways, which are nonspecic
and neither acidic nor digestive (Nam et al. 2009). Internalization studies have been
performed in this respect in order to elucidate whether the size and type of
nanoparticles affect their entry route (Frana et al. 2011). An alternative is to nd a
nanostructure that could be resistant to acidic pH even when it is clathrin-mediated
endocyted (Harding et al. 1991; Dominska and Dykxhoorn 2010). In contrast,
pH-sensitive nanocarriers can be desirable for prodrug delivery (Shenoy et al. 2005).
When NPs are used as a vector for antigen delivery, they usually target APCs to
modulate the generated immune response. Different APCs have different prefer-
ences regarding NP size. For instance, DCs preferentially uptake virus-size particles
(20200 nm) while macrophages use the phagocytosis pathway to uptake larger
particles (>500 nm) (Zhao et al. 2014). Lymph-node DCs are a common target for
antigens when therapeutic tolerance is sought (i.e., autoimmune diseases and organ
transplantations); in these cases small nanoparticles (<40 nm) are very promising
candidates since they show signicant uptake into lymphatic vessels (Reddy et al.
2006). Particles with a diameter of less than 200 nm are endocyted by
clathrin-mediated endocytosis, whereas particles greater than 500 nm in size will
preferentially undergo caveolae-mediated endocytosis (Duan and Li 2013). It has
also been proved that NPs larger than 500 nm arrive to the lymph nodes in a
DC-dependent manner, whereas NPs smaller than 200 nm can drain freely to the
lymph nodes, where they are taken up by local cells (Klippstein and Pozo 2010).
Very small particles (below 10 nm) are internalized by pinocytosis.
Many NPs tend to aggregate in biological fluids, so their overall sizes change,
and this would influence their uptake by phagocytes. Thus, NPs must be tested for
aggregation in biological media prior to be used in further tests.
Particle shape is a determining factor in phagocytosis. Shape influences the
attachment and the internalization of the NP. The local geometry of nanoparticles at
the point of interaction with a macrophage denes the complexity of actin structures
required for the engulfment and the formation of the phagosome (Sahay et al. 2010;
Caron et al. 2013). For example, a macrophage attached to an ellipse can internalize
it in a few minutes or over 12 h depending on whether they are in contact with a
pointed end or a flat region, respectively (Duan and Li 2013). If the angle between
the membrane at the point of initial contact and the line dening the particle
curvature is <45, macrophages will successfully internalize the NP via actin-cup
and ring formation. However, if the angle is greater than 45, internalization is
inhibited (Sahay et al. 2010; Duan and Li 2013). Therefore, NPs with a shape
characterized by an angle >45 could be used to evade internalization by immune
innate cells. Phagocytosis is also dependent on the surface chemistry of the
nanoparticle and the proteins that form its corona. NPs opsonized by
immunoglobulins or iC3b will attach to FCR or CR, respectively, while SRs are
molecular pattern recognition receptors that are opsonin-independent. The receptor
ligand interaction promotes the rearrangement of the actin laments and the for-
mation of a phagosome, the size of which depends on the size of the
NP. Opsonization by plasma serum proteins can be avoided by surface modication
through the addition of natural (albumin, polysaccharides) (Moros et al. 2012) or
synthetic polymer coatings such as PEG and PEG containing copolymers (Owens
and Peppas 2006).
Zeta potential is another important factor that affects the phagocytosis of
nanoparticles. NPs with the lowest absolute Z potential value can avoid uptake by
innate immune cells (Oh and Park 2014; Duan and Li 2013). Positively charged
NPs exhibit a higher uptake, mostly by the clathrin-mediated pathway
(Harush-Frenkel et al. 2007), and this is probably due to a better interaction with the
anionic cell membrane. Negatively charged NPs can potentially bind to cationic
sites on the macrophage surface and be recognized by scavenger receptors. Neutral
and PEGylated NPs have the lowest uptake efciency, a much longer circulating
time, and better biocompatibility. Several studies have proved that hydrophobic
NPs induce a higher immune response than hydrophilic ones (Zhao et al. 2014).
Finally, conformation of polymer chains in the coating layer is another factor that
may be used to modulate the exposure of different groups on the nanoparticle, and
hence, the interactions of the nanoparticles with cells and proteins.
3.3.1 Methods to Measure NP Uptake
As indicated before, internalization of nanoparticles is dependent on their charac-

teristics such as particle size and shape, surface charge, surface composition, and
conformation. Consequently, when nanoparticles are intended for drug or antigen
delivery systems, the rational design of nanostructures and the subsequent evalu-
ation of the internalization process should be carried out.
There are several in vitro methods to evaluate cell internalization depending on
the type of NP. These assays are important when design nanovaccines (a high
uptake by antigen-presenting cells will help to provide a good immune response),
for the evaluation of the in vivo biodistribution (a high in vitro uptake by macro-
phages suggests that many NPs will be captured by liver and spleen macrophages
after in vivo administration), or for the study of intracellular localization (endo-
somes, lysosomes, cytoplasm, nucleus).
From all these methods, uptake of fluorescently labeled NPs can be easily
evaluated by flow cytometry after quenching of extracellular fluorescence to avoid
interference by the signal from particles attached to the cell membrane
(Dobrovolskaia and McNeil 2007). Moreover, the equipment can quantify the
fluorescence intensity, allowing a good comparison of the level of uptake of dif-
ferent fluorescently labeled NPs or of several cells.
Intracellular fluorescence NPs can be analyzed by conventional fluorescence
microscopy and also by confocal laser scanning microscopy (Fig. 3), which allows
the construction of 3D images by combining several z-stacks (Vicente et al. 2013a).
Colocalization studies can be performed using tracking probes that specically stain
different subcellular structures, such as lysosomes, mitochondria, or endoplasmic
reticulum (Moros et al. 2012).
In the case of nonfluorescent but electrondense NPs, they can be visualized by
transmission or scanning electron microscopy (Daz et al. 2008) and also in
combination with focus ion beam technology (Pastoriza 2008). Using this tech-
nology, it is possible to see gold nanoparticles inside a macrophage (Fig. 4).
Fig. 3 Cellular uptake of fluorescent protamine nanocapsules. Raw264.7 cells were incubated
30 min (a) or 3 h (b) with 50 g/ml of protamine nanocapsules labeled with rhodamine. Cell
nucleus was stained in blue with DAPI
Fig. 4 Uptake of gold NPs by a macrophage. Images taken by SEM-FIB technology at different
resolutions: a macrophage; b detail of a section; c, d same section at higher resolutions
Alternatively, quantication of the endocytosis can be performed by the com-

parison of the cell density between control and NP-incubated cells by optical
microscopy (Frana et al. 2011), or evaluated indirectly by the luminol-based
detection method, in which the increase in luminescence is measured upon luminol
oxidation in phagolysosomes.
Several different techniques can be applied to understand the mechanisms of NP
uptake and these are mainly based on the specic inhibition of each of the endocytic
routes, either by blocking antibodies or by chemicals, such as cytochalasin D, 5-(N,
N-dimethyl) amiloride hydrochloride, chlorpromazine hydrochloride, or lipin III,
which inhibit phagocytosis, macropinocytosis, and caveola and clathrin-mediated
endocytosis, respectively (Frana et al. 2011). However, the large number of
possible endocytic routes (Table 3), makes very difcult, or even impossible, to
block all of them (Doherty and McMahon 2009).
3.4 Evaluating Toxic Effects from the Determination

of Oxidative Stress Markers
Reactive Oxygen Species (ROS) and Nitric Oxide (NO) play an important role in
the inflammatory response (Rehman et al. 2012). The toxicity of NPs may be due to
an excess of ROS induction, which leads to membrane damage and inflammation
(Daz et al. 2008). Assessment of ROS generation induced by NPs is, therefore,
useful to evaluate their potential toxicity (Xia et al. 2006; Foucaud et al. 2007).
Due to their ability to uptake particles actively, phagocytic cells (neutrophils,
macrophages, or dendritic cells) are the most widely used cells as experimental
models to test in vitro ROS production (Jones and Grainger 2009), although other
cell lines can also be selected (AshaRani et al. 2008; Wason et al. 2013;
Chompoosor et al. 2010; Ahamed et al. 2011). Elevated ROS may induce cell death
or the expression of certain genes involved in the activation of cell signaling
pathways (Hancock et al. 2001). ROS produced by the cells after interaction with
foreign particles can damage the surrounding tissue. As the biochemical reactivity
of ROS is indiscriminate, the vast majority of cells have defense mechanisms to
neutralize it, for example, using ubiquitous glutathione and glutathione peroxidase,
which can also be used as a stress marker (Jones and Grainger 2009).
3.4.1 Methods to Measure ROS
ROS can be measured directly in the producer cells or indirectly by studying their
effects on cell behavior or other biochemical reactions, such as glutathione per-
oxidase or lipid peroxidation assays (Jones and Grainger 2009; Roesslein et al.
2013).
The most commonly used in vitro tests to measure levels of ROS are based on
colorimetric, fluorescent, or chemiluminescent dyes, although there are other
methods that involve the use of electron spin resonance (ESR). Controls with NPs
alone are very important because some NPs are able to produce ROS by themselves
in cell-free systems (Dufn et al. 2007), they could interfere with the detection
systems, or NPs may agglomerate in culture media and absorb light or produce
signals that could alter the measurement (Roesslein et al. 2013; Kroll et al. 2009).
Fluorescence methods are very useful to monitor oxidative activity as they are
extremely sensitive (Foucaud et al. 2007). The most common assay for the direct
measurement of ROS can be performed with different nonfluorescent salts, such as
2,7-difluorescein-diacetate (DCFH-DA) or dichlorodihydrofluorescein diacetate
(H2 DCFDA), which is converted to fluorescent dichlorofluorescein (DCF) upon
intracellular oxidation (Keston and Brandt 1965). The emitted fluorescence is
directly proportional to the concentration of hydrogen peroxide. There are
numerous examples in the literature concerning the use of these assays to evaluate
the effects of NPs on cells (Daz et al. 2008; Roesslein et al. 2013; Kroll et al. 2009)
The detection can be measured by flow cytometry (Daz et al. 2008) but it can
also be quantied using a microplate reader, which allows the measurement of
multiple well plates to provide a large amount of data with low variability (Ahamed
et al. 2011; Wang and Joseph 1999; Sharma et al. 2012).
Optical imaging, such as confocal microscopy, is also used to detect the intra-
cellular production of ROS (Lee et al. 2011; Kuznetsov et al. 2011; Colon et al.
2010) and commercial kits are available for this purpose, e.g., MitoSOX Red (Xia
et al. 2006).
The GSH assay is based on the important role that reduced glutathione
(GSH) has in the antioxidant activity as this is one of the main mechanisms of
defense against oxidation (Sebasti et al. 2003). GSH can be measured by colori-
metric or fluorescence assays in in vitro cultures and it is considered as an oxidative
stress marker (Ahamed et al. 2011; Roesslein et al. 2013). One mechanism of action
of NPs could be to cause a decrease in the activity of GSH, which would lead to an
excess of ROS (Piao et al. 2011).
Immunocytochemistry is used to detect specic DNA lesions due to the presence
of ROS; for example, the OH radical-specic 8-hydroxydeoxyguanosine (8-OHdG)
is a good marker of oxidative DNA damage. Briefly, the assay uses cells incubated
with a monoclonal antibody against 8-OHdG and samples are then analyzed using
digital imaging analysis software (Knaapen et al. 2000; Schins et al. 2002).
Lipid peroxidation involves the oxidation of polyunsaturated fatty acids by ROS at
the cellular or organelle level (e.g., mitochondria). As a result, products such as
malondialdehyde (MDA) are formed. The measurement of lipid peroxidation is one
of the most widely used indirect indicators of the production of ROS and it is generally
performed with the detection of MDA, which can be measured by the fluorescent
thiobarbituric acid reactive substances (TBARS) assay (Potter et al. 2011).
3.4.2 iNOS Detection
Inducible nitric oxide synthase (iNOS) is a pro-inflammatory enzyme that regulates

the expression of NO and it is involved in the pathogenesis of inflammatory diseases
(Rehman et al. 2012). Activation of iNOS in macrophages in response to
lipopolysaccharide (LPS) stimulus or ROS results in the production of NO and tumor
necrosis factor- (TNF-) (Hirst et al. 2009), inducing inflammatory responses. It has
been seen that this inflammatory response, mediated by iNOS, can be modulated by
different polymer NPs. These NPs can suppress the expression of iNOS, reducing the
production of NO and thus working as a potential anti-inflammatory agent (Yoo et al.
2013; Shen et al. 2013). NO is involved in different physiological processes,
including regulation of blood pressure, neural communication, and immune response
(Bryan and Grisham 2007; Palazzolo-Ballance et al. 2007).
iNOS can be analyzed by ELISA (Koh et al. 2011), fluorescent labeling
(Pascarelli et al. 2013), or chemiluminescent Western Blot (Brady et al. 1997). The
iNOS mRNA expression levels can also be measured by reverse transcription
polymerase chain reaction analysis (Ma et al. 2010). Recently, an electrochemical

immunosensor was developed to detect directly the presence of iNOS in neuronal
cell culture without a probe or labeling (Koh et al. 2011).
3.5 Cytotoxic Immune Cells
During the immune response, several cytotoxic cells such as Natural Killer,
lymphokine-activated killer cells, and cytotoxic T lymphocytes (CTL) can be
activated. These systems contribute to the immunity against viral pathogens, par-
asites, and neoplastic transformation, as well as hosting pathology associated with
tissue and bone marrow graft rejection and acting in a variety of autoimmune
diseases (Heusel et al. 1994). These cytotoxic cells depend primarily on the
perforin/granzyme system to kill their cell targets (Shresta et al. 1998). CTL (CD8+
lymphocytes) are characterized by typical lysosomal granules and by the expression
of a characteristic pattern of surface molecules (Fig. 5). They target specic cells on
the basis of cell-surface antigen recognition, presented in context with molecules of
class I major histocompatibility complex (MHC) (Groscurth 1989). However, the
CTL response against T-dependent antigens requires the cooperation of CD8+ T
effector lymphocytes with CD4+ T helper lymphocytes, mostly due to their
dependence on IL-2, which is provided by activated T helper cells.
Fig. 5 Recognition of infected/tumoral cell by a cytotoxic T cell. A pre-cytotoxic cell recognizes

the foreign peptide through its specic TCR. After activation, it becomes cytotoxic cell with the
help of IL-2 provided by T helper cells, releasing now lytic granules able to kill the target cell
For this reason, the in vitro cytotoxic T lymphocyte assay is an excellent can-
didate for the evaluation of potential immunotoxicity that may be induced by
polymer nanoparticles (Burleson et al. 2010). Moreover, the CTL activity induced
by an antigen may be enhanced by some nanomaterials and this should be explored
in vaccines based on nanocarriers (Vicente et al. 2013a, b, 2014; Cui et al. 2003) for
infectious or therapeutic purposes.
3.5.1 Methods to Measure Cytotoxic Activity
Several methods are available to measure cytotoxic activity and these are based on
the characteristics of these cells and the mechanisms of cell lysis:
Common methods to measure the cytolytic activity are based on readily
detectable compounds that are generated or released after cell lysis. In this case,
the target cells are labeled with radioactive sodium chromate (51Cr). This has
been the most widely used method over the last few decades and it is the gold
standard to measure cytotoxicity induced by NK and CTL (Lee-MacAry et al.
2001; Jerome et al. 2003).
Other compounds that can be quantied in damaged cells include lactate
dehydrogenase (LDH) release (Wonderlich et al. 2006). In these cases, the
addition of cytotoxic cells to target cells leads to the release of chromate or LDH
and this provides a measure of cytotoxicity.
A classical method that is extensively used is the measurement of the IFN-
released by the CTL. Nevertheless, this method cannot be employed to evaluate
the cytotoxic activity but only the differentiation and maturation of CD8+ T cells
(Parronchi et al. 1992).
DNA fragmentation assay by labeling target cells with [3H]thymidine. After the
addition of the CTL, the target cells will present DNA fragmentation.
These DNA fragments are washed away and the total radioactive DNA is
measured in a beta radioactive counter and compared with that in the intact cells
(Wonderlich et al. 2006).
Detection of granzyme B by enzyme-linked immunosorbent spot (ELISPOT).
Granzyme B is a serine protease that is released by CTL and NK cells and it is
involved in the mechanism of lysis of the target cells (Shafer-Weaver et al.
2003).
Detection of CD107a and b molecules with a fluorescent specic antibody by
flow cytometry. These molecules will be present on the cell surface of CD8+
cells after the degranulation of cytotoxic granules (Betts et al. 2003).
Flow cytometry can also be used to analyze CTL-induced cell death by labeling
the target cells with a membrane dye, such as PKH-26, and a DNA intercalating
marker, such as TO-PRO-3 iodide (TP3) (Lee-MacAry et al. 2001). The cyto-
toxicity is determined by the relative number of live target cells (PKH26+)
compared with the dead, permeabilized cells (PKH26+TP3+).
The measurement of activated caspase-3 in target cells is another method that is

used to study CTL-induced cell death using flow cytometry (Jerome et al. 2003).
Finally, flow cytometry also allows the responsive CTL to be measured using
multimeric forms of the MHC I or HLA molecules bound to the antigenic
peptide (Wooldridge et al. 2009; Yagi et al. 2006; Yao et al. 2008a).
3.6 Cytokine Production
Immune responses are affected and mediated by the release of cytokines (Table 2),
which play an important role in T cell-mediated immunity, inflammatory responses,
cancer, autoimmunity, and allergy (Katial et al. 1998). Cytokines produced by
cultured immune cells represent a widely accepted way to estimate the inflamma-
tory properties of a test material, including polymer nanoparticles (Dobrovolskaia
2013).
Among innate cells, macrophages can be divided into pro-inflammatory classi-
cally activated M1 macrophages, which mainly produce IL-1 and IL-1, IL-6,
IL-12 and TNF-, and anti-inflammatory alternatively activated M2 macrophages,
which secrete tumor growth factor (TGF)- and IL-10 and regulate the immune
response. Natural killer cells secrete principally TNF- and IFN-; neutrophils and
mast cells, IL-12 and TNF-, respectively, and dendritic cells secrete IL-12 and
IFN- (Gonzlez et al. 2006; Steinman et al. 2003).
Lymphocyte activation is strongly influenced by the cytokine environment and
these activated cells are subsequently able to produce more cytokines (Table 2).
Measurement of the cytokines released by activated cells in the presence of NPs will
help to determine the type of immune response induced by the tested nanomaterials
including polymer nanoparticles. Thus, the type of secreted cytokine determines the
type of cellular response. For example, Th1 polarization is induced by IFN-, IL-2,
and TNF- cytokines, and this mainly induces cell-mediated immunity and
phagocyte-dependent inflammation. Th2 cells, which produce IL-4, IL-5, IL-6, IL-9,
IL-10, and IL-13, induce B cell proliferation/differentiation, secretion of antibodies
(including those of the IgE class) and eosinophil accumulation (Romagnani 2000;
Constant and Bottomly 1997). Antigen-specic B cells primed by Th1 cells also
produce cytokines associated with type 1 immune responses, such as IFN and
IL-12, while B cells primed by Th2 cells produce IL-2, IL-13, and IL-4 cytokines
that are often associated with allergic responses (Lund 2008).
Modulation of the immune response through cytokines is one of the roles played
by many designed NPs, either for the design of vaccines, treatment of immune
diseases, or induction of tolerance (Zolnik et al. 2010). For instance, it has been
demonstrated that myelin antigen-coupled to poly (lactide-co-glycolic acid (PLGA)
NPs were able to ameliorate signicantly the ongoing disease and subsequent
relapses in a mouse model of multiple sclerosis by decreasing the inltration of Th1
(IFN-) and Th17 (IL-17) cells as well as inflammatory monocytes/macrophages
(Aggarwal et al. 2009).
In addition, NPs can also be used to deliver cytokines directly. Encapsulation of
the leukemia inhibitory factor (LIF), a known tolerogenic cytokine, in PLGA- NPs
functionalized with a CD4 antibody showed efcient polarization of the CD4+ T
cells to the desired phenotype (Park et al. 2010). These NPs were tested in mice and
they showed a good efciency for the delivery of the cytokine, the prevention of
IL-6-driven Th17 cell development, the expansion of regulatory FOXP3+CD4+ T
cell numbers, and the survival of a heart allograft that was otherwise rejected 7 days
after implantation.
3.6.1 Methods to Measure Cytokines
Several types of immune cells (peripheral blood mononuclear cells, macrophages,

puried T cells, etc.) can be incubated at different time points with NPs and the
individual secreted cytokines can be directly measured on the cell supernatants by
traditional enzyme-linked immunosorbent assay (ELISA) (Prach et al. 2013; Wang
et al. 2014b; Zhang et al. 2014).
Multiplex kits for the simultaneous detection of several cytokines have also been
designed. Multiplexed ELISA assays are microarrays that are built by printing
nanospots of distinct capture antibodies in each well of a multiwell plate (Sehgal
et al. 2014; Lonez et al. 2014; Young et al. 2014). Another alternative is the bead-
based assay, which involves the use of special beads coated with specic antibodies
against different cytokines. The beads are differentiated by their sizes and spectral
signatures, and they can be measured by flow cytometry or by Luminex technology.
Some of these kits allow the simultaneous quantication of up to 100 analytes
within a single sample (Vicente et al. 2013a, 2014; Climent et al. 2014; Dube et al.
2014; Jorquera et al. 2013).
Intracellular cytokines can also be evaluated by flow cytometry using specic
antibodies after cell permeabilization (Lee et al. 2013; Schlitzer et al. 2013; Stano
et al. 2011). This methodology can be combined with the use of specic antibodies
against surface markers that dene cell populations, thus allowing the characteri-
zation of the cytokine producing cell (Rosalia et al. 2013; Stano et al. 2013;
Demento et al. 2012; Macho Fernandez et al. 2012).
It is also possible to detect cytokine production at gene expression level by
measuring mRNA expression by real-time polymerase chain reaction (PCR)
(Halminen et al. 1999; Trojan et al. 2007). However, since gene upregulation does
not always correlate with the amount of protein, it is important to corroborate these
results by protein assays (Jones and Grainger 2009).
3.7 Changes in Cell Surface Markers and Methods

of Analysis
Immune cells (lymphocytes, macrophages, dendritic cells, etc.) have different

proteins on their surfaces, which dene their phenotype and activation state. When
NPs enter the body, they may modify the expression level of some of these proteins
on the cells, influencing the functional immune activities.
Studies on the surface markers are usually performed by flow cytometry after
incubation of the immune cells with fluorochrome-conjugated antibodies. For
example, our group measured changes in membrane expression of different inte-
grins and selectins (LFA-1, integrin 1, L-selectin CXCR4) in human peripheral
blood mononuclear cells incubated with NPs (Lozano-Fernndez et al. 2014). Other
groups have determined the maturation/activation status of dendritic cells by
evaluating markers such as HLA-DR, CD83, CD86, and CD54 after exposure to
NPs (Pfaller et al. 2010; Torres et al. 2011).
An alternative method to dene cell status is by the evaluation of the tran-
scriptional prole or the use of quantitative qPCR (Fruchon et al. 2009).
A novel biosensor with anti-CD-antibodies, called cytosensor, was recently
developed and this allows the quantication of the activation/signaling molecules
expressed in the membrane of immune cells (Wang et al. 2013).
3.8 Cell Migration and Methods of Analysis
Cell migration is crucial for the functional activity of the immune cells. It is a
physical process that requires changes in the morphology of the cell and the
dynamic interactions between a cell and the extracellular matrix. The
spatial-temporal coordination of many proteins involved in this process, such as
membrane receptor, kinases, components of cytoskeleton, adhesion molecules etc.,
is needed for efcient movement (Vicente-Manzanares and Horwitz 2011;
Huttenlocher and Horwitz 2011; Gardel et al. 2010).
Cell directional migration or chemotaxis plays a key role in different immune
mechanisms such as leukocyte inltration (migration of cells from blood to tissues),
antigen presentation (migration of dendritic cells from inflamed tissues to the
draining lymph nodes to present the antigen to helper T cells), tumor control, or
angiogenesis (vessels formation). As NPs may influence this cell migration, it is
important to study their effects on immune cells.
Cell migration is mediated by adhesion molecules and, as a consequence,
changes in their surface expression can be analyzed as explained above, but also by
direct migration studies. The in vitro studies to check cell migration are commonly
performed in 2D cultures but these do not really represent the in vivo situation
regarding cell adhesion, signaling or even morphology (Huttenlocher and Horwitz
2011; Olsen et al. 2013).
New alternatives are emerging and these include migration studies that are more
focused on recreating a realistic 3D environment (Friedl et al. 2012), microfluidic
platforms to analyze real-time cell migration and intravasation of tumor cells
(Chung et al. 2010), platforms to integrate cell-substrate impedance sensors to
investigate single cancer cell migration in 3D matrixes (Nguyen et al. 2013), or
polymer chips with three-dimensional microporosity to analyze the migration of
DCs (Olsen et al. 2013).
Numerous studies on the visualization of cell migration have benetted from the
intrinsic properties of NPs. For instance, mouse skeletal myoblasts (SkMs) were
loaded with fluorescent nanoparticles to track their movement by time-lapse con-
focal microscopy (Idris et al. 2009). Micron-sized iron oxide (MPIO) particles were
used to visualize the in vivo migration of dendritic cells using cellular magnetic
resonance imaging (MRI) (Rohani et al. 2011). Further studies are needed to
integrate the NP properties into new technologies.
3.9 Methods to Analyze Activation Pathways and Cellular

Responses Induced by NPs
A deeper characterization of all cellular mechanisms that are activated by NPs,

whether they are relevant for their activity or for their toxicological and immuno-
logical effects, is possible using -omics. High-throughput approaches allow the
measurement of changes induced by a treatment at gene (genomicstranscrip-
tomics) or protein expression (proteomics) levels. These methods can be used to
evaluate the safety of the nanoparticles, but also provide the possibility to analyze
cell behavior or potential biological activities in the presence of nanomaterials (v.g.
cells growing on 3D articial structures).
Genomic techniques applied to study the genomic expression induced by NP
treatment include RNA or cDNA arrays. In these techniques a solid surface, usually
a glass slide or a nylon membrane, is used and a library of nucleic acid probes are
attached on individual spots for each tested gene. This surface is hybridized with
the RNA or cDNA obtained from cells previously incubated with NPs. Control and
NP-treated samples are labeled with a fluorescent dye in the reverse transcription
step or during the RNA amplication step. Detection can also be achieved by
radiolabeling. The most commonly used dyes are Cy3 and Cy5, which are green
and red, respectively. The hybridization of the cDNA or RNA into the array is
performed with a mixture of both samples (dual channel) or with each sample in a
different array (single channel). After several washing steps, the fluorescence is read
using an array scanner and analyzed using specic software. Depending on the size
and the number of spots on each slide, they can be categorized as macroarrays,
arrays, or microarrays. This technique enables the monitoring of a large number of
genes at the same time, and one of the main advantages is that it can be applied for
the detection of unsuspected effects (overexpression or inhibition of some genes)

induced by NPs.
Another technique that is used to study gene expression is real-time quantitative
PCR (qPCR), a variant of the polymerase chain reaction that allows not only many
DNA copies to be obtained, but also allows their quantication with a fluorescent
reporter. This technique can also be carried out in high-throughput mode using
plates or arrays that are able to detect changes in the expression of multiple genes,
or on a smaller scale by analyzing a limited number of preselected genes. The
number of genes that can be monitored in a single experiment using a qPCR array is
not as high as in the RNA arrays, but the data analysis is much faster and the data
are more reliable (Knight 2001; VanGuilder et al. 2008). In fact, qPCR is con-
sidered to be the gold standard technique for gene expression measurements and it
is sometimes used to conrm the results obtained with the arrays.
There are numerous examples in the literature concerning the use of cDNA
and/or RNA microarrays followed by qPCR to study differential gene expression
induced by NPs. The most frequently affected genes include those involved in the
cell cycle, apoptosis, chemokines, complement cascade, or metabolic stress.
Ronzani et al. studied cell viability and gene expressions in rat NR8383 and
human THP-1 monocytic cell lines in the presence of polymer nanomaterials. They
measured the expression of genes involved in oxidative damage (NCF1), inflam-
mation (NFB, TNF, IL6, IL1), autophagy (ATG16L), and apoptotic balance
(PDCD4, BCL2, CASP8). A decrease in the viability of the rat NR8383 cells was
observed along with an upregulation of ATG16L, BCL2, and TNFA genes. On the
other hand, an increased viability of the THP-1 cells was observed and this effect
was accompanied by NCF1, NFB, and IL1 downregulation (Ronzani et al. 2014).
Omidi et al. employed a microarray gene expression proling methodology and
reported genotoxicity induced by cationic lipid oligofectamine nanoliposomes in
human alveolar epithelial A549 cells. They targeted 200 genes and found changes
in several cell defense and apoptosis-related genes (Omidi et al. 2008).
In addition to the applications described above, qPCR is also used to conrm the
presence of siRNA or DNA on the target cells upon transfection using NPs loaded
with these nucleic acids for gene therapy. For example, in vitro transfection studies
conrmed that the incorporation of poly(propyl acrylic acid) (PPAA), a polymer
designed to disrupt lipid bilayer membranes within a sharply dened pH range, into
chitosan-DNA nanoparticles enhanced gene expression in both HEK293 and HeLa
cells compared to chitosan nanoparticles alone (Kiang et al. 2014). The delivery of
the POSTN siRNA into the 3 integrin-positive tumor endothelial cells reduced
the expression of the gene, as shown by qPCR.
Another example is the use of dendrimers to transfect siRNA into human
immunodeciency virus (HIV)-infected lymphocytes (Weber et al. 2008). The
efcient transfection of a glyceraldehyde 3-phosphate dehydrogenase GAPDH
siRNA, using amino-terminated carbosilane dendrimers (CBS) as carriers, was
assessed by qPCR. The siRNA/CBS dendriplexes were able to silence GAPDH
expression and reduce HIV replication in SupT1 (human leukemia T lymphocytes)
and in peripheral blood mononuclear cells (PBMC).
Conrmation of gene silencing can also be carried out by Western blot. For
instance, gene silencing using Arg-Gly-Asp(RGD)-labeled chitosan NPs with
siRNA to target several growth factors has proven to be efcient in vitro in certain
ovarian cell lines, with a signicant reduction in the levels of protein expression
determined by Western blot (Han et al. 2010).
4 Nanovaccines
Some NPs can be engineered to serve as vaccine delivery systems (Xiang et al.
2013). In addition to the attenuated or dead pathogens that are conventionally used
in vaccines (Daniel et al. 1992; Wyatt et al. 2004), in recent years several vaccines
containing recombinant proteins, synthetic peptides, carbohydrates, lipids, or DNA
have increasingly been used in new prototypes of vaccines (Xiang et al. 2006;
Rabinovich et al. 1994). These subunit vaccines have advantages compared to the
traditional ones but they are not immunogenic enough in their own right and they
need to be coadministrated with a potent adjuvant to achieve a protective immune
response. Very few adjuvants are currently approved for human use in vaccines.
The most widely used is Alum (aluminum salts) and, more recently, some
oil-in-water emulsions like MF59 and AS03, liposomes and AS04 (composed of
monophosphoryl lipid A adsorbed to Alum) have also been approved (Xiang et al.
2013). Other NPs (liposomes, chitosan, gold, etc.) are under investigation for either
parenteral or mucosal administration. All of these materials are used in attempts to
increase the stability and immunogenicity of the antigen by different mechanisms of
action (Mbow et al. 2010).
The use of NPs in vaccines could have several advantages, including protection
of the associated antigen from degradation, enhanced antigen internalization by
antigen-presenting cells, and the release of antigen in a controlled and sustained
manner (De Temmerman et al. 2011). In addition, the physicochemical character-
istics of NPs can be tuned in order to modulate the immune system towards an
antibody or T cell-mediated immune response, or even towards a balanced
response. It is important to design an appropriate vaccine for each case since
different types of responses are required depending on the type of pathogen. For
instance, in the case of extracellular bacterial infections an antibody-mediated
response is preferred, whereas a T cell-mediated response is usually necessary for
viral and other intracellular pathogens (Xiang et al. 2013).
The use of nanocarriers has expanded the administration routes and needle-free
strategies such as intranasal (Vicente et al. 2013a) or intradermal (Cui et al. 2003)
routes have been explored. In both cases, the presence of surveillance immune cells
in the barrier tissues increases the efcacy of antigen presentation and stimulation of
the immune system and, hence, the efcacy of the vaccines.
A multifunctional antigen nanocarrier consisting of a hydrophobic nanocore,
which can allocate lipophilic immunostimulants such as imiquimod, a TLR-7
agonist, and a polymer corona made of chitosan (CS), intended to associate antigens
and facilitate their transport across the nasal mucosa, has been shown to induce a
high immune response against the hepatitis B surface antigen in mice after intra-
nasal administration (Vicente et al. 2013a). This immune response was Th1/Th2
balanced (cellular/humoral) in comparison to the conventional alum adjuvant,
which induces a biased Th2 (humoral) response. This balanced immune response
has also been achieved with other core-shell nanocarriers such as the squalene core
and polyglucosamine (PG) shell nanocapsules (Vicente et al. 2014).
Polymer nanocarriers also allow the use of DNA for vaccination and a balanced
immune response is again obtained (Cui et al. 2003; Xu et al. 2004). In this case,
the DNA itself is able to induce both Th1 and Th2 responses, but the use of a
nanocarrier to transport the DNA and other co-stimulatory molecules could enhance
the immune response and increase the efcacy of the genetic vaccination (Cui et al.
2003).
4.1 Nanoparticles as Antigens
It has been reported that some NPs, such as certain liposomes, may be antigenic by
themselves and could induce the production of specic antibodies. However, most
of these studies were performed in the presence of an adjuvant (Alving et al. 1996;
Banerji and Alving 1981, 1990; Richards et al. 1983; Fogler et al. 1987; Wassef
et al. 1984, 1990) or with a prior inflammatory environment (Dobrovolskaia 2013;
Richards et al. 1983). In general, most NPs behave as haptens and they are not
immunogenic unless they are conjugated to a protein carrier like bovine serum
albumin (BSA) and are administrated with a strong adjuvant. This is the case of
poly(amido amine) (PAMAM) dendrimers and some liposomes (Lee et al. 2001,
2004a).
Surface coating of NPs with poly(ethylene glycol) (PEG) and other components,
such as amphiphilic polymers, has been investigated in an effort to reduce im-
munogenicity. PEG is a polymer that is commonly used as a coating to decrease the
immunogenicity of modied protein drugs and drug-delivery vehicles
(Dobrovolskaia 2013). The case of PEG is of particular interest since there are
controversial studies in which the antigenic properties of PEGylated nanostructures
have been described. It is still not well established whether PEG is immunogenic by
itself or whether the PEGylation method could be responsible for this immuno-
genicity. However, there are numerous reports that show the rapid clearance of a
second dose of PEG-coated liposomes from the circulation (Ishida and Kiwada
2008; Laverman et al. 2001). It is not clear if this clearance, called accelerated
blood clearance (ABC), is mediated by the generation of anti-PEG-specic IgM
antibodies or if other proteins could be contributing to this phenomenon. Many
factors are involved in the ABC process and these include doses, interval between
rst and second dose, particle size, charge, liposomal composition, and PEG density
(Dobrovolskaia 2013). Splenic and hepatic macrophages could also contribute to
this ABC effect (Abu Lila et al. 2013). Nevertheless, it has been demonstrated that
NPs could improve the therapeutic effect of some drugs by avoiding the production
of antibodies against these systems (Perkins et al. 1997; Ramani et al. 2008a, b;
Kosloski et al. 2010).
4.2 Protocols to Evaluate the Immune Response to Vaccines
The parameters which should be measured for the evaluation of a nanovaccine are
summarized in Table 4.
Induced cellular and humoral responses have usually been measured by different
methods, such as ELISA (Vicente et al. 2014; Mottram et al. 2006; Kasturi et al.
2011; Fairley et al. 2013), ELISPOT (Enzime-Linked InmunoSpot) (Kasturi et al.
Table 4 Steps in the evaluation of a nanoparticle

Parameters Methods
In vitro (before and after addition of antigen)
Physicochemical Composition HPLC, MS
characterization (in Particle size and polydispersity TEM, SEM, DLS, STPS
different physiological
Shape TEM, SEM
media)
Zeta potencial DLS, PCD, STPS
Surface area BET, STPS
Coating TEM, SEM, DLS
Solubility/aggregation HPLC, MS/DLS, TEM, SEM
Percentage of associated antigen ELISA, Western blot, UVVis
Simulate the antigen release under ELISA, Western blot, UVVis
physiological conditions
Stability studies at different DLS, BET, TEM, SEM,
temperatures/times/after
lyophilization or sterilization
Protein interaction Protein binding Ultracentrifugation,
2D-electroforesis, MS, SPR, SEC,
ITC, QCM
Conformational changes Spectroscopy techniques (circular
dichroism, fluorescence, FTIR)
Toxicity studies Cell viability Colorimetric test (MTT, LDH),
xCELLigence, Anexin V/IP
Hemocompatibility (Hemolysis, UVVis (detection of hemoglobin
aggregation, agglutination, or CMH) Aggregometer, OM
coagulation studies, platelet Coagulometer, ELISA, brinolytic
activation) activity, Cell counter, PCLM,
TEM, SEM,
Oxidative stress (ROS, iNOS) Fluorescent labelling (plate reader,
FC, CM), GSH assay, TBARS,
ELISA, western blot, RT-PCR
Genotoxicity Comet assay, bacterial reverse
mutation assay, micronucleus assay
(continued)
Table 4 (continued)
Parameters Methods
Activation/inhibition of Phagocytosis Flow cytometry
immune components Confocal microscopy
Optical microscopy
Routes of uptake Chemical inhibitors
Monoclonal antibodies
siRNA
Complement activation Western blot, ELISA
Cytokine release ELISA, bead-based assay (FC,
Luminex), FC
Changes on molecular Flow cytometry
markers/migration studies 2D-3D cell cultures
Cytotoxicity by CTL and NK cells Cr release, LDH, IFN release,
51
Cell activation Genomics and proteomics, western

blot, RT-PCR
Presence of Sterility Bacterial, yeast and fungus tests
contaminants Endotoxin content RPT, LAL,
In vivo (animal models: mouse, rat, rabbits, non-primate humans)
Toxicity studies Acute and chronic exposure Histology
Hemocompatibility Changes in the number of RBC RBCC
Humoral immune Primary and secondary responses ELISA, ELISPOT
response Antibody prole (IgM, IgG, ELISA, lateral flow test, bead-based
subclasses of IgG, IgA) assays
Routes of administration ELISA
(parenteral, intranasal)
Cellular response CTL Assay Cr release, LDH, IFN release,
51
Cytokine production by specic ELISA, bead-based assays

CD8+ cells
Modulation of immune Ratio of IgG1/IgG2a ELISA, bead-based assays
response Cytokine prole (Th1/Th2) ELISA, bead-based assays
T cell dependent Antibody or plaque forming cells PFC
antibody responses Antibody prole (IgM, IgG) against ELISA
(T-DAR) a known antigen
BET BrunauerEmmettTeller (BET) surface area analysis, CD cluster of differentiation, CM confocal
microscopy, CMH cyanmethemoglobin, CTL cytotoxic T cells, DLS dynamic light scattering, ELISA
enzyme-linked immunosorbent assay, ELISPOT Enzyme-linked immunoSpot, FC flow cytometry, FTIR
Fourier transform infrared spectroscopy, GSH gluthatione, HPLC high performance liquid
chromatography, ITC isothermal titration calorimetry, LAL limulus amebocyte lysate, LDH lactate
dehydrogenase release, MS mass spectrometry, OM optical microscopy, PCD particle charge detector,
PCLM phase contrast light microscopy, PFC plaque-forming cells, QCM quartz crystal microbalance,
RBC red blood cells, RBCC red blood cell counting, RPT rabbit pyrogen test, RT-PCR real time
polymerase chain reaction, TEM transmission electron microscopy, SEC size-exclusion chromatography,
SEM scanning electron microscopy, SPR surface plasma resonance, STPS Size-Tunable Pore Sensors,
TBARS thiobarbituric acid reactive substances assays, UVVis ultravioletvisible spectroscopy
2011; Walsh et al. 2013), or CTL assays (Yoshikawa et al. 2008; Zhang et al.
2015).
Nowadays, novel techniques have arisen, such as surface plasmon resonance
(SPR) (Brakha et al. 2014; Pedersen et al. 2014) to evaluate the presence of
antigen-specic antibodies, or the xCELLigence system that enable a real-time
measurement of cytotoxic activity (MacLean et al. 2014; Pham et al. 2014).
While in vivo results provide more physiological and realistic probes of the
response to the vaccine, in vitro data can be useful as a prior screening method
when different prototypes have to be evaluated, and they can even provide infor-
mation about the molecular mechanisms behind the immune response.
Prior to the use of NPs in a vaccine, it is important to carry out a physico-
chemical characterization of the formulation by measuring the particle size and zeta
potential of the formulations before and after the antigen association process, since
antigen incorporation could modify important properties of the prototype. The
percentage of association of the antigen to the nanomaterial must be evaluated,
either directly or by measuring the concentration of free antigen remaining in the
supernatant by ELISA or Dot Blot (Vicente et al. 2013a, b, 2014). In addition, it is
also useful to simulate the antigen release under physiological conditions and to
carry out stability studies at different temperatures (Fairley et al. 2013).
These formulations are frequently lyophilized and studies on the stability of the
dried powder are very important. The usual protocol involves measuring the particle
size, polydispersity index, and antigen association at different times after a pro-
longed period (Vicente et al. 2013b; Prego et al. 2010).
4.2.1 Humoral Response to Vaccines
In vivo experiments designed to evaluate immune response to vaccines (Table 4)

are usually based on the immunization of different animal models such as rodents,
rabbits, and non-primate humans (mainly monkeys) (Vicente et al. 2013b; Bok
et al. 2011; Kim et al. 2014a; Muttil et al. 2010).
The evaluation of the humoral response is based on the analysis of the antibody
levels produced in serum or mucosa at different time points after immunization of
the animals with the nanovaccine. Different numbers of doses would be adminis-
tered depending on the type of vaccine and the route of administration. This
approach allows the evaluation of both the primary and secondary immune
responses, which are mostly represented by an increment in specic IgM and IgG
antibodies directed against the antigen carried out by the nanoparticles,
respectively.
The type of NP and the route of administration can influence the response and it
is interesting to evaluate different antibody isotypes. Thus, the IgG subclasses
(IgG1 and IgG2a) should be measured in order to determine the type of response,
and in the case of mucosal administration, IgA should also be quantied (Okamoto
et al. 2009).
The ratio IgG1/IgG2a helps to analyze the main type of adaptive immune
response activated by the vaccine (mainly mediated by Th1 or Th2 lymphocytes).
While a predominant Th2 activation induces a humoral response characterized by
IgG1 production, the presence of the IgG2a subtype is related to cellular responses
mediated by Th1 lymphocytes (Vicente et al. 2013b; Kasturi et al. 2011; Fairley
et al. 2013). It is worth noting that inbred mouse strains with the Igh1-b allele
(C57BL/6, C57BL/10, SJL and NOD) do not have the gene for IgG2a and they use
the IgG2c isotype instead (Martin et al. 1998).
The most common method to evaluate the humoral immune response is ELISA.
In this technique, the antigen of interest is immobilized and serum samples are
tested to quantify specic antibodies by comparison with a standard curve of known
amounts of an antigen-specic antibody (Vicente et al. 2014; Kasturi et al. 2011) or
expressed as serum titer, i.e., the greatest dilution of the serum that still gives a
positive result (Mottram et al. 2006; Fairley et al. 2013).
Another conventional method that is used to evaluate the humoral response
involves the detection of B cells that produce antibodies by a technique called
ELISPOT, also known as the antibody-secreting cell (ASC) assay. This technique
was rst described in 1983 by Czerkinsky et al. (1983) and Sedgwick and Holt
(1983) and it is based on an ex vivo culture of splenocytes or lymph node cells from
immunized mice in special plates coated with the antigen of interest. After dis-
carding cells, the presence of specic antibodies is detected by an enzymatically
labeled secondary antibody and the number of spots per well is then quantied
(Kasturi et al. 2011; Walsh et al. 2013).
The methods described above could also be useful for the quantication of
specic antibodies induced by the nanostructure itself. Both competitive and
indirect ELISAs are used to measure the specicity of antibodies against several
nanostructures such as liposomes or PAMAM dendrimers (Alving et al. 1996;
Banerji and Alving 1981, 1990; Richards et al. 1983; Fogler et al. 1987; Wassef
et al. 1984, 1990; Lee et al. 2001, 2004a).
4.3 T Cell-Dependent Antibody Response Tests
The potential immunotoxicity of nanoparticles can be evaluated in vivo using the T

cell antibody response (TDAR). These assays measure the humoral response to a
known antigen that requires the involvement of T and B lymphocytes and
antigen-presenting cells together with cell products such as cytokines. If an alter-
ation in the level of antibody production is detected, further studies are required in
order to identify the cell population involved (Piccotti 2008). In the case of
nanomaterials, specic assays have not yet been designed to analyze the TDAR, so
standard protocols used for other pharmaceuticals can be applied to NPs since they
do not seem to interfere with this assay (Herzyk and Gore 2004). Two assays can be
performed to assess the primary TDAR: antibody-forming cell (AFC) and ELISA
(Table 4)
The AFC or plaque-forming cell (PFC) technique is considered to be the gold

standard. Briefly, mice or rats receiving NPs are immunized with a model antigen,
such as sheep red blood cells (SRBCs) and their spleens are removed 45 days
later. Splenocytes are harvested in agar plaques that contain SRBCs and comple-
ment. The in vitro production of specic IgM directed against SRBCs is measured
by the formation of plaques (clear areas) due to hemolysis produced around a single
antibody-forming B cell. The number of plaques corresponds to the number of
antibody-producing cells, and can be altered if NPs affects to the generation of these
cells. However, this assay is limited because only evaluates the number of specic
antibody-producing plasma cells in the spleen.
An alternative method has been developed that measures the presence of
SRBC-specic antibodies in the serum, namely the SRBC-specic IgM ELISA. In
this case, the blood from animals receiving NPs is collected 56 days after
immunization with SRBCs, when the peak antibody production is detected. This
assay is easier to perform than the PFC and the serum can be frozen and used
several times (Piccotti 2008; Ladics 2007; Dobrovolskaia et al. 2009).
To perform a TDAR assay, a recognized T-cell-dependent antigen must be used
to obtain a robust antibody response. In recent years, the SRBCs antigen has been
replaced by the keyhole limpet hemocyanin (KLH), a very immunogenic and stable
antigen that can be used in multiple species (mice, rats, etc.) (Piccotti 2008; Herzyk
and Gore 2004; Dobrovolskaia et al. 2009). Besides SRBCs and KLH, other anti-
gens have also been proposed and these include tetanus toxoid, hepatitis B surface
antigen, and bacteriophages (Lebrec et al. 2014). A routine TDAR assay takes
28 days, with the NPs administered daily during the whole assay. There are two
variants of this assay depending on the endpoint. The rst variant involves a single
immunization with KLH at day 24 and serum is collected at the end of the assay to
measure the specic anti-KLH IgM levels (early response) (Piccotti 2008). In the
second variant, the immunization with KLH is carried out on day 14 and specic
IgM and IgG are measured in serum at days 19 and 29, respectively (early and late
responses), by ELISA or another appropriate immunoassay. Gore et al. established a
protocol using rats immunized with KLH and they demonstrated that this assay can
be comparable to the one using SRBCs (Herzyk and Gore 2004; Gore et al. 2004).
A detailed protocol to perform the TDAR assay in rodents has been described by
Plitnick and Herzyk (2010). The TDAR should be performed when unintended
immunotoxicity is detected in other assays or when a specic target is not well
identied, in order to evaluate alterations in the immune function (Piccotti 2008;
ICH 2006). The TDAR assay should be conducted in the same species and strain of
animal in which the toxicity effect was analyzed, and both sexes should be used.
A positive immunosuppressant control is included in which cyclosporine A,
dexamethasone, cyclophosphamide, azathioprine, or prednisolone are employed. The
suppression may be more effective for IgG than for IgM antibodies, so both Ig classes
should be measured in order to increase the sensitivity of the assay (Gore et al. 2004).
TDAR is designed to detect immunosuppression but, in some cases, NPs could
have immunostimulatory effects, although the signicance of these activation
effects remains controversial (Lebrec et al. 2014).
4.4 Role of Size, Charge, and Shape
Different physicochemical parameters dene the biological behavior of nanoma-

terials. Among these, particle size, charge, and shape have proven to be the main
factors that determine the immune response. Numerous studies have shown the
relation between nanoparticle size or charge and the immune response elicited.
However, particle shape has not been investigated as thoroughly as the other fac-
tors, probably due to the limited availability of techniques to produce nonspherical
polymer particles (Champion et al. 2007). It is, however, crucial for the rational
design of delivery systems to understand how size, charge, and shape can affect the
type of response generated.
4.4.1 Influence of Size
Size influences the type of APC that will internalize the nanostructure (Xiang et al.
2013). As indicated previously, small particles (20200 nm) are generally inter-
nalized through endocytosis via clathrin, caveola, or by their independent receptors.
This NP size range is also supposed to induce a stronger immune response than the
larger counterparts (Mottram et al. 2006; Pelkmans 2005; Fis et al. 2004a, b;
Manolova et al. 2008). Different groups have postulated that the optimal size for
NPs to be uptaken by DC is 50 nm (Aoyama et al. 2003; Nakai et al. 2003).
Regarding the elimination of NPs, it has been reported that most NPs are
eliminated from circulation by liver and spleen (they are actively internalized by
macrophages) (Owens and Peppas 2006; He et al. 2010; Banerjee et al. 2002), and
this effect increases with larger nanoparticles (>200 nm). In addition, it has also
been shown that nanoparticles with a size of 30110 nm accumulate in lungs, heart,
kidney, and stomach (Banerjee et al. 2002).
The type of immune response could also be modulated by the NP size. Despite
the fact that there is some controversy in this eld, it has been reported that 40 nm
NPs induce Th1 and CD8+ type responses, whereas 100 nm NPs promote Th2
responses (Mottram et al. 2006; Fis et al. 2004a, b).
4.4.2 Influence of Surface Charge
As explained above, charged nanoparticles are taken up more efciently than

neutral nanoparticles of the same size (Chrastina et al. 2011), and it has been
reported that, in general, cationic NPs induce stronger responses than anionic or
neutral NPs (Foged et al. 2005; Thiele et al. 2003).
4.4.3 Influence of Shape
There is a relationship between NP shape and cellular uptake and tissue biodistri-
bution (Xiang et al. 2013). It has been reported that spherical particles and oblate
ellipsoids are more efciently internalized than rod-shaped or prolate ellipsoids
(Verma and Stellacci 2010; Sharma et al. 2010). Cylindrical and discoidal NPs have
been shown to evade internalization by macrophages (Champion and Mitragotri
2006, 2009). Nonspherical NPs are accumulated to a greater extent on tumoral
tissues than their spherical counterparts and disc-shaped NPs accumulate more in
most organs, except for the liver, than the hemispherical NPs (Tao et al. 2011;
Decuzzi et al. 2010).
4.5 Correlation Between In Vitro and In Vivo Assays

Regarding Vaccines
Although vaccine efcacy is usually evaluated by in vivo challenge assays, in vitro

data can be useful for a prior screening when different prototypes are to be eval-
uated, and they can also contribute to a better understanding of the mechanisms
involved in the interaction between nanostructures and biological components.
A good correlation has been found in several studies between in vivo and in vitro
methods, including hemolysis induction, determination of pyrogenicity, cytokine
release, complement activation and leukocyte procoagulant activity (Dobrovolskaia
and McNeil 2013a).
Despite the difculty associated with in vitro assays in terms of reproducing the
in vivo physiological environment (containing fluids, matrix, cell interactions,
cell-NPs), there are several examples that indicate how in vitro assays could provide
similar data to those obtained in vivo. This assertion is supported by our results
using a multifunctional chitosan nanocapsule including the TLR7 agonist imiqui-
mod and the hepatitis B surface antigen. The NPs that induced high levels of
specic IgG after intranasal immunization in mice had also induced an in vitro
response after their uptake by macrophages, followed by the secretion of
pro-inflammatory cytokines (IL-6 and TNF-) (Vicente et al. 2013a). Similar
behavior was observed with a vaccine against hepatitis B or influenza virus con-
taining polyglucosamine. This versatile vaccine was able to potentiate and modulate
the immune response upon intramuscular administration in mice, but also enhanced
the intracellular delivery of imiquimod and antigen, as registered by the release of
cytokines (Vicente et al. 2014).
Chitosan NPs containing hepatitis B surface protein also proved to have positive
effects on the DC maturation in vivo; intranasal and intramuscular immunizations
with chitosan NPs induced high serum antibody titers (Tafaghodi et al. 2012). The
same behavior has been described for PLGA nanocapsules incorporating the
rMOMP antigen from C. trachomatis, which induced the secretion in vitro of IL-12
(Th1 type cytokine) and triggered in vivo elevated Th1 antibody responses and
cytokine release (IL-12 and INF) (Fairley et al. 2013).
5 Interaction of NPs with Other Blood Components:

Hemocompatibility
An understanding of the interactions of NPs with blood components is very

important not only when they are to be administered into the systemic circulation,
but also when any other route is selected since the NPs could reach the blood.
Blood is composed of plasma and several types of cells. Among the cellular
components of the blood, erythrocytes are the major component and they have a
central role in blood functions. Thus, it is important to understand the interactions
between erythrocytes and NPs (Dobrovolskaia et al. 2008a, b; Barshtein et al.
2011).
5.1 Effects on Erythrocytes
The effect of NPs on the structure, function, and lifespan of red blood cells (RBCs)
is a critical factor for their biocompatibility. Current methods to determine hemo-
compatibility induced by nanomaterials are outlined below (Bridget Wildt and
Brown 2013).
5.1.1 Hemolysis
The purpose of this common test is to determine the potential of a NP to damage the
RBC. When hemolysis occurs, the hemoglobin is released after rupture of the
erythrocyte plasma membrane. A typical assay involves the incubation of the NPs
with diluted whole blood or washed RBCs and subsequent centrifugation of the
samples and collection of the supernatant. If hemolysis has occurred, the hemo-
globin released gives a reddish color to the supernatant (Bridget Wildt and Brown
2013), which can be directly measured at 405 nm with a spectrophotometer, or the
hemoglobin can be reduced to the stable compound cyanmethemoglobin (CMH),
which is measured at 540 nm.
In general, small NPs show greater hemolytic activity than larger particles,
probably because the total surface area that is in contact with the cell is larger. In
fact, Mayer et al. have described that NPs smaller than 60 nm hydrodynamic
diameter were the most hemolytic ones from several tested (Mayer et al. 2009).
A possible mechanism for hemolysis is due to changes that increase permeation of
the plasma membrane caused by NP adhesion on the RBC surface (Dobrovolskaia
et al. 2008a, b; Barshtein et al. 2011; Mayer et al. 2009). Surface characteristics of
the NPs, such as charge, solubility, or coating, are also critical factors that affect
hemolytic properties. The hemolytic activity may increase or decrease depending
on the surfactants used for the synthesis. Water-soluble PLGA derivatives increase
activity (Kim et al. 2005), whereas the presence of PEG reduces the degree of
hemolysis and enhances, for instance, the hemocompatibility of PAMAM den-
drimers (Bridget Wildt and Brown 2013; Wang et al. 2010).
Numerous parameters can influence the results. Modication of the NP surface
with the protein corona may modify its interaction with the RBCs. The presence of
adsorbed plasma proteins on the NP surface may increase the size of the particles or
lead to the formation of aggregates, which may inhibit adhesion to RBC. A lower
hemolytic activity is therefore often found when complete blood is used. This
suggests that the hemolysis assay should be performed in the presence of whole
blood rather than using washed erythrocytes (Aggarwal et al. 2009; Barauskas et al.
2010; Chen et al. 2008; Chambers and Mitragotri 2007). Another issue is the
possible interference from the NPs themselves due to their optical properties. This
problem can be partially solved by centrifugation of the sample prior to its eval-
uation (Hall et al. 2007). Other NPs, such as silver NPs, agglomerate in the presence
of phosphate-buffered saline (PBS), thus decreasing the sensitivity and reliability of
the hemolysis assay (Choi et al. 2011; Zook et al. 2010). Other NPs may adsorb the
hemoglobin released after damage of the RBC and this may give false negative
results.
Other factors related to experimental parameters such as protocol details, the
type of anticoagulant used for the collection of the blood, the origin of the blood,
etc., can also affect the results (Dobrovolskaia et al. 2008b; Dobrovolskaia and
McNeil 2013b).
As far as NP charge is concerned, the results of several studies have shown that
positively charged NPs are more hemolytic than negative ones, with a good cor-
relation found between the hemolytic potential and the zeta potential (Barauskas
et al. 2010; Cho et al. 2014).
A possible explanation is that cationic NPs interact with the negatively charged
plasma membrane, leading to holes and erosion, and this allows the release of
cytosolic material (Bridget Wildt and Brown 2013; Jain et al. 2010). On the other
hand, hemolytic activity was also found with negatively charged NPs such as
mesoporous silica nanoparticles. However, in this case the hemolytic properties
were related to the number of silanol groups that were accessible to the cell
membranes of RBCs (Slowing et al. 2009) and this can be reduced by
amine-modication of SiO2-modied NPs.
In order to allow comparison between laboratories, a NP is considered
non-hemolytic if the level of hemolysis induced is below 2 % (Dobrovolskaia et al.
2008b; Dobrovolskaia and McNeil 2013b).
The results of various studies suggest that each kind of NP should be fully
evaluated due to the numerous parameters that must be taken into account. For
example, lipid-based liquid crystalline nanoparticles (LCNPs), intended for use as
drug-delivery and diagnostic agents, have hemolytic properties that are strongly
related to the chemical structure of the monomers. These properties are mostly due
to the specic lipid composition and are independent of the stabilizing polymer or
surfactant (Barauskas et al. 2010).
Many other polymer structures proposed for drug delivery, such as poly
(3-hydroxybutyrate)poly(ethylene glycol)poly(3-hydroxybutyrate) (PHBPEG
PHB) (Chen et al. 2008), N-acyl chitosan nanoparticles (Lee et al. 2004b) and
chitosan--glycerophosphate (CS--GP) hydrogel, an injectable chitosan-based
thermosensitive hydrogel (Zhou et al. 2011), have proven to be safe regarding
hemocompatibility. More examples can be found in the paper by Mocan (Mocan
2013), who assessed the hemolytic impact of several nanoparticles on red blood
cells.
5.1.2 Oxidative Stress
Oxidative stress is considered to be one of the mechanisms that induces hemolysis

as it damages the plasma membrane (Bridget Wildt and Brown 2013). RBCs have a
continuous intracellular production of ROS (imen 2008), so they contain enzy-
matic (superoxide dismutase, glutathione peroxidase) and nonenzymatic (vitamin E,
C, glutathione) pathways to combat high levels of reactive oxidants. In vitro assays
for ROS analysis have already been described in the Sect. 3.4 of this chapter and
can also be used to evaluate any alteration of ROS in RBCs.
Another common marker of oxidative stress is the peroxidation of membrane
lipids. Malondialdehyde (MDA) is an end product of membrane lipid peroxidation.
MDA can cross-link erythrocyte phospholipids and proteins, thus decreasing the
functionality of the membrane and therefore decreasing the survival (imen 2008).
Lipid peroxidation is usually determined by the reaction of MDA with thiobarbi-
turic acid (TBA), which forms a colorimetric product proportional to the presence
of MDA (Janero 1990).
5.1.3 Aggregation and Agglutination
The presence of sialic acid residues in the membrane of RBC creates a negative
charge that repels the adhesion to other blood cells or to the vascular endothelium
(Bridget Wildt and Brown 2013). If RBCs are under low shear force or stasis, they
form reversible aggregates. If the aggregation becomes irreversible, the process is
known as agglutination. The specic mechanisms involved in RBC aggregation are
not completely understood, neither how this aggregation could be reverted.
Changes in surface charge due to alterations in plasma membrane proteins may lead
RBCs to aggregate and increase their binding with other cell types such as
monocytes (Bridget Wildt and Brown 2013).
Elevated levels of brinogen, other large plasma proteins and neutral polymers,
such as dextran, may also cause RBC aggregation, the extent of which is propor-
tional to its molecular mass and concentration (Neu and Meiselman 2002).
Enhanced RBC aggregation plays an important role in blood viscosity and has
negative effects on blood flow dynamics. This parameter is commonly evaluated
using the erythrocyte sedimentation rate, which is measured in an aggregometer
(Marton et al. 2001), or by simple visual analysis under an optical or electron
microscope. Some examples of NPs that affect (or not) RBC aggregation can be
found in the literature. Differences in the aggregation, with respect to the control
RBCs (not incubated with NPs), were not observed by microscopy for a novel
hyperbranched PG-based cationic polymer (Kainthan et al. 2006). In another study,
agglutination and sedimentation of erythrocytes was observed for TiO2 NPs (Li
et al. 2008).
This aggregation capacity could be modied using surfactants such as
Poloxamer 188, which may prevent erythrocyte aggregation and decrease the vis-
cosity of whole blood (Moghimi and Hunter 2000).
5.1.4 Membrane Interaction, Uptake, and Morphology Changes
RBCs are usually used as a model of non-phagocytic cells since they do not have
phagocytic receptors. A study carried out with different particles revealed that size
is the critical factor for entry into a RBC, suggesting that the mechanism of uptake
is different from phagocytosis and endocytosis (Rothen-Rutishauser et al. 2006).
The absence of hemolysis does not guarantee the safety of NPs. Some interac-
tions between NPs and RBCs may affect the normal functionality of the erythro-
cytes without destroying them (Hudson et al. 2008). For instance, NPs may modify
the deformability of RBCs, thus interfering with their flow through microcapillaries.
Interactions between differently sized mesoporous silica nanoparticles and RBC
membranes were studied by Zhao et al. (2011b). Small NPs (*100 nm) were
adsorbed onto the membrane without disruptions or morphological changes
whereas large ones (*600 nm) induced a strong local membrane deformation,
which led to spiculated RBCs, internalization of the particles, and eventual
hemolysis (Zhao et al. 2011b).
There are several useful methodologies that can be used to study the effects of
NPs when they interact with the RBC membrane. In some studies erythrocyte
rheology is analyzed by measuring the elongation index (EI) with a microfluidic
ektacytometer (Kim and Shin 2014). Other groups have described morphological
changes in RBCs observed by light microscopy or have conrmed the results using
electron microscopy (SEM) and atomic force microscopy (AFM) (Asharani et al.
2010). Special care should be taken, mostly in the SEM technique, because the
integrity of the samples could be affected by the dehydration process.
In vivo studies to analyze the effects of NPs on RBCs are focused on red blood
cell count (RBCC), hematocrit blood level, mean corpuscular volume, and hemo-
globin concentration, amongst others, after the animal model has been exposed to
nanoparticles (Bridget Wildt and Brown 2013).
A review by Dobrovolskaia and McNeil (2013b) suggests a good correlation
between in vitro hemolysis assays and in vivo toxicity studies. Since in vitro
hemolytic properties may be predictors of in vivo behavior, which is very relevant
for safety reasons, more studies are required to compare the two situations (Bridget
Wildt and Brown 2013).
5.2 Interactions of Nanoparticles with the Coagulation

System
The coagulation pathway is an important mechanism to maintain blood homeostasis

as it helps to prevent blood clotting under normal conditions and facilitates coag-
ulation in cases of vascular injury.
Activated platelets release their granules content, which will activate endothelial
cells, leukocytes, and other platelets to form a thrombus. Endothelial cells play an
important role in coagulation when a blood vessel is damaged as they induce the
coagulation cascade at the site of injury (Ilinskaya and Dobrovolskaia 2013).
Regarding blood proteins, thrombin is the main enzyme of the coagulation pathway
(Fig. 6) and it is involved in many functions, including the activation of platelets,
conversion of brinogen to brin and positive feedback of the coagulation cascade.
Nanomaterials can interact with the components of the coagulation system once
they enter the bloodstream, and this interaction is influenced by the physico-
chemical properties of the NPs and the surface composition. NPs can be designed to
interact with the coagulation system, either for the promotion of thrombus forma-
tion or for its inhibition by acting like antithrombotic drugs.
Regarding NPs designed to induce coagulation, some groups have focused their
attention on stopping hemorrhage complications. For instance, Bertram and col-
leagues developed and tested engineered nanospheres composed of 170 nm poly
(lactide-co-glycolide)-poly-L-lysine to augment platelet aggregation at injury sites,
with the nanospheres able to interact with activated platelets but not with inactive
ones (Bertram et al. 2009). Modication of the NP surface can also be used for
specic targeting. Okamura et al. used PLGA nanosheets conjugated with H12 (a
specic region of brinogen which is a ligand of GPIb-IIIa, a platelet integrin) to
bind and activate platelets (Okamura et al. 2009). Other approaches include the
conjugation of coagulation factors to NP vectors in order to prolong the circulation
time of the factors, thus reducing the number of injections required and the price for
Fig. 6 Intrinsic and extrinsic pathways of the coagulation cascade
a specic treatment (Ilinskaya and Dobrovolskaia 2013). This approach has been
tested to treat hemophilia patients (Spira et al. 2010, 2012; Yatuv et al. 2010).
In other cases NPs can inhibit coagulation by working as antithrombotic drugs.
NPs can be used as carriers for anticoagulant drugs and this results in fewer doses
and reduced side effects while maintaining efcacy and increasing circulation time
(Ilinskaya and Dobrovolskaia 2013). One example is the development of aptamers
against thrombin. Aptamers are short oligonucleotides that bind target proteins
(e.g., thrombin) and impede the binding of the natural ligands, in this case resulting
in the inhibition of coagulation. The main drawback with aptamers is their very
short half-life in plasma. Binding of a thrombin-specic aptamer to the surface of
15 nm gold NP stabilizes the aptamer in plasma and allows the attachment to
different exosites of thrombin, which impedes the future binding of several ligands
of thrombin, such as brinogen, platelet domains, FV, FVII, and heparin (Shiang
et al. 2011; Hsu et al. 2011).
This intended benecial effect induced by specic aptamers could be deleterious
if unspecic adsorption of coagulation proteins occurs with NPs.
5.2.1 Factors that Affect the Coagulation Pathway
The physicochemical properties and surface composition of NPs affect the way in
which they interact with the components of the coagulation pathways. NP size has
proven to be one of the most influential properties that affect this interaction. For
instance, Sanns et al. used carboxyl-modied polystyrene NPs of different sizes,
surface areas, and curvatures to analyze their effect on the initiators of the intrinsic
coagulation pathway (Sanns et al. 2014). Large NPs (from 60 up to 220 nm) were
able to trigger the production of thrombin from prothrombin in citrated stabilized
plasma, while small ones (24 and 26 nm) were unable to do so. Also, the absorption
of FXIIa on large negatively charged polystyrene NPs can be stabilized and its
enzymatic activity can be enhanced, thus proving that surface charge can also be
used to potentiate the coagulation enzyme effects (Sanns et al. 2014).
The induction of the coagulation pathway could be useful in some disease
conditions with affectation in the coagulation cascade or in hemorrhagic situations,
but it could be very harmful if NPs activate the formation of undesired thrombus.
5.2.2 Methods of Analysis
Evaluation of the effects induced by NPs on the coagulation cascade usually focuses
on three of the most important effector enzymes of the coagulation pathway:
(1) thrombin, (2) plasmin, and (3) kallikrein (Kal). Another set of assays can be
performed to study how NPs interfere with the conversion of brinogen to brin.
The most commonly used methods of analysis are listed below:
Clot-based plasma coagulation tests measure the time taken for a plasma sample to
clot after the addition of calcium and an activator (Samama and Guinet 2011). The
time to clot formation is monitored either manually or using a coagulometer (Simak
2013a).
Thromboelastography is a global hemostatic test in which changes in the blood
components are analyzed from the initiation of coagulation until the end of bri-
nolysis (Samama and Guinet 2011; Simak 2013a). It is largely used in sanitary
centers and it could also be applied on the NP eld, but it is worth noting that the
ability of thromboelastography to determine the effect on coagulation induced by a
nanomaterial is limited because this is a complicated assay with a high coefcient of
variation because numerous blood components can interfere with the results.
Synthetic chromogenic substrate-based assays are used to determine the activity of
endogenous proenzymes, enzymes, cofactors and inhibitors (Simak 2013a).
Fibrinolytic and thrombolytic activity assays are based on the quantication of
brolase activity (Patton et al. 1993). In addition, commercially available ELISA kits
exist for the detection of different factors and enzymes of the coagulation pathways.
For in vitro testing of platelet aggregation, it is important to consider different
factors, such as type of NPs, the pretended in vivo exposure of the NPs (route of
administration, dose), the platelet (PLT) source, and method of platelet collection
(whole anticoagulated blood, platelet-rich plasma).
A detailed explanation of the different techniques to evaluate aggregation, dis-
integration, activation, etc., of the PLTs is provided by Simak (2013b). These
techniques are summarized here but it is anticipated that a standardized panel of

in vitro tests is still needed.
In vitro methods to analyze platelets and platelet activation
Cell counters are widely used to count PLTs and measure size, volume, and
aggregation. NPs are incubated with PRP under static conditions during 15 min
at 37 C and then PRP is analyzed using a Coulter analyzer. The decrease in the
PLT count is related to the percentage of aggregation since aggregates are not
counted by the analyzer. This assay is very simple and can be useful to identify
NPs that induce a high activation of PLTs, but it does not differentiate between
aggregation and disintegration, so other methods are needed.
Different microscopy techniques are useful to evaluate different parameters
related to PLT activation:
Phase contrast light microscopy is useful for counting PLTs and assessing the
basic morphology of PLT-NP aggregates. Transmission electron (TEM) and
Scanning electron microscopy (SEM) provide a detailed morphology of PLTs
and their aggregates, and atomic force microcopy (AFM) allows visualization of
the interactions between NPs and PLTs.
Fluorescence microscopy is useful to visualize PLTs previously labeled with
specic dyes and/or exposed to fluorescent NPs. However, due to the low number
of PLTs analyzed by microscopy and the artifacts that can appear due to the sample
processing, these techniques should be complemented with other studies.
Light transmission aggregometry (LTA) is used to detect aggregates using
PRP. This assay is based on the changes in light absorbed as aggregates are
formed. Light absorbed by PRP incubated with NPs is compared to PRP
incubated with an agonist (considered 100 % aggregation) and platelet poor
plasma (PPP) as a negative control (0 %).
A similar assay can also be performed with whole blood, but, in this case,
impedance, which is proportional to the formation of aggregates, is detected.
Another alternative is measurement of the adenosine diphosphate (ADP)
released during the aggregation process of PLTs using a lumi-aggregometer,
where the luminescence is proportional to the level of aggregate formation (Li
et al. 2009; Naeye et al. 2011).
PLT can be analyzed by flow cytometry using antibodies against specic
markers, such as surface activation markers (i.e., P-selectin or GPIIn-IIIa b-
rinogen binding-site).
Leukocyte-PLT aggregation, such as circulating monocyte CD14+-PLT aggre-
gates, can also be evaluated using monoclonal antibodies not only against PLT
but also against leukocyte surface markers. Moreover, flow cytometry is very
useful when fluorescent NPs are used in these assays.
Many other techniques can be used: ELISA to measure markers or different
proteins that are released into the supernatant after the activation of PLTs,
photometry, luminometry, and scintillation colorimetric assays to determine the
integrity of PLTs, and also platelet function analyzers, perfusion chambers, etc.
Proteomics, genomics, and transcriptomics are also useful to analyze the effects
induced on PLTs by NPs.
Very recently, a new analytical platform on a chip was proposed to detect
platelet adhesion and aggregation (Kim et al. 2014b).
In vivo assays to analyze platelet activation
Although the in vitro assays indicated above are really very useful to understand
the potential effects of NPs on the PLTs function, the in vivo assays are essential for
a direct safety evaluation. For in vivo assays, several animal models with arterial,
venous, or microvascular thrombosis have been designed. These models allow
analysis of the enhancing or inhibitory effects of the NPs on the PLTs.
The analysis of different polymers on PLT revealed that only cationic polymers
were able to activate PLTs. This activation was proportional to the number of
surface amines and occurred through the disruption of membrane integrity
(Dobrovolskaia et al. 2011). These results are consistent with those obtained in
another study performed with polystyrene latex NPs with different surface modi-
cation and charge: neutral, carboxylated, and aminated NPs. The aminated NPs
also induced platelet aggregation by disruption of the membrane, whereas the
carboxylated ones induced aggregation through the classical coagulation pathway
(Fig. 6). In contrast, neutral NPs did not induce platelet aggregation at all
(McGuinnes et al. 2011).
The effect of liposomes was also studied. It was conrmed by flow cytometry
that liposomes interact with PLTs (Constantinescu et al. 2003) and that surface
charge and the presence of plasma components are critical factors for PLT acti-
vation (Simak 2013b).
6 Studying the Interaction of Nanomaterials with Plasma

Proteins: Conformational Changes
The proteins that are attached to the surface of the NPs form the so-called protein
corona. Albumin, brinogen, apolipoproteins, immunoglobulins, and complement
factor C3b are the most common plasma proteins found attached to different NPs
(Aggarwal et al. 2009). This protein corona will compromise the fate of the NPs
regarding biodistribution and interaction with cells and tissues (Dobrovolskaia et al.
2008a). The binding of plasma proteins to the NP surface induces their opsonization
and enhances their uptake by immune phagocytic cells, which is one of the main
disadvantages of the use of NPs as drug carriers. However, the use of some
molecules with hydrodynamic properties to cover the surface, such as poly(ethylene
glycol) (PEG) (Peracchia et al. 1999) or galactose (Moros et al. 2012), reduces
opsonization and uptake.
The most widely used methods to measure NP-bound proteins are gel elec-
trophoresis, mass spectrometry (MS), fluorescence correlation spectroscopy, and
surface plasmon resonance (SPR) (Aggarwal et al. 2009; Lundqvist et al. 2008,
2011; Rocker et al. 2009; Cedervall et al. 2007; Cohavi et al. 2011). However, the
identication of proteins attached to NPs is not sufcient in its own right because
the interaction of NPs with plasma proteins can induce conformational changes in
those proteins and have signicant consequences such as the loss of the protein
functionality, modication in the interactions with other elements, or even to the
initiation of inflammatory/allergic or autoimmune responses. For instance, it has
been shown that the interaction of negatively charged poly(acrylic acid)-conjugated
gold nanoparticles with human plasma induces the unfolding of brinogen (Deng
et al. 2011). Consequently, an inflammatory response was induced through the
interaction of the unfolded brinogen with the Mac-1 receptor on the surface of a
monocyte cell line (THP-1) and activation of the NF-B signalling pathway of these
immune cells occurred.
For safety reasons, the conformational changes induced by NPs (such as the
polymer ones), in human proteins should be correctly studied before their use
in vivo.
6.1 Evaluation of Interactions Between NPs and Proteins:

Proteomic Approaches
Proteomic techniques have been used to study the protein corona and to assess the
cellular changes induced by NPs. The huge improvement in mass spectrometry
(MS) techniques, such as the development of new ionizers, assisted by the use of
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate com-
plex protein mixtures, has made possible the broad use of proteomics (May et al.
2011; Mann and Kelleher 2008). The molecules are ionized into a gaseous state
either by matrix-assisted laser desorption ionization (MALDI) or by electrospray
ionization (ESI). The ions are subsequently accelerated into a mass analyzer to
determine their precise molecular masses. Proteins from a sample can also be
labeled in vitro by introducing isotopes in some reactive groups during protein
synthesis (May et al. 2011; Mann and Kelleher 2008). Such isotope labeling will
also allow multiplexed and comparative proteomic analysis of the mixed samples
by liquid chromatography-mass spectrometry (LC-MS).
Surface plasmon resonance (SPR) is another technique which allows the study
of direct molecular interactions. Although SPR is commonly used to identify
interactions between antibodies and analytes, it can also detect direct interactions
between NPs with proteins or even with other NPs. Moreover, NPs can act as
coating surfaces to attach other NPs (Canoa et al. 2015).
Changes in protein expression were also studied ex vivo in cells excised from
mice after NP treatment. A proteomic approach to characterize the effect of chitosan
nanoparticles in hepatic cells identied several proteins involved in the
PI3K/AKT1/mTOR pathway, a pathway responsible for the proliferation and sur-
vival of the majority of cancer cells (Omidi et al. 2008).
The study of human proteins deregulated by the interaction between cells and
different NPs has been conducted with STRING (Search Tool for the Retrieval of
Interacting Genes/proteins) (Lai et al. 2012), which identied some common
proteins involved in cellular stress response pathways and apoptosis, such as
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Thioredoxin 1, and
Periredoxin 1.
Protein labeling is another technique that is used to study nano-bio interactions
in vivo. One example is the stable isotope labeling by amino acids in cell culture
(SILAC), in which cells grow in a media supplemented with heavy, stable
isotope-labeled amino acids, producing metabolically labeled proteins. In parallel, a
control culture grows in normal media, producing non-labeled proteins. After
labeling, one cell population is stimulated by the addition of an external agent,
which may affect the cell metabolism. Then the lysates of both the stimulated and
the control cells are combined and the precipitated proteins are then identied and
quantied by mass spectometry at once. The proteins from one or another culture
will be differentiated due to the labeled isotopes. Although it is not a commonly
used technique as yet, it has already been carried out in some animals by feeding
them with isotope-labeled amino acids in a diet-based approach, a technique known
as stable isotope labeling in mammals (SILAM), working in a similar way as
SILAC does (Zanivan et al. 2012; Brown et al. 2012; Zhang and Neubert 2009).
There are also protein arrays, similar to the RNA or cDNA arrays, that could be
used for proteomic screening, but their use is not widespread due to some signif-
icant limitations (Joos and Bachmann 2009). For instance, the need to screen a large
number of capture molecules (either antibodies, aptamers, etc.) to nd the most
suitable for each protein and the lack of a commercial source for these capture
molecules are two of the problems associated with this technique.
7 Other Important Issues to Take into Account Regarding

Immunotoxicity
Besides the parameters mentioned before, affecting toxicity (size, shape, solubility,
protein binding, and route of administration), other questions must be considered.
Nanomaterials that are synthesized for in vivo diagnosis or therapy must be ltered
or sterilized prior to in vivo administration. Characterization of their therapeutic
potential and toxic effects should be carried out with the sterilized NPs in order to
avoid effects that could be due to the presence of microorganisms or endotoxin
(Dobrovolskaia et al. 2009; Vallhov et al. 2006).
7.1 Endotoxin Content
Endotoxin is the main component of the outer membrane of Gram-negative bac-

teria. It is composed of proteins, lipids, and lipopolysaccharides (LPS), the latter of
which are responsible for most of the biological properties of the endotoxin
(Liebers et al. 2008). LPS consists of a lipophilic component, lipid A, which is the
biologically active part that might differ from bacteria strain to strain, and a
polysaccharide component, which is responsible for the LPS antigenic properties.
Lipid A is a glucosamine-based phospholipid and the polysaccharide component is
commonly divided into the terminal O-specic chain and the core region, which is
further subdivided into the outer and inner core (Alexander and Rietschel 2001).
LPS is a highly immunostimulant molecule. Several proteins participate in the
recognition of LPS by the immune system: LPS is recognized by CD14 and its
soluble form (sCD14), or lipopolysaccharide-binding protein (LBP) delivers
endotoxin to MD2, which is an extracellular adaptor protein associated with TLR4.
The binding of LPS to MD2 and the oligomerization of TLR4 triggers the initiation
of intracellular signaling that would result in the production of pro-inflammatory
cytokines and type I interferons (Dobrovolskaia and Vogel 2002; Triantalou and
Triantalou 2005). Finally, fever, hypotension, blood coagulation, and other
pathophysiological events are produced as a consequence of the secretion of these
cytokines, which are mostly secreted by monocytes and macrophages (Alexander
and Rietschel 2001).
Endotoxin is a very stable molecule that tolerates very high temperatures
(Sharma 1986). It is frequently found in water, laboratory water ltration systems,
reagents, and tools. Therefore, it is a very common contaminant in
laboratory-synthesized engineered nanomaterials. Moreover, due to their elevated
surface-to-volume ratio, the adsorption of endotoxin onto nanomaterials is con-
siderably higher than in larger particles. For this reason it is very important to
evaluate the presence of endotoxin in formulations and devices (Dobrovolskaia and
McNeil 2013c). It has been reported that some nanoparticles can strongly potentiate
the inflammatory responses induced by LPS (Dobrovolskaia et al. 2012; Inoue
2011).
The United States Pharmacopeia (USP) published a document (General Chapter
85, Bacterial Endotoxin Test (BET)) that sets out guidelines for endotoxin testing
and the upper limits of endotoxin contamination in medical products (Pharmacopeia
2010).
7.1.1 Methods to Measure Endotoxin Content
Since endotoxin may have different molecular weight and activity, endotoxin levels
should be expressed in Endotoxin Units (EU), and it depends on the composition of
the lipid A. An endotoxin unit is a standardized unit of biologic activity, measured
with the LAL test and calibrated to the USP/FDA reference endotoxin; it is equal to
the international unit (IU) of activity used by the World Health Organization
(WHO) (Pharmacopeia 2010; Poole and Mussett 1989).
Commonly used methods
Rabbit pyrogen test (RPT)

This test is based on measuring the fever caused by endotoxin. It involves
injecting the material into the rabbit ear vein and monitoring the animal temperature
before and after the injection, every 30 min for 3 h. Different protocols are followed
in different countries and these are described in their particular pharmacopeias
(Dobrovolskaia and McNeil 2013c; Du et al. 2011).
Limulus Amebocyte Lysate (LAL)
The LAL test has been widely used for more than 30 years for the detection of
endotoxin in injectable drugs and medical devices. It is based on the interaction of
the protein extract of amebocytes of the horseshoe crab Limulus polyphemus with
endotoxin and the consequent formation of a clot (Ding and Ho 2010). This
reaction has been harnessed to design three different formats known as Gel clot
(Fig. 7), chromogenic, and turbidity tests. The latter two tests are available in
endpoint and kinetic formats and the turbidity kinetic LAL assay is the most sen-
sitive, with a limit of detection of 0.001 endotoxin units/mL (Dobrovolskaia and
McNeil 2013c).
An important issue to be considered when detecting endotoxin in nanoformula-
tions is the possible interference induced by the nanomaterials. NPs may have cat-
alytic properties, they may absorb at the wavelength used in the chromogenic LAL
(405 nm) or they may bind to antibodies or LPS-Binding Proteins. In fact, it has been
reported that many engineered nanomaterials interfere with one or more LAL assays
(Dobrovolskaia et al. 2009, 2010). Since this interference can give confusing results,
it is important to evaluate possible effects using inhibition/enhancement controls
(IECs). This method involves spiking known amounts of endotoxin into a test
sample. In addition to IECs, quality controls (QCs) must be prepared by spiking
Fig. 7 Type of endotoxin test LAL (gel clot): presence (a) and absence (b) of clot formation
known amounts of endotoxin into endotoxin-free water and, since water does not
interfere with LAL, the level of recovery should be 100 %. It is important to perform
separate IECs with each LAL assay because some nanoparticles could interfere with
one but not all of the assays (Dobrovolskaia and McNeil 2013c). Many nanoparticles
interfere with LAL assays (Dobrovolskaia et al. 2010) and alternative methods
should therefore be considered to evaluate the endotoxin content in the formulations.
Alternative methods
Gel staining-based methods

Several kits are available that are based on the electrophoretic separation of
endotoxin (negatively charged), followed by oxidation and incubation with a
staining solution that binds to the oxidized endotoxin. The disadvantage of this
method is that glycoproteins other than endotoxin can also be detected
(Dobrovolskaia and McNeil 2013c).
Methods based on the use of anti-LPS monoclonal antibodies or LPS-binding
proteins: ELISA, Western blot, CECA, SPR ELISA, and Western blot can be used to
detect endotoxin with anti-LPS monoclonal antibodies or LPS-binding proteins.
CECA is an amperometric competitive assay based on the use of an endotoxin
standard conjugated to horse rabbit peroxidase (HRP), which competes with
endotoxin present in the sample for binding to endotoxin neutralizing
protein-coated electrodes (Priano et al. 2007). An alternative method to CECA has
also been described in which polydiacetylene liposomes are used as colorimetric
sensors to detect various types of endotoxin (Rangin and Basu 2004).
Surface plasmon resonance (SPR), a method that detects changes in the refractive
index caused by structural alterations in the proximity of a thin lm metal surface,
can also be used to detect endotoxin with anti-LPS antibodies (Das et al. 2014).
Mass spectrometry-based methods
Both capillary zone electrophoresis and gas chromatography coupled to mass
spectrometry have been proposed for the detection of O-deacylated LPS and
3-hydroxy fatty acids of the lipid A core, respectively. However, the sensitivity of
these methods is quite low (400 EU/mL) and this explains why this technique is
more frequently used in agricultural and environmental sciences, where the allowed
endotoxin levels are higher (Dobrovolskaia and McNeil 2013c; Li et al. 2004, 2005;
Binding et al. 2004; Jenske and Vetter 2008).
Macrophage activation test
This assay is based on the induction of cytokines as a consequence of the presence
of endotoxin or other pyrogens in the samples. The test involves the incubation of
the monocytic cell line MM-6, human whole blood, or puried peripheral blood
mononuclear cells (PBMCs) with the test sample and appropriate controls, during
24 h, followed by quantication of the cytokine levels (IL-1, IL-7 or TNF-) by
ELISA (Hoffmann et al. 2005a, b).
7.1.2 Working in the Absence of Endotoxin
Endotoxins are not easy to remove by ltration or sterilization methods because

they are small molecules with a high resistance to thermal decomposition. To avoid
the presence of endotoxins in NP preparations, the best strategy is to use
endotoxin-free materials and buffers. In order to minimize possible contamination it
is important to prevent it by taking particular care at the stage of nanomaterial
synthesis (Dobrovolskaia and McNeil 2013c). Ideally, the sterility should be
maintained during all synthesis steps, but in most cases it is difcult or unfeasible to
achieve this in a standard research laboratory. In addition, it is possible to purify
and depyrogenate the formulations after synthesis by baking at 200 C for 30 min
or using reagents such as ethylene oxide, acid hydrolysis, alkylation, gamma irra-
diation, hydrogen peroxide gas plasma, or a soft hydrothermal process (Magalhaes
et al. 2007; Aida and Pabst 1990; Petsch and Anspach 2000; Miyamoto et al. 2009).
In the case of applying a purication process, the most widely used methods are
Triton X114 extraction, ultraltration, anion-exchange chromatography, afnity
adsorption, immunoafnity chromatography, or hydrophobic interaction chro-
matography (Magalhaes et al. 2007; Aida and Pabst 1990; Petsch and Anspach
2000; Miyamoto et al. 2009). However, not all of these techniques are applicable to
the entire range of nanomaterials. In fact, the integrity and composition of the NPs
and their active compounds could be altered on applying some of the sterilization
methods and this aspect must be characterized in depth on a case by case basis
(Frana et al. 2010).
It is also important to mention that although some NPs have antimicrobial
activity (Padmavathy and Vijayaraghavan 2008; Tayel et al. 2011; Hajipour et al.
2012), the majority of these antimicrobial NPs are not able to protect against a
broad spectrum of pathogens (Hajipour et al. 2012) and most NPs do not have any
antimicrobial activity at all. For this reason, NPs must be submitted to a sterilization
or ltration process that will remove any potential contamination by
microorganisms.
7.2 Sterilization Methods
There are both physical and chemical methods of sterilization. The main physical
methods are ltration, autoclave, ultraviolet (UV), or gamma irradiation (Table 5).
The most common chemical methods of sterilization use ethylene oxide, gas
plasma, or formaldehyde as chemical agents.
7.2.1 Physical Methods
Filtration is a gentle method and is the best choice for thermolabile or chemically
sensitive NPs or active compounds (Nayak et al. 2010). However, ltration is not
Table 5 Summary of sterilization methods

Sterilization method Recomendation Precautions
Physical
Filtration Thermolabile NPs NPs with large diameter
Chemically sensitive NPs
Autoclave Solid lipid NPs Sensitive to heat
Ultraviolet NPs sensitive to heat Chemical reactions
Gamma irradiation NPs sensitive to heat Drug-delivery systems
Chemical
Ethylene oxide Good microbicide Carcinogenic, very reactive and irritant
Gas plasma Tiopronin-gold NPs High toxicity (free radicals)
Do not use with PEG-coated NPs
possible in all cases. While the conventional lters for sterilization have a cut off of
220 nm, some NPs have a larger diameter. Another limiting factor is the compo-
sition of the NPs or the molecules that are on the surface. Quite often NPs cannot be
ltered without secondary effects such as modication of their concentration or loss
of active compounds (Mehnert and Mder 2001).
Autoclaving has proven to be efcient for solid lipid NPs. These NPs were
freeze-dried after sterilization (Cavalli et al. 1997), maintaining their stability for at
least 1 year (Cavalli et al. 1997; Heiati et al. 1998).
For those compounds that are sensitive to heat, the use of ultraviolet (UV) or
Ionizing gamma () radiation could be more convenient for sterilization. UV is a
nonionizing radiation that has been largely used to disinfect surfaces and materials
that are designed for medical devices, but it might cause some undesirable chemical
reactions in some pharmaceuticals and surgical supplies (Gopal 1978). The com-
bination of UV radiation with another photocatalytic agent, such as Ag or TiO2
NPs, can be used to eliminate bacteria and spores in medical devices (Lee et al.
2005; Prasad et al. 2009; Rai et al. 2009; Yao et al. 2008b).
Ionizing radiation has a greater penetrating power than UV and it can therefore
be used on the nal packaged product or at some stages during the synthesis
process, thus avoiding further risk of microbial contamination (Gopal 1978).
Sterilization by radiation is also an effective method that is accepted by the
European Pharmacopeia. For instance, it has been shown that for the sterilization of
cyclodextrin NPs, irradiation is an efcient method and it induces fewer
physicochemical modications than autoclaving (Memisoglu-Bilensoy and Hincal
2006). However, radiation may also have some negative effects on drug-delivery
systems. The energy transferred could induce fragmentation of covalent bonds and
lead to the production of free radicals that, in turn, will cause damage to the
irradiated materials as a consequence of chemical attack (Sintzel et al. 1997). In
some cases the radiation might decrease the amount of active ingredient by partial
decomposition or produce toxic fragments due to decomposition of the material
(Sintzel et al. 1997; Boess and Bgl 1996).
7.2.2 Chemical Methods
Chemical methods are useful for nanomaterials that are sensitive to heat or radiation
and that cannot be ltered due to their size or viscosity.
Chemical sterilizers tend to be highly reactive gases that are commonly used at
low temperature and come into direct contact with the object. Because they are
harmful, these agents need to be used under controlled conditions and with special
chambers designed for sterilization. Sterilizing agents are constituted by chemically
reactive substances that could react with both NPs and active substances carried by
the NPs, and this possibility needs to be extensively investigated. Ethylene oxide is
the most commonly used chemical agent but other compounds, such as gas plasma
and formaldehyde, are also frequently utilized (Table 5). Ethylene oxide is a col-
orless flammable gas at room temperature and it is a good microbicide because it is
a very reactive, carcinogenic, mutagenic, irritant, and anaesthetic gas.
Formaldehyde is the simplest aldehyde and it is also a gas. In a similar way to
ethylene oxide, formaldehyde is toxic and carcinogenic.
Gas plasma or hydrogen peroxide gas plasma is formed by the vaporization of
hydrogen peroxide, a highly reactive compound, using a gas chamber under an
electrical eld. This electrical eld induces ionization of the gas and leads to the
formation of free radicals, which are responsible for the high toxicity of this
chemical sterilizer.
These chemical methods were used by our group to sterilize gold NPs func-
tionalized with two different molecules, tiopronin and thiolated-poly(ethylene
glycol) (PEG), and its efcacy was compared with that of physical methods (Frana
et al. 2010). The use of ethylene oxide and UV radiation seems to be the most
efcient methods to induce the smallest physicochemical alterations on both types
of NPs, as determined by transmission electron microscopy (TEM), UVVis
spectroscopy, Fourier transform infrared spectroscopy (FTIR) and thermogravi-
metric analysis (TGA). Moreover, cells incubated with these sterilized NPs did not
show changes when compared to the non-sterilized ones in terms of viability and
ROS production.
Gas plasma has also proved to be a convenient method to sterilize the
tiopronin-gold NPs, although it should not be used when NPs are functionalised
with PEG. Our group described how gas plasma strongly affects to Au@PEG NPs
(Frana et al. 2010) because it is a highly oxidative procedure that could have a
direct influence on PEG chemical stability.
Other procedures, such as lyophilization or drying, may also alter the NPs or the
molecules present on their surface (Frana et al. 2010). For this reason, the selection
of the best sterilization method should include complete physicochemical and
biological characterization in order to identify any potential change in the NPs and
their surface or active molecules.
8 Concluding Remarks
This chapter has summarized the type and functions of the immune components, the
endocytic routes, the inflammatory role of complement activation and the type of
pro-inflammatory and anti-inflammatory cytokines produced by several immune
cells. NPs can affect the immune system in harmful, but also in benecial ways, like
in the nanovaccines, being this issue also addressed in the text.
Most of the important aspects of the potential interactions between NPs, proteins
and components of the immune system are included, with special attention to the
physicochemical characteristics of the NPs, not only for the understanding of their
biological activities in contact with the immune system, but also for safety issues.
The effect of NPs on proteins, such as the induction of conformational changes and
the way to measure them, is mentioned. Special attention is dedicated to the ster-
ilization methods, coagulation cascade, platelet activation, and techniques to
evaluate the endotoxin content on NPs. Finally, the chapter includes a large variety
of in vitro and in vivo assays that can be used to study several aspects of the
interaction NPs-immune components.
Acknowledgments This work was nancially supported by Xunta de Galicia (INBIOMED

2012/273, DXPCTSUG-FEDER and GPC, Potentially growing groups), Ministerio de Educacin
y Ciencia (Nanovac project SAF2011-30337-C02-02) and the BIOCAPS project (316265,
FP7/REGPOT-2012-2013.1). We thank Isabel Pastoriza and Luis Liz for providing gold NPs, and
Maria Jos Alonso for the protamine nanocapsules. The confocal and SEM-FIB images were taken
in the University facilities of the Centre for Scientic and Technical Support (CACTI).
Conflict of interest The authors declare no conflict of interest.
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Chapter 10
Investigating Interactions Between
Nanoparticles and Cells: Internalization
and Intracellular Trafcking
Herv Hillaireau
Abstract Nanoparticles used as drug nanocarriers offer unique possibilities to

overcome cellular barriers in order to improve the delivery of various molecules,
including biomacromolecules such as nucleic acids or proteins. Depending on
nanoparticle characteristics and the type of cells considered, various mechanisms of
internalization may occur, as well as subsequent intracellular trafcking pathways.
Understanding these pathways may have important pharmacological implications.
This chapter will review the main nanoparticle internalization and trafcking
mechanisms and their experimental characterizations, allowing to understand how
they are affected by nanoparticle physicochemical properties. The phagocytosis
pathway will rst be described, being increasingly well characterized and under-
stood, which has allowed several successes in the treatment of some cancers and
infectious diseases. In contrast, other non-phagocytic pathways encompass various
complex mechanisms, such as clathrin-mediated endocytosis, caveolae-mediated
endocytosis and macropinocytosis. Although more challenging to control for
pharmaceutical drug delivery applications, they are actively investigated in order to
tailor nanocarriers able to deliver anticancer agents, nucleic acids, proteins, and
peptides for therapeutic applications.
Keywords Caveolae
Caveolae-mediated endocytosis Clathrin
Clathrin-mediated endocytosis
Endocytosis Endosome Internalization
Intracellular trafcking
Lysosome
Macropinocytosis Opsonization

Phagocytosis Pinocytosis Receptor-mediated endocytosis
H. Hillaireau (&)
Institut Galien Paris Sud, Faculty of Pharmacy, UMR CNRS 8612, Univ. Paris-Sud,
Universit Paris Saclay, 5 Rue J.B. Clment, 92296 Chtenay-Malabry, France
e-mail: herve.hillaireau@u-psud.fr

DOI 10.1007/978-3-319-41421-8_10
292 H. Hillaireau
1 Introduction
In order to be active, a drug has to reach the relevant pharmacological target, which
means undergoing a complex series of interactions with the cells of the body. When
this target is located inside diseased cells, the therapeutic molecule must generally:
(1) cross one or various biological membranes (e.g., mucosa, epithelium,
endothelium) before (2) reaching the target cell and diffusing through the plasma
membrane to (3) nally gain access to the appropriate organelle where the bio-
logical target is located. Deviating from this ideal path may decrease the drug
efciency but also entail side effects and toxicity.
More than 30 years ago, the idea emerged to tailor carriers small enough to ferry
the active substance to the target cell and its relevant subcellular compartment. In
the seventies, the proof of concept has been done that submicronic lipid vesicles
known as liposomes (Black and Gregoriadis 1974), as well as synthetic polymer
nanoparticles (Couvreur et al. 1977), were able to concentrate into cells, molecules
that did not diffuse intracellularly. It became clear that such nanocarriers had a
great potential for the targeted delivery of drugs. Today, such approach is exploited
to optimize the intracellular delivery of many small molecules, but also macro-
molecules like nucleic acids, peptides, or proteins.
Depending on nanoparticle characteristics and the type of cells considered,
various mechanisms of internalization may occur, as well as subsequent intracel-
lular trafcking pathways. Understanding these pathways may have important
pharmacological implications. This chapter will review the main nanoparticle
internalization and trafcking mechanisms and their experimental characterizations,
allowing to understand how they are affected by nanoparticle physicochemical
properties. The phagocytosis pathway will rst be described, being increasingly
well characterized and understood, which has allowed several successes in the
treatment of some cancers and infectious diseases. In contrast, other non-phagocytic
pathways encompass various complex mechanisms, such as clathrin-mediated
endocytosis, caveolae-mediated endocytosis and macropinocytosis. The intracel-
lular fate will vary accordingly, which has important implications for intracellular
drug delivery.
2 Nanoparticle Internalization and Trafcking Routes

Occurring in Cells
Depending on the nature of the cells concerned, and the physicochemical properties
of the nanocarriers, different endocytosis modes may occur (Fig. 1): phagocytosis
or other endocytic routes, i.e., mainly macropinocytosis, clathrin- and
caveolae-mediated endocytosis.
10 Investigating Interactions Between Nanoparticles and Cells 293
Fig. 1 Overview of the major internalization pathways of nanoparticles by cells, and

corresponding trafcking routes
2.1 Phagocytosis
Phagocytosis occurs mostly in specialized cells, also called professional phago-

cytes: macrophages, monocytes, neutrophils, and dendritic cell (Aderem and
Underhill 1999). In particular, macrophages of the reticuloendothelial system (RES,
also known as mononuclear phagocytic system) are the major phagocytes involved
in the uptake of nanoparticles and have been by far the most studied for drug
delivery purposes.
The phagocytic pathway of entry into cells can be described using three distinct
steps:
1. Recognition by opsonization in the bloodstream. Opsonization is an important
process occurring before the phagocytosis itself. It consists in tagging the for-
eign nanoparticles by proteins called opsonins, making the former visible to
macrophages. This typically takes place in the bloodstream rapidly after intro-
duction of the particles (Alexis et al. 2008). Major opsonins include
immunoglubulins (Ig) G (and M) as well as complement components (C3, C4,
294 H. Hillaireau
C5) (Frank and Fries 1991; Vonarbourg et al. 2006), in addition to other blood
serum proteins (including laminin, bronectin, C-reactive protein, type-I col-
lagen) (Owens and Peppas 2006).
2. Opsonized particles then attach to macrophage surface through specic
receptor-ligand interactions. The major and best-studied receptors for this pur-
pose include the Fc receptors (FcR) and the complement receptors (CR). FcRs
bind to the constant fragment of particle-adsorbed immunoglobulins, while CRs
mostly bind to C3 fragments (Aderem and Underhill 1999; Groves et al. 2008).
Other receptors including the mannose/fructose and scavengers receptors can be
involved in the phagocytosis (Aderem and Underhill 1999). Receptor ligation is
the beginning of a signaling cascade mediated by Rho-familiy GTPases (Caron
and Hall 1998) which triggers actin assembly, forming pseudopodia that zipper
up around the particle to engulf it.
3. The resulting phagosome, having a minimum size of 250 nm (Alberts et al. 2002),
ferries the particle throughout the cytoplasm. As actin is depolymerized from the
phagosome, the newly denuded vacuole membrane becomes accessible to early
endosomes (Swanson and Baer 1995). Through a series of fusion and ssion
events, the vacuolar membrane and its contents mature, fusing with late endo-
somes and ultimately lysosomes to form a phagolysosome. The rate of these
events depends on the surface properties of the ingested particle, typically from
half to several hours (Aderem and Underhill 1999). The phagolysosomes acidify
and acquire many enzymes, including esterases and cathepsins (Claus et al. 1998),
leading to the degradation of biodegradable or bioerodible particle material.
2.2 Other Endocytic Pathways
The non-phagocytic endocytosis has been traditionally referred to as pinocytosis,

literally cell drinking, i.e., uptake of fluids and solutes, as opposed to cell eating,
i.e., uptake of solid particles for phagocytosis. This terminology may not be rele-
vant for the study of the nanoparticle-cell interaction, since solid particles, due to
their small size, can be internalized through these non-phagocytic pathways. Unlike
phagocytosis, restricted to specialized cells, other endocytic pathways occur in
virtually all cells by four main mechanisms: clathrin-mediated endocytosis,
caveolae-mediated endocytosis, macropinocytosis and other clathrin- and
caveolae-independent endocytosis.
2.2.1 Clathrin-Mediated Endocytosis
Endocytosis via clathrin-coated pits, or clathrin-mediated endocytosis (CME),

occurs constitutively in all mammalian cells, and fullls crucial physiological roles,
including nutrient uptake and intracellular communication (Schmid 1997; Di Fiore
and De Camilli 2001). For most cell types, CME serves as the main mechanism of
internalization for macromolecules and plasma membrane constituents. CME via
specic receptors-ligand interaction is the best-described mechanism (to such extent
that it was previously referred to as receptor-mediated endocytosis (RME)); how-
ever, it is now clear that alternative nonspecic endocytosis via clathrin-coated pits
also exists (as well as receptor-mediated but clathrin-independent endocytosis).
The CME internalization pathway (either receptor-dependent or independent) is
associated to degradation of the endocyted material in lysosomes. This should be
taken into account with respect to degradation of the nanocarrier but also of fragile
cargo molecules.
Receptor-dependent CME is one of the best-characterized endocytic mecha-
nisms. It is a shared pathway for the internalization of a variety of ligand-receptor
complexes (Mukherjee et al. 1997). Many ligands have been used for this purpose,
including low-density lipoprotein (LDL), transferrin, epidermal growth factor
(EGF) (Bareford and Swaan 2007; Chavanpatil et al. 2006).
The endocytosis typically occurs in a membrane region enriched in the clathrin
protein. The formation of an endocytosis vacuole is driven by assembly of a
basket-like structure (Kanaseki and Kadota 1969) formed by polymerization of
clathrin units, which deforms the membrane into a coated pit. Some receptors, like
the LDL receptors, are concentrated in these clathrin-coated pits, while others
receptors like the transferrin and EGF receptors become concentrated upon ligand
binding (Mukherjee et al. 1997). The ssion of the vesicle, requiring the GTPase
dynamin, leads to so-called clathrin-coated vesicles. The uncoating of the vesicles
later allows the recycling of clathrin units (Conner and Schmid 2003). Some ligands
are also recycled, as transferrin and riboflavin (Bareford and Swaan 2007). The
resulting endocytic vesicle may have an average size of 100 (Bareford and Swaan
2007) or 120 nm (Conner and Schmid 2003). This vesicle delivers its cargo to early
endosomes, which are acidied (pH *6) (Al-Awqati 1986). Some receptors and
ligands dissociate at this stage and are recycled for another round of delivery (e.g.,
LDL receptor, transferrin and its receptor). The early endosomes then mature into
late endosomes (pH *5) which, after fusion with prelysosomal vesicles containing
acid hydrolases, generate a harsh environment prone to degradation of the inter-
nalized cargo (Mukherjee et al. 1997; Bareford and Swaan 2007).
In the case of polarized cells, the recycled molecules can either return to the
membrane from which they were internalized, or they can cross the cell and be
delivered to the opposite membrane in a process called transcytosis (Matter and
Mellman 1994). Transcytosis of transferrin is of particular importance in the case if
endothelial cells forming the bloodbrain barrier (BBB) (Jones and Shusta 2007).
Another CME mechanism, involving nonspecic adsorptive pinocytosis, has
been simply referred to as fluid-phase endocytosis by some authors (Sun et al.
2004). Compounds absorbed by this pathway avoid direct binding with membrane
constituents, but often display nonspecic charges and hydrophobic interactions
with the cell membrane. Fluid entry occurs via clathrin-coated vesicles as described
above, internalizing also receptor-ligands located in these pits, together with
extracellular fluid and its content (Bareford and Swaan 2007). Apart from the
296 H. Hillaireau
different modes of interaction with the membrane, the major specicity of this
pathway is a slower internalization rate compared to the receptor-dependent CME
(Strmhaug et al. 1997).
2.2.2 Caveolae-Mediated Endocytosis
Although CME is the predominant endocytosis mechanism in most cells, alternative

pathways have been also identied, in particular the caveolae-mediated endocytosis
(CvME). Caveolae are characteristic flask-shaped membrane invaginations, having a
size generally reported in the lower end of the 50100 nm range (Mukherjee et al.
1997; Bareford and Swaan 2007; Conner and Schmid 2003; van Oss 1978), typically
5080 nm. They are lined by caveolin, a dimeric protein, and enriched with choles-
terol and sphingolipids (Mayor and Pagano 2007). Caveolae are particularly abundant
in endothelial cells, where they can constitute 1020 % of the cell surface (Conner and
Schmid 2003), but also smooth muscle cells and broblasts (Chen et al. 1997). CvME
are involved in endocytosis and trancytosis of various proteins; they also constitute a
port of entry for viruses (typically the SV40 virus) (Cheng et al. 2006), and receive
increasing attention for drug delivery applications using nanocarriers.
Unlike CME, CvME is a highly regulated process, involving complex signaling,
which may be driven by the cargo itself (Bareford and Swaan 2007; Conner and
Schmid 2003). After binding to the cell surface, particles move along the plasma
membrane to caveolae invaginations, where they may be maintained through
receptor-ligand interactions (Bareford and Swaan 2007). The ssion of caveolae
from the membrane, mediated by the GTPase dynamin (Parton and Simons 2007),
then generates the cytosolic caveolar vesicle which does not contain any enzymatic
cocktail (this pathway is employed by many pathogens to escape degradation by
lysosomal enzymes) (Chen et al. 1997). The use of nanocarriers exploiting CvME
may therefore be advantageous to by-pass the lysosomal degradation pathway when
the carried drug (e.g., peptides, proteins, nucleic acids, etc.) is highly sensitive to
enzymes.
On the whole, the uptake kinetics of CvME is known to occur at a much slower
rate than that of CME (Marsh and Helenius 2006). Ligands known to be inter-
nalized by CvME include folic acid (Oh et al. 1998), albumin (Rejman et al. 2005)
and cholesterol (Bareford and Swaan 2007).
2.2.3 Macropinocytosis
Macropinocytosis is another type of clathrin-independent endocytosis pathway

(Chang et al. 1992), occurring in many cells including macrophages (Mukherjee
et al. 1997). It accompanies the membrane ruffling and is induced especially upon
stimulation by growth factors or other signals (Mukherjee et al. 1997; Conner and
Schmid 2003). Macropinocytosis occurs via the formation of actin-driven mem-
brane protrusions, similarly to phagocytosis. However, in this case, the protrusions
do not zipper up along the ligand-coated particle; instead, they collapse onto and
fuse with the plasma membrane (Conner and Schmid 2003). This generates large
endocytic vesicles, called macropinosomes, which sample the extracellular milieu
and have a size generally higher than 1 m (Conner and Schmid 2003) [and
sometimes as large as 5 m (Mukherjee et al. 1997)]. The intracellular fate of
macropinosomes varies depending on the cell type, but in most cases, they acidify
and shrink. They may eventually fuse with lysosomal compartments or recycle their
content to the surface (Mukherjee et al. 1997). Macropinosomes have not been
reported to contain any specic coating, nor to concentrate receptors (Schnitzer
2001). This endocytic pathway does not seem to display any selectivity, but is
involved, among others, in the uptake of drug nanocarriers.
2.2.4 Other Endocytic Pathways
Various clathrin- and caveolae-independent endocytosis have also been described. In

particular, pathways similar to CvME involving cholesterol-rich microdomains called
rafts, having a 4050 nm diameter, have received increasing attention (Conner and
Schmid 2003). A classication for the clathrin- and caveolae-independent pathways
has been proposed (van Oss 1978). However, the understanding of their implications
in the interactions with drug delivery nanosytems is still in a nascent stage.
3 Investigation of Nanoparticle Uptake

and Internalization
3.1 Phagocytosis
3.1.1 Experimental Techniques
Nanoparticles are mainly characterized by (1) their size, measured directly by

various microscopy techniques or indirectly by dynamic light scattering; (2) their
surface properties such as charge, indirectly evaluated from their zeta potential in a
reference electrolyte, hydrophilicity/hydrophobicity and chemical composition
(chromatography and surface spectroscopy); (3) their morphology, assessed by
microscopy. The visualization of their interaction with cells can be achieved
through incorporation of a fluorescent tracer which can be either covalently bound
to the nanocarriers or to the encapsulated drug or simply incorporated into the
nanoparticles (simulating the encapsulated drug). Confocal microscopy has shown
invaluable usefulness to monitor the intracellular trafcking, for example when
performed on unxed living macrophages (Swanson and Watts 1995). The quan-
tication of phagocytosis can be precisely performed using radiolabeled polymers
(in case of nanoparticles) or lipids (in case of liposomes) as well as radiolabeled
drugs, and in some cases through an extraction and chromatography process. The
298 H. Hillaireau
characterization of phagocytosis as an active internalization pathway is often

achieved through inhibition by lowering temperature or using metabolic inhibitors
(e.g, sodium azide) or inhibitors of actin polymerization (e.g, cytochalasin D). The
modern tools of proteomics and live imaging are also used to characterize the
molecular mechanisms of phagocytosis (Groves et al. 2008). Finally, the comple-
ment activation induced by the nanocarriers surface can be investigated by mea-
suring the residual hemolytic capacity of the serum after incubation with the
particles (Racoosin and Swanson 1992).
3.1.2 Nanocarrier Characteristics Influencing Phagocytosis
Size
Although a minimum size of 0.5 m for a particle to undergo phagocytosis is often

put forward (Aderem and Underhill 1999; Groves et al. 2008), this statement is not
always (Martina et al. 2007) justied and generally used to highlight the wide size
tolerance for phagocytosis [macrophages can eat bigger than their head (Labarre
et al. 1993)] compared to other endocytosis modes. Model polystyrene particles in a
range of around 250 nm to 3 m have actually been shown to have an optimal
in vitro phagocytosis rate (particle weight per cell), merely increasing with the
particle size (the number of particles by cell decreases by three orders of magnitude
at the same time), whereas nanoparticles smaller than 250 nm were less efciently
internalized (wt/cell) (Desjardins and Grifths 2003; Aderem 2002). Similarly,
particles based on other polymers [human serum albumin (Korn and Weisman
1967), modied cellulose (Roberts and Quastel 1963; Schfer et al. 1992), poly
(methylmethacrylate) (Korn and Weisman 1967; Tabata and Ikada 1988) and poly
(alkylcyanoacrylate) (PACA) (Korn and Weisman 1967)] exhibited a higher uptake
when their size increased from around 200 nm to several microns. However, in the
case of drug carriers intended for an intravenous administration (requiring small
particle size to avoid embolization), a size of 200 nm can be considered as optimal
(Korn and Weisman 1967). Liposomes generally display the same pattern: larger
(>100 nm) and multilamelar ones are less numerous but deliver a higher payload to
macrophages, compared to the smaller (and unilamelar) ones (Tabata and Ikada
1990; Mller et al. 1992; Rahman et al. 1982; Schwendener et al. 1984; Harashima
et al. 1995). Some studies reported a more balanced (Moghimi and Szebeni 2003)
or even opposite (Rudt and Muller 1993) impact of the liposome size, suggesting
that other factors like surface properties may get the upper hand.
In the presence of serum, the size of the nanocarriers was observed to have a
strong influence on the opsonin adsorption, and therefore on the phagocytosis.
Indeed, the in vitro consumption of complement proteins was demonstrated to
increase with the size of lipid nanocapsules (the total surface exhibited by the
particles being constant) (Heath et al. 1985). This was explained by the fact that on
the most curved surface of the smallest particles, the proper geometric conguration
for efcient complement activation could be achieved less easily than on larger ones
(Vonarbourg et al. 2006). Noteworthy, in vitro and in vivo studies on 200800 nm

liposomes suggested two kinds of uptake by phagocytes: a size-dependant one
involving opsonization, increasing with the size of liposomes, and a
size-independent one, corresponding to unopsonized particles (Allen et al. 1991).
Finally, it is worth mentioning that some macrophages have shown a decrease in
the phagocytosis of small particles (200 nm) and a shift to other endocytosis
pathways like clathrin-mediated endocytosis (Vonarbourg et al. 2006). But in
general, a low uptake by macrophages is observed for nanoparticles smaller than
100 nm, whatever the mechanism involved (Vonarbourg et al. 2006).
Surface Properties
Early studies performed on nanocarriers shown that liposomes (Harashima et al.

1994; Koval et al. 1998) as well as polymer nanoparticles (Gregoriadis 1978;
Torchilin et al. 1980; Grislain et al. 1983) are rapidly cleared from the bloodstream
by macrophages of the RES, virtually irrespective of the particle composition.
Indeed the presence or not of a proper surface coating able to repel opsonins proved
to be the bottom line in entering the phagocytosis pathway or not. The physico-
chemical characteristics of the particle surface are thus critical and determine the
interaction with opsonins prior to cell surface.
The most important driving forces for protein adsorption are often regarded to be
ionic and hydrophobic interactions (combined with entropic gain caused by con-
formational changes of the protein during adsorption) (Lenaerts et al. 1984; Kreuter
et al. 1979). In the case of nanocarriers, highly charged particles have proven to x
complement proteins, especially liposomes (Claesson et al. 1995; Verrecchia et al.
1993), either negatively or positively charged, whatever the complement activation
pathway (Devine et al. 1994; Gabizon and Papahadjopoulos 1992). The observation
that the former often activate even more complement than the latter may originate in
differences in the amount of adsorbed proteins and the opsonins/dysopsonins ratios
(Juliano and Stamp 1975; Chonn et al. 1991) (dysopsonins decrease recognition by
phagocytes). More specically, apolipoproteins have been proposed to contribute
specically to the uptake by hepatocyte (Juliano and Lin 1980). In the case of
polymer nanoparticles, although negative charges can be related to a higher uptake
(Vonarbourg et al. 2006; Moghimi et al. 1993; Scherphof and Kamps 1998), the
surface hydrophobicity appears to be the key factor for opsonization. Nanoparticles
prepared from hydrophobic polymers poly(styrene) (Mosqueira et al. 1999), poly
(lactide) (PLA) (Roser et al. 1998; Norman et al. 1992), poly(lactide-co-glycolide)
(PLGA) (Leroux et al. 1994) and poly(alkylcyanoacrylate) (PACA) (Leroux et al.
1995) undergo important adsorption of Ig, complement proteins and other plasma
proteins like albumin, either in vitro or in vivo. To account for these observations, a
higher level of protein adsorption on hydrophobic surfaces than on hydrophilic ones
has been proposed (Esmaeili et al. 2008; Bertholon et al. 2006), as well as high
afnity of IgG and albumin for hydrophobic regions (Gabizon and Papahadjopoulos
1992; Jeon et al. 1991).
300 H. Hillaireau
However, as a general rule, it is considered that the in vivo fate of exogenous

nanoparticles is opsonization and phagocytosis by RESs protagonists, be they
liposomes or polymer nanoparticles, with little discrimination regarding their
composition, unless the particles possess a very small size (lower than 50100 nm)
or, more importantly, a specic hydrophilic coating able to repel opsonins. Poly
(ethylene glycol) [PEG, also known as poly(oxyethylene)] has been extensively
described for this purpose (Vonarbourg et al. 2006; Owens and Peppas 2006). In
general, PEGylated nanocarriers dramatically decreased the in vitro opsonin
adsorption and macrophage uptake, as compared to their non-PEGylated counter-
parts. After intravenous administration, PEGylation results in a decreased RES
uptake and a prolonged circulation half-life, from typically few minutes to several
hours. Besides PEG, polysaccharides have also been proposed as alternative
hydrophilic polymers (Ilium et al. 1986).
When opsonized nanocarriers encounter macrophage surface, it is noteworthy
that polymer nanoparticles and liposomes, whose structure and chemical compo-
sition strongly differ, still show similar interactions with macrophages, based on
their surface electric charge. Liposomes displaying a negatively charged surface,
generally containing the negatively charged phospholipids phosphatidylserine and
phosphatidylglycerol, have a much higher binding to and phagocytosis by mac-
rophages as compared to neutral vesicles (Moghimi and Szebeni 2003; Cullis et al.
1998; Labarre 2012; Lee et al. 1992a, b, 1993); the same is true for positively
charged liposomes (Tabata and Ikada 1990; Mller et al. 1992). A similar pattern
was found for negatively and positively charged polymer nanoparticles compared to
neutral ones (Roberts and Quastel 1963; Schfer et al. 1992; Moghimi et al. 1993;
Scherphof and Kamps 1998). Additionally, hydrophobic nanoparticles are more
readily captured than hydrophilic nonionic ones (Roberts and Quastel 1963).
Several mechanisms have been proposed to account for the preferential uptake of
charged particles: existence of high charge density areas at the cell surface able to
mediate endocytosis of positively charged particles (Cullis et al. 1998; Hsu and
Juliano 1982) and involvement of nonspecic interactions with nonspecic
receptors by electrostatic interactions for negative particles (Mller et al. 1992),
especially with type B scavenger receptor (Raz et al. 1981). Taking advantage of
macrophage receptors to enhance phagocytosis has been further achieved by cou-
pling specic ligands to nanocarriers. For example, grafting rabbit Ig (Vtvicka and
Fornsek 1987) as well as mouse monoclonal antibody (Rigotti et al. 1995) to
liposomes greatly enhanced their uptake by rat and human macrophages, respec-
tively, most probably through increased FcR binding. Mannose receptors have also
been exploited by the introduction of mannose residue and neoglycoprotein on
liposome surface, enhancing uptake by murine Kupffer cells and peritoneal mac-
rophages (Derksen et al. 1988; Betageri et al. 1993; Barratt et al. 1986). Other ways
have also being explored, as attested by a study on the plasma membrane glyco-
protein CD44 and the possible involvement of scavenger receptors (Muller and
Schuber 1989). However, if the aforementioned studies provide decisive insight in
the nanoparticle surfacemacrophage interaction, it should be kept in mind that, as
soon as in vivo is concerned (or even in vitro studies in presence of serum), the
opsonization process is likely to take precedence.
Shape
The vast majority of nanoparticles developed for drug delivery have a spherical
shape. However, the control of particle shape is receiving increasing attention in
order to control phagocytosis. First, maintaining or not the particles spherical
shape, i.e, rigidity, can be a signicant factor. As far as interaction with the cell
membrane is concerned, macrophages tend to show a strong preference for rigid
particles. One study showed that soft polyacrylamide particles were unable to
stimulate the assembly of actin laments required for the formation and closure of
phagosomes, as opposed to rigid particles (having the same total polymer mass and
surface properties) (Kole et al. 1994). On the other hand, particle rigidity can have
an opposite effect on opsonization. Rigid liposome membranes, composed of
cholesterol and saturated phospholipids with a high melting point, are indeed
known to decrease complement activation and thus phagocytosis (Rudt and Muller
1993; Jeon et al. 1991). Similarly, core-shell nanoparticles having a rigid poly-
styrene core were signicantly less prone to uptake by RES than nanoparticles
made of a more flexible core, based on fluid-like poly(methyl acrylate) (PMA) (Yu
et al. 1997): a flexible particle is thought to provide a greater number of surface
interactions with the biological environment (Alexis et al. 2008). Thus, no clear
relationship emerges between nanocarrier rigidity and phagocytosis.
Besides particle rigidity, other works have focused on the control of the shape
itself. The rationale behind this approach can be found in the well-known exposure
of macrophages to exogenous particles of a high geometrical variety, like
rod-shaped bacteria, Escherichia coli and Bacillus anthracis, disk-shaped senescent
erythrocytes or multiple culpate and solpate airborne pollen grains. Advances in the
synthesis techniques now allow a more precise control of particle geometry
(Beningo and Wang 2002; Sun et al. 2005). Lipid disks have been developed as
alternatives to liposomes, having a diameter of 120 nm (Champion et al. 2007) to
250 nm (Champion et al. 2007), and a thickness of only a few nanometers, showing
efcient uptake by RES macrophages. Other lipid assemblies, either bilayer disks
(Guo 2001) or cube-shaped so-called cubosomes (Larabi et al. 2003) have been
proposed as new drug nanocarriers. However, the impact of the shape of such
systems on phagocytosis as compared to liposomes remains to be fully elucidated.
As demonstrated for polystyrene particles of various shapes (ellipsoids, disks,
UFO-like), the local particle shape at the point of contact dictates whether mac-
rophages initiate phagocytosis or simply spread on particles (Carmona-Ribeiro
2006). For example, a macrophage attached to an ellipse at the pointed end will
internalize it in a few minutes, while a macrophage attach to a flat region of the
same ellipse will not internalize it for over 12 h. This effect, originating in the
complexity of actin structure required to initiate uptake, was even prevailing on
particle size (Carmona-Ribeiro 2006).
302 H. Hillaireau
3.1.3 Theoretical Models of Nanocarrier Phagocytosis
Several theoretical models have been proposed in order to understand and ulti-
mately predict phagocytosis, depending on the nanoparticles characteristics. Based
on the idea that nanoparticle phagocytosis is primarily an interaction between two
surfaces, a rst model has been anticipated using the so-called wettability
hypothesis. According to this model, the probability of phagocytosis is related to
the wettability (measured by the contact angle) of the cell membrane in comparison
to that of the particle surface (Drummond and Fong 1999). In other words, this
model is limited to the hydrophilicity/hydrophobicity characteristics of the
nanoparticles. A more recent theory is based on the hypothesis that the lm tension
existing between the particle and the cell during the early and intermediate stages of
phagocytosis plays a critical role in the mediation of the particle engulfment. This
more comprehensive model allows to take additional forces into account, such the
as electrostatic, van der Waals, receptor-ligand (FcR-Fc) and cytoskeletal (actin
polymerization) forces, allowing rened predictions based on nanoparticle size and
surface properties (Champion and Mitragotri 2006).
3.2 Other Endocytic Pathways
3.2.1 Experimental Techniques
In addition to the above-described methodologies to study the phagocytosismost of

which are also applicable to investigate various endocytosis pathways, specic markers
(Table 1) can be tracked using various inhibitors to characterize CME [e.g, chlorpro-
mazine, known to disrupt the assembly of clathrin-coated pits (Wang et al. 1993)],
CvME [e.g, nystatin and lipin (Schnitzer et al. 1994)] and macropinocytosis [amilorid
Table 1 Major markers of subcellular compartments

Markers Subcellular compartments
Caveolin Caveolae
EAA1 Early endosomes
ESCRTs Late endosomes
GM130 or giantin Cis-Golgi or cis/mid-Golgi cisternae
LAMP-1 Lysosomes
LDL Clathrin-coated pits
LysoTracker Lysosomes and late endosomes (pH < 5.2)
Rab 7 Late endosome
Rab 11 Recycling compartment
TGN46 Trans-Golgi
Transferrin Clathrin-coated pits; early and recycling endosomes
(Swanson 1989; Hoffmann et al. 2001)]. The specic inhibition of CME and CvME has
also been recently achieved through infection with adenoviruses encoding mutant
specic endocytic peptides, prior to incubation with nanoparticles (Harush-Frenkel
et al. 2007). The characterization of RME is often performed using competition studies,
while techniques like surface plasmon resonance allow to quantify the interaction
between receptors and ligand-decorated nanoparticles (Stella et al. 2000). Atomic force
microscopy was also recently used to quantify the interaction between a cell and
nanoparticles deposited on the probe tip (Vasir and Labhasetwar 2008).
Finally, the uptake studies performed on cocultures of different cell lines require
the identication of each cell type simultaneously to the detection of the
nanocarriers, which can be achieved using internalized fluorescent dyes in confocal
fluorescence microscopy, or using specic antibodies in flow cytometry (Grabowski
et al. 2016) (Fig. 2).
3.2.2 Nanocarrier Characteristics Influencing Non-phagocytic

Endocytosis
Contrary to phagocytosis, it is difcult to describe a thorough and consistent prole

of nanoparticle matching each of the above-mentioned endocytic pathway. Indeed,
unlike phagocytosis occurring primarily in professional phagocytes, other endocytic
mechanisms may take place in virtually all types of cells and vary accordingly;
differences may also occur between the apical and basolateral membranes of a
polarized cell (von Bonsdorff et al. 1985). Moreover, several endocytic mechanisms
often take place simultaneously.
Size
Nanoparticle size is a relevant parameter regarding the endocytic pathway, although

its impact may vary upon the type of cells. For example, the same polystyrene
nanoparticles (varying from around 201000 nm) were not preferably endocyted
according to their size by the HUVEC endothelial, the ECV 304 bladder carcinoma
and the HNX 14C squamous carinoma cell lines, whereas the 20100 nm particles
were preferentially internalized by the Hepa 1-6 hepatoma and the HepG2 human
hepatocyte cell lines and the 20600 nm particles by the KLN 205 squamous
carcinoma cell line (Zauner et al. 2001). Some trends can however be drawn.
Cells from the gastrointestinal epithelium [Caco-2 cell line (Desai et al. 1997) as well
as rat gastrointestinal tissue (Desai et al. 1996)] display a greater uptake for 100 nm
PLGA particles compared to 0.510 m ones, both in terms of number and total mass.
The same size-dependency was observed on conjunctival epithelial cells in vivo for
PLGA particles (Qaddoumi et al. 2004), as well as for poly(e-caprolactone)
(PCL) particles (Calvo et al. 1996). In the 1100 nm range, explored more recently, the
highest uptake (in number) was found with 50 nm gold beads (Devika Chithrani et al.
2006; Jiang et al. 2008) for Hela cells, occurring via receptor-mediated endocytosis.
304 H. Hillaireau
Fig. 2 Investigation of nanoparticle uptake kinetics by two cell subpopulations cocultured

simultaneously. a, b Microscopic observations in phase contrast (a) and confocal fluorescence
(b) of THP-1 macrophages (labeled by orange Lyzotracker, colored in red) in contact with A549
pulmonary epithelial cells (labeled by carboxyfluorescein diacetate succinimidyl ester, colored in
green) exposed to PLGA nanoparticles (labeled by Dyomics DY-700, appear as white dots). All
fluorescent probes have been selected to avoid emission spectrum overlap. c, d Flow cytometry
quantication of nanoparticle uptake. Cell subpopulations are identied by fluorescent CD14
antibodies (c) simultaneously with particle uptake by DY-700 dye (d). e, f Nanoparticle uptake is
represented as the increase of mean fluorescence intensity (MFI) compared to corresponding
untreated cells, for both cell subpopulations (Grabowski et al. 2016)
The size may also directly affect the mode of endocytosis. Although the typical
endosome sizes reported (i.e., 100 nm for CME, 5080 nm for CvME) (Bareford
and Swaan 2007; Conner and Schmid 2003; van Oss 1978; Chen et al. 1997) do not
perfectly match the sizes of drug nanocarriers (most of the time higher than
100 nm) (Desai et al. 1996, 1997; Calvo et al. 1996), some size-dependant endo-
cytosis pathways have been reported in the non-phagocytic murine melanoma B16
cells, using 501000 nm polystyrene beads devoid of ligands (Rejman et al. 2004).
Internalization of nanoparticles having a diameter below 200 nm was found to
involve CME. As the size of the particle increased, a shift to the CvME internal-
ization pathway became apparent and turned to be the predominant pathway for
particles as big as 500 nm. Thus, CME occurred for nanoparticles with a size limit
of around 200 nm and kinetic parameters seemed to determine internalization of
these particles along CME rather than CvME (Rejman et al. 2004). More studies are
required to understand the CvME uptake of the biggest particles. On the other hand,
looking at the lower end of nanocarrier size, alternative pathways to CME and
CvME have recently been proposed. In particular, studies on polystyrene
nanoparticles internalization by HeLa cells showed that, while beads of 40 nm in
diameter entered cell through well-known CME, particles smaller than 25 nm were
internalized via a novel non-clathrin and non-caveolae-mediated pathway, being
also cholesterol-independent (Lai et al. 2007), which may open the door to the
design of new drug delivery nanocarriers.
Finally, macropinocytosis corresponds to a poor size-selective endocytosis
pathway, generally occurring in complement of CME or CvME (Harush-Frenkel
et al. 2008; de Rieux et al. 2007). In some cases, size may however have little
influence on uptake (Prabha et al. 2002), compared to surface properties (e.g.
charge and presence of ligands).
Surface Charge
Due to the negatively charged character of the cell plasma membrane, positively
charged drug nanocarriers generally display better association and internalization
rates. Such nanoparticles are generally based on (or coated with) cationic polymers,
the most widely used being the polysaccharide chitosan. Several studies report an
efcient uptake by Caco-2 cells of cross-linked chitosan nanoparticles [e.g., parti-
cles having a zeta potential f +15 to +30 mV (Ma and Lim 2003; Mao et al.
2005)] through adsorptive endocytosis, and possibly involving CME. Similar pat-
terns were observed on A-549 epithelial cells (Huang et al. 2002, 2004). Other
cationic nanocarriers similarly impact endocytosis, such as PLA-PEG based
nanoparticles coated with the cationic lipid stearylamine (f +35 mV) which
showed increased and faster uptake by HeLa cells compared to the negatively
charged parent PLA-PEG nanoparticles (f 35 mV). The former was processed
through the CME pathway, contrarily to the latter (Harush-Frenkel et al. 2007).
Some authors, using quaternary ammoniums to modulate the surface charge of
mesoporous silica nanoparticles (f 5 to +20 mV), suggested a threshold of
306 H. Hillaireau
positive surface charges on endocytosis, depending also on the cell line used
(Chung et al. 2007). Interestingly, a positive charge resulting from drug adsorption
may also impact on the nanocarriers endocytosis, as shown with PCL nanoparticles
loaded with tamoxifen on MCF-7 breast cancer cells (Chawla and Amiji 2002);
however, the drug leakage should be taken into account.
Nonionic Surface Coating
Coating nanoparticle by nonionic polymers like PEG can also influence endocy-
tosis, as suggested by several studies focused on the interaction between various
PEGylated nanoparticles and brain endothelial cells. It was shown that nanospheres
prepared from a poly(methoxyethyleneglycol cyanoacrylate-co-n-hexadecyl
cyanoacrylate) (PEG-PHDCA) copolymer were able to accumulate in both heal-
thy rat brain and brain glioma (Calvo et al. 2001a, b, 2002; Brigger et al. 2002) not
only owing to a prolonged blood circulation time, but also to a specic afnity of
the surface of these nanoparticles for the endothelial cells of the bloodbrain barrier
(BBB) (Brigger et al. 2002). Using an original in vitro model of rat BBB
(Garcia-Garcia et al. 2005), the authors shown that PEG-PHDCA were internalized
through the CME pathway after specic recognition by LDL receptors, and accu-
mulated in endosomal/lysosomal compartments (Kim et al. 2007). While the total
amount of adsorbed proteins was lower onto the PEG-PHDCA nanoparticles than
on their PHDCA counterparts, a preferential adsorption of apolipoprotein E (apo E)
onto PEG-PHDCA nanoparticles was correlated with their increased cell uptake,
thus suggesting the critical role of this protein in the endocytosis of these particles
by the rat brain endothelial cells (Kim et al. 2007a, b). Similar conclusions were
drawn from parallel studies performed on PACA nanoparticles PEGylated by the
single adsorption of polysorbate 80. Such nanoparticles have indeed shown efcient
endocytosis by brain endothelial cells using various labels (Kreuter 2001). Here
also, a preferential adsorption of apo E (and/or apo B) on these particles suggested
that the particles may undergo endocytosis after specic binding to LDL receptors
(Kreuter 2001, 2002). Adsorption of polysorbate 80 onto solid lipid nanoparticles
(SLN) and PLA nanoparticles has also been investigated along the same line
(Gppert and Mller 2003, 2005; Sun et al. 2004). Although some controversy
arose about possible interactions between the desorbed surfactant molecules and the
cell tight junctions (Olivier et al. 1999; Kreuter et al. 2003), this was not the case
with nanoparticles prepared from the PEG-PHDCA copolymer where the PEG
chains are chemically linked, thus rmly bound at the surface of the particles.
Decoration by Targeting Ligands
The decoration of nanoparticles by targeting ligands, i.e, molecules able to rec-

ognize a specic biological target, has been investigated in order to promote
delivery to a specic cell population and/or to control the intracellular trafcking of
the nanocarriers. This strategy relies on the idea that ligand-bearing nanocarriers
will be internalized through the same pathway as the ligand alone. Moreover, the
concentration of ligands on the nanoparticles surface offers potential for stronger
cell interactions as compared to ligand alones. Indeed, the entropic gain in the
formation of multivalent complexes may increase the binding constants by a factor
of 1000 for bivalent interactions and even by 108 for tri- and pentavalent ones
(Haag and Kratz 2006).
The folic acid (FA) vitamin has been widely studied as a targeting ligand for
nanocarriers, especially for anticancer strategies (Chavanpatil et al. 2006). Indeed,
FA binds with a low afnity to the reduced folate carrier present in virtually all
cells, but with a high afnity (in the nanomolar range) to the
glycosylphosphatidylinositol-linked folate receptor (FR), which exhibits highly
limited distribution (Haag and Kratz 2006). In particular, FR is often overexpressed
on the surface of cancer cells but highly restricted in normal tissues (Hilgenbrink
and Low 2005). Moreover, FR has the ability to transport both FA and the
FA-linked cargo by RME with subsequent endosomal escape into the cytosol (Haag
and Kratz 2006; Weitman et al. 1992), thus avoiding lysosomal degradation.
Although CvME appears to be involved in the uptake of FA in some cases
(Rothberg et al. 1990; Pelkmans and Helenius 2002), the complete mechanism is
complex and remains debated (Haag and Kratz 2006; Dauty et al. 2002). In the case
of polymer nanocarriers, plasmon surface resonance revealed that FA covalently
bound to PEG-PHDCA nanoparticles had a tenfold higher apparent afnity for FR
compared to free FA (Stella et al. 2000). Liposomes were also decorated with FA
by incorporating a phospholipid-anchored FA (Sabharanjak and Mayor 2004) or a
FA-PEG-phospholipid conjugate (Lee and Low 1995) into the liposome bilayer.
Such liposomes have shown a preferential uptake by FR-expressing cells. Similar
cell uptake data were obtained with PLGA nanoparticles coated with the poly
(L-lysine)-PEG-FA conjugate (Gabizon et al. 2004), with albumin nanoparticles
coated with activated FA (Chavanpatil et al. 2006), as well as with polymer
micelles prepared from a mixture of poly(L-histidine)-PEG-FA and PLA-PEG-FA
(Kim et al. 2005).
Transferrin (Tf) has also been studied as targeting ligand to specic cell popu-
lations in order to increase cellular uptake of nanocarriers. Indeed Tf receptors
(TfR) are overexpressed in several malignant tissues compared to healthy ones (Lee
et al. 2005) [typically twofold to tenfold more (Qian et al. 2002)]. PLGA
nanoparticles conjugated with Tf exhibited a twofold greater in vitro uptake by
MCF-7 cells as well as a reduced exocytosis, compared to unconjugated PLGA
particles; competition experiments with free Tf conrmed the involvement of TfR
in the uptake process (Vasir and Labhasetwar 2007). In vivo studies performed in
S-180 solid tumor-bearing mice showed a promising accumulation in the tumor of
paclitaxel after intravenous administration of Tf-conjugated to PEG-PACA
nanoparticles loaded with this drug (Sahoo and Labhasetwar 2005). TfR are also
known to be highly expressed in some healthy tissues like brain capillaries where
they are known to mediate transcytosis (Jones and Shusta 2007). Interestingly,
Tf-conjugated to PEG-coated albumin nanoparticles signicantly increased the
308 H. Hillaireau
delivery of AZT to rat brain, the proportion of the drug located in this tissue being
doubled as compared to the same nanoparticles devoid of ligand (Xu et al. 2005).
The use of ligands like Tf for nanocarrier functionalization may however be
hindered by a competition with the corresponding endogenous pool of ligands
(Jones and Shusta 2007). This is the reason why monoclonal antibodies
(MAb) have been employed, as for instance the mouse OX26 directed against the
rat TfR. This MAb binds to a TfR epitope dinstinct from the Tf binding site, thus
preventing competition with endogenous Tf (Mishra et al. 2006). In this context,
OX26 has been conjugated to PEGylated liposomes to increase the brain delivery of
the encapsulated drug daunomycin to rats (Lee et al. 2000; Huwyler et al. 1997).
The transcytosis mechanism of such OX26 immunoliposomes was demonstrated
using an in vitro model of BBB consisting in a monolayer of rat brain endothelial
cells RBE4 (Maruyama et al. 1995). Similar studies were carried out on mice, but
using another MAb, the rat 8D3 MAb to the mouse TfR (Mishra et al. 2006). It was
also observed that PEGylated immunoliposomes decorated with the Fab fragments
of antibodies reduced the RES uptake that is observed when using the whole
antibodies (Cerletti et al. 2000) whose Fc fragment may be recognized by macro-
phages (Maruyama et al. 1997). Using the avidin (SA)-biotin (BIO) technology,
chitosan nanospheres were also conjugated with PEG bearing the OX26 MAb.
These functionalized chitosan-PEG-BIO-SA/OX26 nanoparticles were able to
translocate into the brain tissue after intravenous administration (Harding et al.
1997). A high density of antibodies at the nanocarrier surface may, however,
increase hydrophobicity, thus limiting the ability to escape the recognition by the
RES. To combine efcient targeting and minimal RES uptake, an optimal coating of
1030 antibody molecules per particle has been suggested in the case of liposomes
(Akta et al. 2005; Andresen et al. 2005). For example, a density of around 30
OX26 antibody molecules per (100 nm) liposome was found optimal for brain
delivery to rats in two independent studies (Huwyler et al. 1997; Maruyama et al.
1999).
Lectins have also attracted interest because of their inherent ability to provide
specic binding to carbohydrates located at the surface of epithelial cells (Huwyler
et al. 1996). Tomato lectins (TL) have been intensively utilized for the delivery to
the intestinal mucosa after oral administration, as TL-coated particles were shown
to avidly adhere to enterocytes both in vitro (Ponchel and Irache 1998) and in vivo
(Lehr et al. 1992). Other lectins like wheat germ agglutinin (WGA) have the ability
to bind cancer cells preferentially to normal cells (Florence et al. 1995).
Interestingly, PLGA nanoparticles coated with WGA exhibited a preferential uptake
by A549 and H1299 cancer cells (Aub et al. 1963; Mo and Lim 2005).
Ligands of cell adhesion molecules (CAMs) have been more recently investi-
gated for the targeting of various endothelial cells. In particular, RGD peptides have
been used to target tumor cells with increased expression of specic CAM integrins
(Mo and Lim 2005). For example, PEGylated liposomes conjugated with the RGD
peptide were found to form clusters on endothelial microvessels of tumors in mice,
contrary to control liposomes conjugated with a RAD peptide (Dunehoo et al.
2006). Intracellular CAM-1 (ICAM-1) is another particularly interesting target for
perturbed endothelial cells (Schiffelers et al. 2003). Polystyrene nanoparticles

bearing MAb to human and mouse ICAM-1 were developed for this purpose (Ding
et al. 2006). Interestingly, endothelial cells did not internalize ICAM-1 MAb but
well MAb-coated nanoparticles or multivalent MAb conjugates. Indeed, the uptake
was found to require ICAM-1 clustering. The endocytosis pathway was indepen-
dent from CME, CvME, macropinocytosis and phagocytosis (Muro et al. 2003,
2006). The nanoparticles nally trafcked to lysosomes (Muro et al. 2003). In vivo
studies showed that nanoparticles conjugated with ICAM-1 MAb enabled also
vascular delivery to pulmonary and vascular endothelium (Muro et al. 2006).
Shape
The influence of particle shape on endocytosis is being increasingly investigated. In

some cases, it was found that spherical nanoparticles had a higher and faster rate of
endocytosis compared to rods or disks, as demonstrated using gold nanoparticles
(Devika Chithrani et al. 2006) as well as ICAM-1- and TAT-coated nanoparticles
(Christodou-Solomidou et al. 2000; Muro et al. 2008). On the contrary, other
studies suggested preferential uptake of rod-shaped (Zhang et al. 2008) or cylin-
drical (Gratton et al. 2008) particles.
3.2.3 Theoretical Models
Several models have been proposed to describe the non-phagocytic endocytosis of

viruses and nanoparticles. A rst model of RME based on the hypothesis of a cell
membrane containing diffusive mobile receptors, wrapping around a particle coated
with corresponding ligands, leads to a threshold particle radius of around 30 nm,
above which endocytosis is likely to occur (Gratton et al. 2008). Then, a more general
mechanism has been proposed, which includes also nonspecic attractive/repulsive
forces. This contribution, which was found to be as important as specic interactions,
leads to more general predictions (Gao et al. 2005). The importance of elastic
deformation of the cytoskeleton for the engulfment has also been shown to be of
signicant contribution (Decuzzi and Ferrari 2007). However, the variety of the
mechanisms of non-phagocytic internalization pathways seems to complicate the
elaboration of a general model, able to t the multiple experimental data.
4 Investigation of Nanoparticle Intracellular Trafcking
In addition to above-mentioned techniques, the intracellular trafcking can be

monitored using fluorescent markers specic for vesicles (e.g, Lysotracker for the
endo/lysosomes). Cell fractionation techniques are also used, such as a selective
permeation of membranes, in which sequential treatments with proteases and
310 H. Hillaireau
Fig. 3 Investigation of nanoparticle subcellular distribution following uptake by endothelial cells. a,

b Semi-quantication of internalized PEG-PHDCA nanoparticles labeled by Nile red in primary rat
endothelial cells from the blood brain barrier, showing the involvement of LDL receptors. c Cell
fractionation methodology, based on selective membrane permeation or desorption, allowing access to
cytoplasmic membrane-bound, cytosolic, endo/lysosome and residual fractions (Eboue et al. 2003).
d Quantication using 14C-labeled PLGA over 2 h exposure, showing a progressive endosomal
sequestration. Reproduced from Kim et al. (2007); Garcia-Garcia et al. (2005b). Source: Elsevier
surfactants allow the recovery and the quantication of (fluorescence or radiola-

beled) material associated to the plasmic membrane, cytosol, endo/lysosomal
vesicles and other organelles (Sun and Wirtz 2006) (Fig. 3).
With respect to nanocarrier characteristics, the particle electric charge plays a
crucial role in its intracellular fate. This is of particular importance in its interaction
with endocytic vesicles, in response to the pH decrease during endosome matura-
tion and fusion with lysosomes. Most strategies developed in this eld aim at
promoting endosomal escape in order to limit drug degradation due to low pH and
presence of enzymes, and to ensure the cytosolic delivery of the drug when needed.
pH-sensitive liposomes have been tailored for this purpose. Most of them are
based on dioleyl phosphatidylethanolamine (DOPE), which undergoes a transition
from lamellar to inverted micelles structures at low pH, allowing the fusion between
the liposomal and the endosomal membranes, and the destabilization of the
endosomes (Garcia-Garcia et al. 2005; Fattal et al. 2004). DOPE is often used in
combination with the mildly acidic amphiphils oleic acid (OA) and cholesteryl
hemisuccinate (CHEMS). At neutral pH, OA and CHEMS (ionized) act as stabi-

lizers and allow DOPE to maintain a bilayer structure; at lower pH, OA and
CHEMS get protonation and cause the destabilization of the liposomal bilayer with
the subsequent release of the liposome content (Zuhorn et al. 2007). Typically,
DOPE:OA liposomes become leaky at pH 6.5 and DOPE:CHEMS at pH 5.5 (Ellens
et al. 1984). The transfer of the DOPE molecules to the endosomal membrane is
thought to promote endosome leakage (Garcia-Garcia et al. 2005; Drummond et al.
2000), although the precise mechanism remains to be elucidated (Qian et al. 2002).
Such pH-sensitive liposomes have shown efcient in vitro cytosolic delivery of
model fluorescent probes (Bergstrand et al. 2003) and oligonucleotides
(ODN) (Straubinger et al. 1985; Ropert et al. 1992). However, the in vivo efcacy
is more questionable, mainly due to stability concerns in the presence of serum
(Dzgnes et al. 2001).
Unlike these pH-sensitive anionic liposomes, lipoplexes, resulting from the
complexation of nucleic acids with cationic lipids, exhibit a total net positive charge
(Qian et al. 2002; Fattal et al. 2004). They are often designed using the cationic
DOTMA (Connor et al. 1986) and DOTAP (Felgner et al. 1994). Although they
display some in vitro transfection activity, their efcacy runs short from a thera-
peutic point of view. This was attributed to an inefcient destabilization of the
endosomal membranes (Fattal et al. 2004).
On the contrary, if cationic polymers do not possess fusogenic activity per se,
some of them, like the popular poly(ethylene imine) (PEI), have however the ability
to disrupt endosomal membrane, as a consequence of their important buffering
capacity (Simberg et al. 2004). Indeed, polyplexes resulting from the complexation
of DNA plasmids with PEI show remarkable transfection efciency on various cell
lines, which lead the authors to propose the so-called proton-sponge effect
(Demeneix et al. 2004). According to this hypothesis, the endosomal pH decrease
entails a high protonation of PEI, which results in an osmotic swelling due to water
entry and subsequent vacuole disruption, thus allowing the cytoplasmic release of
the PEI/DNA particles (Demeneix et al. 2004). This was supported by the fact that
PEI transfection efciency was decreased by 100-fold by balomycin-A, a specic
vacuolar H+-ATPase inhibitor (Boussif et al. 1995). Noteworthy, live cell micro-
scopy also allowed to visualize a burst release of polyplexes from the acidic vesicles
(Kichler et al. 2001). Other mechanisms have also been suggested, such as a possible
swelling of the polymer network resulting from the increasing repulsion of the
protonated groups (Merdan et al. 2002). Based on electron microscopy observations,
direct interactions with the lysosomal membrane leading to the formation of holes
have been proposed too (Behr 1997). The precise mechanism of the endo/lysosomal
escape as well as the transport to the nucleus is not yet clearly understood. However,
PEI remains today one of the major transfection agents useful for the design of
nanocarriers able to escape the endosomes. Recent studies aim at clarifying the
impact of the physicochemical characteristics of PEI (structure, molecular weight)
and its polyplexes on the transfection efciency (Bieber et al. 2002).
Combining such features promoting endosomal escape with stealth properties in
the same nanocarriers is often challenging. Some developments of nanocarriers
312 H. Hillaireau
based on modied PEG have paved the way for new pH-responsive systems, the
key feature being the incorporation of acid-labile groups. For instance, polymer
micelles loaded with the anticancer drug doxorubicin were prepared from
PEG-dendrimer hybrids on which hydrophobic groups were attached through an
acid sensitive acetal linkage (Neu et al. 2005). The micelles were stable at pH 7.4,
but upon acidication of endosomes, the loss of hydrophobic groups by hydrolysis
caused the destabilization of the micelles, which enabled drug release (Gillies et al.
2004). Another recent example involves the self-assembling of the amphiphilic
block copolymers PEG-poly(aspartate), to which the anticancer drug adriamycin
was conjugated through hydrazone linkers that were stable at pH 7, but cleavable at
pH 6 and below. The micelles formed by this copolymer were taken up in vitro by
the cells of a multicellular tumor spheroid and the released drug was observed to
accumulate in the cell nuclei, suggesting that escape from endo/lysosomes has taken
place (Gillies and Frchet 2005).
The decoration of nanocarrier surface with ligands also determines its intracellular
fate. Cell penetrating peptides (CPPs), also known as protein transduction domains,
have also raised increasing attention due to their ability to translocate across mem-
branes (Bae et al. 2005; Patel et al. 2007). The most commonly studied CPP for
nanoparticle functionalization is the HIV-1 trans-activating transcriptional activator
peptide (TAT). Remarkably, ultrasmall superparamagnetic iron oxide particles
(USPIO) coated with TAT were shown to efciently tag progenitor cells (Torchilin
2008). An increasing number of examples of conjugation of TAT to liposomes (Lewin
et al. 2000), polymer micelles and polyplexes (Yagi et al. 2007) have been described.
However, the internalization mechanism of CPPs remains to be fully elucidated: it
may involve macropinocytosis (Bae et al. 2005), but also CME and CvME (Patel et al.
2007), as well as direct penetration, although the latter remains debated. Endosomal
escape of CPPs and nuclear targeting also need further investigations.
Other ligands have been recently investigated to address nanocarriers to intra-
cellular organelles, like mitochondria and nucleus. The few examples of mito-
chondrial targeting of nanoparticles include the binding of the peptide sequence
Mito-8 to quantum dots (QDs), which showed strong in vitro mitochondrial
localization (Kleemann et al. 2005). Nucleus targeting of nanoparticles is actively
investigated for gene delivery. Promising experiments have been carried out using
nuclear localization signals (NLS) peptides (Hoshino et al. 2004) or TAT peptides
(Tkachenko et al. 2003) coated onto gold nanoparticles.
Finally, the influence of particle shape on the intracellular trafcking also
deserves more insight. A recent study has compared layered double hydroxides
(LDHs) nanoparticles made of Mg and Al oxides, having hexagonal or rod shapes
(de la Fuente and Berry 2005). Both were internalized by various mammalian cell
lines through CME and were found to escape from endosomes (probably through
their buffering capacity), but hexagonal LDHs remained in the cytoplasm whereas
rod-like LDHs were directed to the nucleus, probably through a
microtubule-mediated active transport mechanism (de la Fuente and Berry 2005).
This opens exciting perspectives, especially for the control of the intracellular gene
delivery.
5 Concluding Remarks
While signicant progress in the understanding of the nanoparticle internalization

by a variety of mammalian cells has already been achieved, many advances in
nanomedicine still rely on phagocytosis, while tailoring nanocarriers targeting the
other endocytic pathways is complicated by the variety of the cells and internal-
ization mechanisms encountered. Modeling such complex biological barriers with
reliable in vitro systems remains a difculty, together with the disparities in the
experimental conditions used to study the nanoparticle-cell interactions. Despite
these hurdles, the expending knowledge of biological markers offers increasing
possibilities to target nanocarriers to and inside the desired cell populations.
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Part III
Turning Polymer Nanoparticle
Technologies into Nanomedicines
Chapter 11
Designing Polymer Nanoparticle
Nanomedicines: Potential Applications
and Challenges
Christine Vauthier
Abstract Several challenges need to be fullled to design polymer nanoparticles

with suitable characteristics to achieve desired applications as nanomedicine. The
part 3 of the book was aimed to discuss different approaches that are used to tune
characteristics of nanoparticles adjusting functionalities needed to maximize the
efcacy of the delivery approach. A chapter is devoted to the synthesis of polymers
that are main components of the polymer nanoparticles and give several func-
tionalities to the nal nanoparticles. The different ways to achieve association of
drugs and approaches that can be developed to control the release of the drug from
the nanoparticles are presented in two distinct chapters. Other aspects included in
this part of the book are devoted to the understanding of interactions between
proteins and nanoparticles that are at the bottom of the control of the biodistribution
and safety of nanoparticles. A nal chapter proposes a prospective view on
developments of polymer nanoparticles as tools for theranostic combining thera-
peutic and diagnostic functionalities. This introduction to the part 3 of the book is
aimed to place these chapters in the more global prospective of designing functional
nanomedicines based on polymer nanoparticles in regard with their potential
applications and challenges to complete.

Keywords Polymer Colloid Chemotherapy Clinical trials Biocompatible

Biodegradable Biodistribution Diagnostic Drug-ability Drug delivery

Imaging Intracellular delivery Intravenous injection Mucosal administration

Nucleic acids siRNA Peptides Personalized medicine Polymer Proteins

Systemic activity Target cells Target tissue Theranostic Therapeutic activ-

ity Treatment
C. Vauthier (&)
Institut Galien Paris Sud, Faculty of Pharmacy, UMR CNRS 8612, University of Paris-Sud,
University Paris Saclay, 92296 Chtenay-Malabry Cedex, France

DOI 10.1007/978-3-319-41421-8_11
328 C. Vauthier
1 Introduction
Polymer nanoparticles are proposed in the arsenal of nanomedicines to improve the

quality of drug delivery treating diseases and the performance of diagnostic
methods based on imaging technologies. Polymers are versatile chemical com-
pounds. Progresses in polymer chemistry of the last decades have paved the road to
methods of synthesis that allow production of polymers with well dene charac-
teristics hence properties. Polymers having a quite complex composition and
architecture can be synthesized with a high degree of precision. For instance, the
polymer can include various domains that each has a different functionality while
they are arranged in the polymer structure in a well-organized manner (Gao and
Matyjaszewski 2009; Le Droumaguet and Nicolas 2010; Hasjichristidis et al. 2011;
Delplace and Nicolas 2015). From the polymer architecture, it is possible to control
the way polymer chains assemble together forming nanoparticles with specic
properties. This opens possibilities for exciting innovations developing multifunc-
tional and intelligent nanomedicines (Mura et al. 2013). Therefore, polymer
chemistry can be seen as an essential support to engineer nanoparticles showing all
properties needed to achieve a well dene clinical need. However, to make a
nanoparticle a nanomedicine, it is extremely important to rigorously identify the
type of functionalities that are needed for the intended application. It is also required
to dene whether the polymer will serve as inert excipient or will give the
nanoparticles the possibility to modulate the biological response (Mahon et al.
2012). Altogether, the number of functionalities should not be overestimated or
underestimated. The way the different functionalities are implemented in the
nanoparticles must remain the simplest as possible. At the end, the designed
nanoparticles should be relevant to answer the predetermined clinical need and to
meet the challenge of translational process toward its regulatory approval for
clinical use (Merkle 2015). In this part of the book, the different chapters report
examples of approaches suggested in the literature to implement a given func-
tionality while engineering polymer nanoparticles designed to answer a clinical
need. Before, this chapter discusses the clinical potentials of the nanoparticles.
Difculties and challenges of implementation are considered at the end of the
present chapter.
2 Clinical Potentials of Polymer Nanoparticle

Nanomedicines
Clinical potentials of nanomedicines including polymer nanoparticles are found in

their possibility to solve most of drug delivery challenges whatever they are related
to the drug molecules or/and delivery issues considering the different possible
routes of administrations used to produce a local or a systemic activity.
Nanoparticles can diffuse in biological tissues thanks to their small dimensions.
11 Designing Polymer Nanoparticle Nanomedicines 329
Having suitable properties that are dened from their composition, method of
synthesis and eventually applying post-synthesis modications, they can carry
many copies of active ingredients up to target cells in a more efcient way than the
free drug is reaching the target cells by itself (Fig. 1). As a second important role
they can help their load to penetrate inside cells to reach intracellular biological
targets when the free drug is unable to go across biological membranes (Fig. 1a).
Many applications were developed considering drugs used in chemotherapies.
Signicant benets were generally obtained from a marked reduction of the inci-
dence of the severe side effects inherent to the chemotherapeutic agent thanks to a
modication of the biodistribution compared with that of the free form of the drug.
Increase in efcacy of chemotherapeutic treatments remains debatable based on an
improved efciency and specicity of drug delivery to the target tissues and cells
(Brambilla et al. 2014). A large number of studies acknowledged the potential of
polymer nanoparticle formulations of drugs increasing the overall efcacy of
chemotherapy based on a modication of the biodistribution of the drug associated
with the carrier. Applications include treatments of cancer and infectious diseases
with already well know molecules. For instance, a small sample of relevant
molecules considered in these treatments includes doxorubicin, paclitaxel, mitox-
antrone for treatments against cancer, amphotericin B, and ciprofloxacin used in
treatments against infections. It is noteworthy that the delivery approach also
showed interesting potential to reboost activity of drugs in cells that became
resistant to treatments (Ganoth et al. 2015; Hemant et al. 2015). In these cases, the
nanomedicine hence the polymer nanoparticle allows to bypass resistance mecha-
nisms that hinder penetration of the drug into cells. Clinical potentials of polymer
nanoparticle-based nanomedicines are not limited to chemotherapy. Much broader
therapeutic potentials have been identied improving drug delivery to
hard-to-access tissues such as the central nervous system and to allow delivery of
molecules that display unfavorable physicochemical characteristics or/and a poor
stability in biological media. For instance, polymer nanoparticles provide a mean to
deliver drugs to the brain carrying the molecules through the blood brain barrier
with a potential to develop efcient treatments against neurodegenerative diseases
such as the Alzheimer and Parkinson diseases (Andrieux and Couvreur 2009; Tosi
et al. 2013). Considering drugs with delivery problems, they are one type of
nanomedicines that have the potential to bring them into clinics despite their poor
drug-ability prole. In general, they are protecting active molecules against
degradation in biological fluid in an efcient way. Then, their endocytosis by cells
provides a means of intracellular delivery of molecule that are incapable to enter
cells by themselves. A particularly relevant example concerns the development of
treatments with nucleic acids including small interfering RNA (siRNA), antisense
oligonucleotides, Micro-RNA (Miele et al. 2012; Kanasty et al. 2013; Zhou et al.
2013; Zuckerman et al. 2014; Xu and Wang 2015). These molecules are active at a
low dose and their biological activity is highly specic due to the complementarity
of sequence requested with that of the target DNA or RNA found in cells that need
to be treated. For instance, siRNA hold promise for the development of innovative
strategies of treatments against cancer and viral infections where abnormal or
330 C. Vauthier
Fig. 1 Drug delivery modalities achieved from the blood compartment to diseased tissues (top
parts) and cells (D) and to healthy tissues (bottom parts) and cells (H) using a nanomedicine such
as a polymer nanoparticle (a) and from a traditional drug formulation (b). The line separating the
blood compartment and the tissues represents the endothelium that is generally more permeable
and less structured in diseased tissues (top parts) compared with heathy tissues (bottom parts).
When drugs are associated with a nanoparticle, drug molecules are ideally expected to be delivered
to diseased cells (D) while they are still associated with the nanoparticles (a). A higher delivery to
disease tissue is expected due to the leaky structure of the blood vessels (top part) while the
nanoparticles should remain in blood vessels irrigating healthy tissues (bottom part). The
nanoparticles can be designed in such a way that they can recognized specically the diseased cells
(D) and deliver the drug with a high efcacy to these cells preserving healthy cells (H) from an
exposure to the drug. When the drug is administered in a traditional formulation, it circulates in the
blood as a soluble molecule (b). It is available to penetrate in any cells if it is capable to diffuse
through the cell membrane or it cannot penetrate in cells when it is unable to diffuse through the
cell membrane by itself. It is also not protected from a degradation that may occur in biological
fluids (b)
exogenous genes are expressed in diseased cells and a down regulation of the
expression of these genes can stop the progression of the disease. However, the
chance for these short fragments of nucleic acids to be translated in clinics is
pending to their entry as intact molecule into cells. Experiments based on a systemic
administration of siRNAs have conrmed that free siRNAs are rapidly degraded by
nucleases in body fluids and are unable to reach the cytoplasm of the target cell in
an active form. Conversely, effective in vivo activities of siRNA were reported
when these molecules were delivered with polymer nanoparticles among other
nanomedicines suggesting that these carriers have the potential to lift locks that
compromised their clinical developments based on the achievement of a systemic
activity. In their mode of action, the nanoparticles protect the nucleic acids against
degradation during transportation in the extracellular media and insure the delivery
of the siRNA into the cell cytoplasm going across the plasma membrane of cells.
Additionally, the nanoparticles can be used to tune the biodistribution of the siRNA
so that a higher amount can reach the target cells. The potential shown by
nanomedicines including polymer nanoparticles to succeed in systemic delivery of
short fragments of nucleic acids is acknowledged by the rapid growth of the amount
of reports published on the subject that has started only few years after the dis-
covery of siRNA (Kanasty et al. 2013; Wue et al. 2014; Xu and Wang 2015). An
exciting eld of research has emerged developing innovative treatments of cancer
and infectious diseases that has already paved the road to a translation in clinics for
the treatment of solid tumors considering an anticancer siRNA (ClinicalTrials.gov
2016a). The molecule produced an efcient knockdown of the target gene
expression in human solid tumor from a blood-mediated nanoparticle delivery
obtained through intravenous administration (Zuckerman et al. 2014; Hubbell and
Langer 2013). Therapeutic peptides and proteins are another type of drugs that are
difcult to deliver in vivo because of their poor stability and their inability to diffuse
across biological membranes by themselves. Polymer nanoparticles can protect
these molecules against degradation including in the harsh conditions found in the
gastro-intestinal tract. A lot of works explore the potential of polymer nanoparticles
for their ability to promote absorption of the transported molecules by mucosal
barriers that are impassable for intact peptides and proteins (Pridgen et al. 2015;
Silva et al. 2015).
Solubility is another well identied bottleneck to clinical translation of drug
molecules. In general, in vivo delivery of drugs that show dissolution problems is
improved after association with polymer nanoparticles. Paclitaxel is a typical
example of drug with which in vivo delivery is complicated by the low solubility of
the molecule. The formulation having considered the formation of a complex with
albumin nanoparticles has improved the systemic delivery of the drug and has also
already led to a clinical translation of the approach (Miele et al. 2009; Yu and Jin
2016). In this example, the success of the delivery method was associated with a
large benet of the reduction of side effects produced by excipients used in previous
formulations and that were needed to achieve the dissolution of the drug and from a
modication of the biodistribution prole of the drug (Chen et al. 2014).
332 C. Vauthier
Being able to improve drug delivery modalities for a broad range of molecules
solving various types of drug delivery problems, a lot of pathologies can benet
from the use nanomedicines including polymer nanoparticle based treatments.
Many concern treatments of cancer based on different modalities that can be pro-
posed with the use of polymer nanoparticles. Other pathologies include neurode-
generative diseases, inflammatory and autoimmune diseases, metabolic diseases
like diabetes, lung, and cardiovascular affections (Couvreur and Vauthier 2006).
Exploration of the potential of polymer nanoparticles to improve diagnostic
methods based on in vivo imaging modalities has generated far less work so far but
investigations has started more recently (see Chap. 17 from Herceg et al.). Similarly
to approaches that are developed to improve drug delivery efcacy to well-dene
tissues or cells, polymer nanoparticles can be used to transport a contrast agent in
the body promoting its accumulation in diseased tissues or cells. Thus, the signal
monitored from the imaging modality can be much increased because of a better
accumulation of the contrast agent in abnormal cells and tissues. Widely applied for
the detection of tumors, the new type of contrast agents should enable the earlier
detection of cancer based on the improvement of contrast produced by the smallest
tumors compared with the application of traditional imaging modalities. In the most
advanced systems, a drug and a contrast agent are conned together in the heart of a
single polymer nanoparticle (Ryu et al. 2014; Mura and Couvreur 2012; Chap.17
from Herceg et al.). The carrier being also a contrast agent can be used for the
diagnostic and to follow simultaneously the delivery of the drug to the target site in
real time. Additionally, it has the clinical potential to evaluate the efcacy of the
applied treatment by monitoring the regression of the volume of the altered tissue
during treatment.
A tremendous amount of work has already demonstrated a broad range of rel-
evant clinical potential of polymer nanoparticles to develop new efcient treatments
for severe diseases and improve the quality of diagnostic methods based on imaging
techniques (Mura and Couvreur 2012; Thorley and Tetley 2013). Translation into
clinics has started and become a reality with applications in chemotherapy to treat
severe forms of cancer (Zhou et al. 2009; Soma et al. 2012; Hubbell and Langer
2013; Yu and Jin 2016) (Table 1). So far, several formulations are in clinical
developments and one product has been approved (Miele et al. 2009; Celgene 2015,
2016; Yu and Jin 2016). The approved product, i.e., Abraxane, consists of
albumin-bound paclitaxel nanoparticles. It is administered by intravenous injection
to patients and concentrates the drug in tumors thanks to the combined effect of two
mechanisms (Miele et al. 2009). Part of the accumulation is due to a passive
accumulation in tumor tissue thanks to the enhanced permeability and retention
effect. This mechanism is particularly found in tumors irrigated by leaky blood
vessels that let nanoparticles to escape the blood compartment. The second
mechanism is specic; it involves a transendothelial transport of the albumin
nanoparticles that is mediated by the albumin-binding protein gp60. In 2005,
Abraxane was approved by the Food and Drug Administration in the United States
of America (FDA) as a second-line treatment for patients with breast cancer
(Abraxane web site 2016). More recently, in 2015, it was approved by the European
11
Table 1 Clinical trials on cancer treatments considering drug formulations occurring as polymer nanoparticles
Disease Drug Type of Name Stage of the Route of Sponsor/Ref.
nanoparticles development administration
Hepatocellular carcinoma Doxorubicin Poly Livatag NCT01655693 Intravenous Onxeo
(isohexylcyano Ongoing Phase Onxeo web site
acrylate) III (2016)
nanoparticles Soma et al.
(2012)
Hepatocellular carcinoma Mitoxantrone Poly(butylcyano Phase II Intravenous Zhou et al.
acrylate) completed (2009)
nanoparticles
Breast cancer, non-small cell lung Paclitaxel Nanoparticle Abraxane Marketed Intravenous Celgene
Designing Polymer Nanoparticle Nanomedicines
cancer, metastatic adenocarcinoma Albumin (Nab-paclitaxel) several ongoing Celgene (2015,

of pancreas nanoparticles clinical trials 2016)
Solid tumors siRNA Cyclodextrin CALAA-01 NCT00689065 Intravenous Calando
polymer Phase I pharmaceuticals
nanoparticles Zuckerman
et al. (2014)
333
334 C. Vauthier
Medical Agency (EMA) as rst-line treatment of patients with non-small cell lung
cancer (Celgene 2015, 2016; European Medical Agency 2016). Clinical trials are
still ongoing with the aim to expend indications of Abraxane for treatments of
other severe forms of cancer including the metastatic adenocarcinoma of the pan-
creas (Celgene 2016; European Medical Agency 2016). Other polymer nanoparticle
formulations of anticancer agents are evaluated for treatment of patients with
hepatocellular carcinoma, a primary liver cancer with severe prognostic (Soma et al.
2012; Zhou et al. 2009; Onxeo web site 2016; ClinicalTrials.gov 2016b). One of the
nanoparticles is intended to deliver doxorubicin (Onxeo web site 2016;
ClinicalTrials.gov 2016b; Soma et al. 2012) and the other is a formulation deliv-
ering mitoxantrone (Zhou et al. 2009). Both are composed of polymers of the poly
(alkylcyanoacrylate) family. The last example consists on polymer nanoparticles
that are designed to deliver an anticancer siRNA by intravenous injection as
treatment for human solid tumors. This system has been evaluated in a clinical trial
phase 1 terminated since 2013 (ClinicalTrials.gov 2016a; Hubbell and Langer
2013; Zuckerman et al. 2014).
As discussed above, polymer nanoparticles can contribute to the development of
innovative approaches that have the potential to diagnose and treat diseases. While
they have the potential to optimize treatments tailoring drug delivery performance
thanks to a higher understanding of disease processes, they are also viewed as drug
delivery platforms with the potential to develop personalized medicine that will take
into account inter-individual variability in therapeutic response to design and
optimize treatment for each patient (Mura and Couvreur 2012; Ryu et al. 2014).
Based on the application of the differentomics strategies that include pharma-
cogenomics and pharmacoproteomics at present, developments of these treatments
require the use of a nanomedicine to lift delivery challenges found with corre-
sponding drugs. Whatever the intended use of polymer nanoparticles, success will
only be achieved at the expense of a rigorous and suitable design of the particle
meeting the different challenges found on the way of the desired application. Their
conception also needs to take into account new issues that could emerge while
using these new technologies as we are still far from the complete understanding of
the safety aspects of their use (see Chap. 16 from Irache et al.).
3 Designing Polymer Nanoparticles to Be Used

as Nanomedicine
Design of polymer nanoparticles to be used as nanomedicines requires the inte-

gration of a series of complex functionalities in a single nano-object (Mura and
Couvreur 2012; Hubbell and Langer 2013; Mura et al. 2013; Kang et al. 2015).
These functionalities are dened from specications that need to be given to the
nanoparticles to achieve the various aspects of their clinical mission. They include
properties linked with the physicochemical colloidal character of the nanoparticles
and those linked with their pharmaceutical activities. Denitions of colloidal

characteristics are generally achieved during preparation of the nanoparticles. They
include the size, shape, and structure of the nanoparticles taken as individual par-
ticles and the colloidal stability of the dispersion that contains a population of
nanoparticles. Pharmaceutical properties are partly dened by the type of materials
that is used to design the nanoparticles. The other part is dened by active ingre-
dients that are composing the cargo. The overall functionalities of nanoparticles are
important as mechanisms of delivery achieved with nanomedicines assumes that the
carrier, hence the whole pharmaceutical formulation, is intended to deliver its cargo
only when it has reached the target cell (Fig. 1a). In certain cases, the delivery
needs to be achieved even at a higher degree of precision considering the inside of
one of the subcellular compartment of the target cell. The delivery concept behind
the use of a nanomedicine is very different compared with that used with con-
ventional drug delivery systems, i.e., capsules, tablets, solutions, etc., that are
designed to release drugs under a molecular form at the site of absorption or at the
site of administration when the formulation is administered parenterally. With
conventional drug delivery systems, drugs circulate in the body under a molecularly
dissolved form and hopefully reach their target cells under this molecularly dis-
solved form (Fig. 1b). Because nanomedicines are intended to deliver the molecular
form of the drug only when it has reached target cells, knowledge on biological
mechanisms that governs the in vivo fate of nanomedicine is needed to design
appropriate particles (Walczyk et al. 2010; Krpetic et al. 2014; Hristov et al. 2015;
Ritz et al. 2015; Setyawati et al. 2015). In parallel, understanding of mechanisms
behind the physiopathology of the disease can also greatly help the design of the
nanomedicine which is aimed to improve efcacy of delivery of the active drug
molecule to the desired biological target while increasing the specicity of delivery
to target cells (Lane et al. 2015; Setyawati et al. 2015). For this reason, design of
nanomedicines is generally following a multidisciplinary approach that includes
polymer chemistry, material sciences, physics, and physicochemistry, pharmaceu-
tical science, biology, immunology, and biomedical sciences.
For their functionality, the primary components of polymer particles designed as
nanomedicines are polymers. Depending on their properties, they can give various
functionalities to nanoparticles (Nicolas 2016). The choice of the chemical nature of
the polymer is determinant to make possible association of drug molecules or
contrast agents. After nanoparticles are loaded with a cargo, stability of the asso-
ciation, and mechanisms of release can also be controlled through the character-
istics of the polymer (Mura et al. 2013; Lehner et al. 2012; Joglekar and Trewyn
2013). For instance, swelling or solubility properties of the polymer hence the
nanoparticles can vary with the pH or the temperature. Properties of the polymer
can be tuned so that it will swell or dissolve at a well dene temperature or pH
triggering the released of its cargo at the place the particles nd this particular
condition in the body. As another function, the polymer is a key material to adjust
the time of residency of the drug delivery system in the organism that depends on
mechanism and rate of degradation/elimination. Most polymer nanoparticles
developed today are formulated with amphiphilic copolymers (Lehner et al. 2012;
336 C. Vauthier
Nicolas et al. 2013). These copolymers are generally composed of at least two parts;
one interacts with the drug insuring drug loading and drug releasing properties. The
second part of the polymer is assumed to locate at the nanoparticle surface. Its role
is to controls the colloidal stability of the nanoparticles and mechanisms that dene
the in vivo fate of the nanomedicine. With such important roles, design of polymers
is one of the keys for the development of efcient nanomedicines. As it is explained
in the Chap. 12 from Cammas-Marion, a large space is available to propose
innovative polymers that will have suitable properties to achieve the success of the
intended delivery method. Methods and approaches that can be used to associate
drugs and control the release of a drug associated with nanoparticles are the subjects
of two distinct chapters (Chaps. 13 from Zandanel and Charrueau, Chap. 14 from
Charrueau and Zandanel). As explained above, the main duty of the nanomedicine
is to transport drugs and/or imaging agents towards target cells or tissues from a site
of administration that is distant from the target site. Systemic activities are mostly
investigated while the nanomedicine is injected intravenously. Mucosal routes of
administration that include oral, nasal and pulmonary delivery are investigated as
well but at a lower extend. Sometimes, the cargo is unable to enter cells by itself
and needs to be delivered in a well dene subcellular compartment of the target
cells. This situation further complicates the delivery method. The delivery system
needs to interact with the target cells according to a very precise mechanism to
trigger the endocytic pathway that will take the cargo into the desired subcellular
compartment of the target cell (Miele et al. 2012; Hristov et al. 2015; Ritz et al.
2015). Thus, success of the delivery method is pending to the achievement of a
series of complex mechanisms that controls the in vivo fate of the nanomedicine, its
interaction with the target cells and its internalization by the target cells.
Contribution of biology and immunology is essential to progress in the develop-
ment of suitable polymer nanoparticles. It helps understanding mechanisms that are
controlling the in vivo fate of nanomedicines and giving polymer nanoparticles
suitable physicochemical characteristics to achieve their delivery mission. From
present knowledge on interactions occurring between nanoparticles and plasma
proteins and their influence on nanoparticle biodistribution and toxicity, the Chap.
15 from LLinskaya and Dobrovolskaia discusses the relation between physico-
chemical properties of polymer nanoparticles and biodistribution. A large part of
the role of the immune system on the biodistribution of the nanoparticles will be
discussed in this chapter. The Chap. 16 from Irache et al. is focused on toxico-
logical aspects of polymer nanoparticles. It points out different problems of safety
that may be found using nanoparticles while they are administered by different
routes of administrations, i.e., intravenous, oral, pulmonary, nasal, and ophthalmic.
The Chap. 17 by Herceg et al. proposes a prospective view on development of
polymer nanoparticles with multiple functions that are included in a single carrier to
achieve diagnosis, treatments and monitoring of the evolution of the disease and
efcacy of treatments. Corresponding technologies assumes that both a therapeutic
agent, a drug, and an imaging agent are conned together in the single nanoparticle.
Development of theranostic agents based on polymer nanoparticles has just started
while it is considered for its potential in the development of personalized medicines
(Mura and Couvreur 2012; Brambilla et al. 2014; Ryu et al. 2014; Chap. 17 from
Herceg et al.). From the various types of multifunctional systems that have already
been tested in vivo, the authors discuss the potential of polymer nanoparticles to
become theranostic agents suitable to be used with different imaging modalities.
Altogether, chapters from this part of the book provide a lot of information to adjust
characteristics and optimize functionalities of nanoparticles regarding the pharma-
cological application for which they are designed to answer a clinical need.
4 Challenges
Clinical development of nanomedicines based on polymer nanoparticles has started

holding great promise for a broad range of applications with the aim to improve
therapeutic treatments and diagnostic by imaging techniques. The success of the
methods is pending to an optimal design and engineering of the nanoparticles. In
turn, this is pending to the resolution of a series of challenge that could explain why
so few systems have been yet translated into clinics (Kang et al. 2015; Hubbell and
Langer 2013).
One of the main challenges for having efcient nanomedicines including poly-
mer nanoparticles is to be able to dene with a high precision the full list of
specications that needs to be given to the nanoparticles. Rational design and
engineering of polymer nanoparticles is still far from ideal because dressing the list
of specications remains a difcult task. The situation is complicated by the lack of
knowledge that is still needed. For instance, lots of information is missing to
understand how nanoparticles are seen by biological systems and what character-
istics of nanoparticles are needed to produce a certain type of response from the
biological systems hence to control the in vivo fate of nanoparticles (Mahon et al.
2012; Krpetic et al. 2014). More knowledge is needed on the understanding of
interactions of nanomedicines with biological molecules found in tissues and cells.
Useful knowledge on physiopathological processus of diseases is also missing. It is
needed to optimize interactions of nanoparticles with target cells hence better
deliver the drug to the right cells and, in parallel, to insure a high safety of the
delivery method. These missing data are keys to improve denition of character-
istics that need to be given to nanoparticles based on a rational approach. The
Chap. 16 from Ilinskaya and Dobrovolskaia that explained how nanoparticles are
interacting with the immune system provides useful information to dene speci-
cations of nanoparticles that are designed to deliver drugs from intravenous
administration. The number of specications also depends on the degree of com-
plexity of the delivery system. The more advanced technologies are considering the
engineering of a multistage delivery system. The nanoparticles are expected to
control the spatiotemporal targeting and delivery of its cargo with a very-high
degree of precision. It may need to respond differently in the various biological
environments it is going through improving the selectivity of the delivery method to
reach the diseased tissue and nally the target cells (Miele et al. 2012).
338 C. Vauthier
Specications that are needed to engineer such kind of nanoparticles can only be set
at the expense of a very good knowledge of the pathophysiological environment of
the condition to be treated. Once specications are identied, many parameters that
need to be given to nanoparticles can be dened from the characteristics of the
polymer. Polymer and colloid sciences are quite advanced and should not be a lock
for the development of nanoparticles having well-dened characteristics. Polymers
can be synthesized with a well control composition and architecture hence prop-
erties (Gao and Matyjaszewski 2009; Hasjichristidis et al. 2011; Nicolas et al. 2013;
Nicolas 2016). Nevertheless, a lot of space is still available for the development of
new biodegradable and biocompatible species of polymers that will have suitable
properties to be incorporated in nanoparticles to give them very precise specica-
tions (Le Droumaguet and Nicolas 2010; Delplace and Nicolas 2015; Merkle 2015;
Nicolas 2016; Chap. 12 from Cammas-Marion). Mechanisms that control the col-
loidal stability of nanoparticle dispersions are also quite well established on the
physicochemical stand point as well as from the side of methods that can be used to
achieve the preparation of stable dispersions. From the chemical and physico-
chemical point of view, synthesis of polymer nanoparticles integrating function-
alities from a list of well-dened specications is an achievable goal (Le
Droumaguet and Nicolas 2010; Delplace and Nicolas 2015; Nicolas 2016). Quality
control of the synthesized nanoparticles is another challenge. Methods suitable to
evaluate the size, size distribution, surface charged, and morphology are quite-well
established but other methods are needed to investigate a series of parameters that
are important to control and insure the quality of the in vivo fate of the nanopar-
ticles hence their safety and efcacy (Ye et al. 2015; Chap. 7 from Clogston et al.).
Another challenge is found designing a nanomedicine that integrates in a single
nano-object all functionalities needed to make the nanoparticles a stable colloid and
to give them suitable equipment to complete the pharmacological activity and that
of the contrast agent. This is about to be solved engineering nanoparticles by
self-assembling of polymers bearing different functionalities and derived from the
same family of polymer (Hasjichristidis et al. 2011, Ryu et al. 2014; Chap. 1 from
Vauthier).
5 Conclusion
Polymer nanoparticles are among the arsenal of nanomedicines that have various
potentials to improve treatments of a broad range of disease and to develop new
diagnostic modalities based on imaging techniques. The design and development of
the nanomedicine is still in the infant age and hold great promises for the medicine
in the future. Huge inroads have already been made and several new challenges
were identied. From these data, it seems now evident that particles will need to be
developed on a case by case basis that will depend on specicities of the phys-
iopathology of the disease, of the drug delivery problem to be solved and on the
therapeutic strategy. It can be expected that every progress made on the
understanding of mechanisms behind the bioreactivity of the nanoparticles and that

will make predictive their safety prole once in vivo will contribute to accelerate
the development of polymer nanoparticles with optimized properties to be applied
as carrier for therapeutic and diagnostic agents. This will also contribute to
rationalize approaches used to design and engineer nanoparticles responding to a
well-identied clinical need.
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Chapter 12
Selecting and Designing Polymers Suitable
for Nanoparticle Manufacturing
Sandrine Cammas-Marion
Abstract Man has always tried to use the materials around him to construct more
and more complex objects for its everyday use, with a particular attention to
materials which can be used to treat diseases and/or injuries. With the technological
progress and the apparition of macromolecular materials, the objects available to
treat the human body improved. Polymers, considered to be powerful building
blocks, attracted and continue to attract a lot of attention from researchers, espe-
cially from those working in the biomedical and therapeutic domains. Moreover,
the advances in medical knowledge about diseases, such as the different types of
cancer, lead to the development of very specic and complex materials, elaborated
specically for the biomedical and therapeutic elds. Therefore, the macromolec-
ular chemist plays a crucial role in the design of such complex polymer structures
obtained by polymerization and/or copolymerization of functional monomers, as
well as by chemical modication of natural or synthetic polymers. To illustrate the
complexity in the design of polymers suitable for manufacturing nanoparticles,
several examples of degradable or fragmentable (co)polymers, specically designed
for application as degradable nanocarriers, are given. These macromolecular
materials are synthesized, either by chemical modication of natural or synthetic
polymers using classical chemical reactions known in organic chemistry, or by (co)
polymerization of functional monomers using the (co)polymerization techniques
known for classical polymer synthesis (radical polymerization, ring-opening
polymerization, etc.). The polymers composition are explained in correlation
with the factors influencing the degradation rate of the corresponding nanovectors,
such as the hydrophilic/hydrophobic balance of the macromolecular materials and
the degradation mechanisms (when known).
S. Cammas-Marion (&)
UMR 6226 CNRS, Institut of Chemical Science of Rennes, Team Organic and
Supramolecular Chemistry, Ecole Nationale Suprieure de Chimie de Rennes (ENSCR),
11 Alle de Beaulieu, CS 50 837, 35 708 Rennes Cedex, France
e-mail: sandrine.marion.1@ensc-rennes.fr

DOI 10.1007/978-3-319-41421-8_12
344 S. Cammas-Marion
Keywords Degradation rate

Amphiphilic oligomers
Biodegradable

Copolymers Click chemistry reaction Nuclear magnetic resonance (NMR)
RAFT studies
1 Introduction
For many years, one tries to develop a more and more specic medicine with the
goal to improve the therapeutic effect of drugs while decreasing the administrated
doses and the side effects together with a simplication of the administration
protocol. Since the end of the twentieth century, signicant progresses were realized
in the design of such medicine leading to the preparation of nanovectors which have
already received the Food and Drug Administration (FDA) approval or are in the
preclinical or clinical phases (Torchilin 2006; Hoffman 2008; Liu et al. 2009;
Malam et al. 2009; Misra et al. 2010). In this context, the combination of nan-
otechnologies and polymer science should lead to the obtaining of nanoparticles
presenting the required properties necessary for an efcient site-specic nanome-
decine; the nanoparticles must: (1) be (bio)degradable or at least biocompatible,
(2) allow the encapsulation of large amount of drugs, (3) lead to a sustainable drug
release which can be controlled by a variation of the pH and/or the temperature or in
response of an external stimulus (ultrasounds, magnetic eld, etc.), (4) have a
controlled surface in order to avoid the recognition by the reticuloendothelial
system (immune system) and to be targeted to the right tissue, cells, or organs by
introducing a targeting moiety (antibody, peptide, vitamin, etc.) on the nanoparti-
cles surfaces (Table 1) (Park et al. 2008; Kumari et al. 2010; Loyer and
Cammas-Marion 2014).
Therefore, the nature and structure of the (co)polymers constituted the
nanoparticles have to be adjusted in order to fulll the specications related to the
use of degradable nanoparticles as site-specic drug delivery systems. Thanks to the
flexibility of polymers chemistry, one can obtain materials showing very diverse
physical and mechanical properties which can be, therefore, adjusted to the selected
application. Consequently, the macromolecular chemist plays a crucial role in the
design of such complex polymer structures obtained by polymerization and/or
copolymerization of functional monomers as well as by chemical modication of
natural and synthetic polymers.
Even if it is theoretically possible to obtain all imaginable structures and
properties, the major difculty is related to the obtaining and the control of prop-
erties required by a given application. Therefore, one primordial element consists in
the denition of very strict specications to which the developed polymers have to
answer, especially for therapeutic applications. The polymers have to be: (1) bio-
compatible meaning that they have to be nontoxic, nonimmunogenic, nonmuta-
genic, and nonthrombogenic; (2) (bio)degradable in biocompatible low molecular
weight molecules; and (3) biofunctional meaning that they need to have adequate
12 Selecting and Designing Polymers Suitable for Nanoparticle 345
Table 1 Specications of nanoparticles

Specications of Characteristics of the Chemistry Examples
nanoparticles (co)polymers
Biocompatible Biocompatible (Co)polymerization of Poly(N-(methacryloyl)-
monomeric units the biocompatible Thr-OEt)-b-PEG-based
monomers nanoparticles (van Dijk
et al. 2010)
Fragmentable bonds Chemical coupling of Modication of low
within the polymer two polymer chains molecular weight PEI
backbone through degradable (Zhao et al. 2011)
bonds
Molecular weight Controlled the Amphiphilic triblock
lower than the one of polymerization HPMA copolymer-Dox
renal threshold conditions, adjust the conjugate (Yang et al.
ratio monomer/initiator 2013)
(Bio)degradable (Bio)degradable (Co)polymerization of Poly(lactides) derivatives
monomeric units (bio)degradable (Nampoothiri et al. 2010;
monomers Belbella et al. 1996;
Guinchedi et al. 1998;
Zweers et al. 2004; Duan
et al. 2007;
Santander-Ortega et al.
2010; Mohammad and
Reineke 2013)
PMLA derivatives (Vert
and Lenz 1981; Cammas
et al. 1996; Stolnik et al.
1996; Martinez Barbosa
et al. 2004; Wang et al.
2006; Nottelet et al. 2010;
Schott et al. 2013)
Encapsulation of large Hydrophobic block Copolymerization of Nanoparticles based on
amount of drug compatible with the suitable comonomers PEG-b-PMLABe
drug copolymers (Huang et al.
2012)
Chemical coupling Classical organic Amphiphilic triblock
of the drug onto the reaction such as HPMA copolymer-Dox
polymer chain coupling using conjugate (Yang et al.
through degradable DCC/NSH 2013)
bounds
Sustainable drug release (Co)polymers (Co)polymerization of Nanoparticles based on
sensitive to a monomers leading to temperature-sensitive
stimulus (variation (co)polymers sensitive polypeptides (Cheng et al.
of the pH, to a stimulus 2011)
temperature,
ultrasounds, etc.)
(Co)polymers Chemical coupling of Amphiphilic triblock
having lateral groups the drug to the polymer HPMA copolymer-Dox
which can be backbone through conjugate (Yang et al.
chemically modied degradable bonds 2013)
using classical
coupling reactions
(continued)
Table 1 (continued)
Specications of Characteristics of the Chemistry Examples
nanoparticles (co)polymers
Controlled surface Copolymers having Chemical coupling of Nanoparticles based on
properties to decrease a hydrophilic block, functional PEG chain polypeptides-grafted OEG
the recognition by the mainly PEG block and functional (Cheng et al. 2011)
immune system hydrophobic block
(Stealth properties) Use of a PEG chain as Nanoparticles based on
initiator for the PEG-b-PMLABe
polymerization of copolymers (Huang et al.
selected monomer(s) 2012)
Targeting properties (Co)polymers with a Polymerization Nanoparticles based on
targeting moiety at reaction in the Biot-PEG-b-PMLABe
the free end of the presence of targeting copolymers (Huang et al.
hydrophilic block moiety as initiator 2012; Loyer et al. 2013)
Chemical modication Nanoparticles based on
of the free end of the Folate modied
hydrophilic block PEG-b-poly
(aspartate-hydrazone-dox)
(Bae et al. 2005)
mechanical, physical, chemical, thermal, and biological properties, to be easy to

use, sterilizable, storable, and to obtain approval (Vert 2003).
Before moving further, let us give the denition of some terms employed just
above. A material can be considered biocompatible if it has the ability to be in
contact with living systems without producing adverse effects (Vert et al. 2012).
Because the nanoparticles designed to be used as drug carriers have to be eliminated
from the body after drug delivery, the polymers which constitute them have to be
either excreted from the body thanks to their molecular weight lower than the
molecular weight threshold of renal excretion (Fox et al. 2009) or (bio) degraded
into biocompatible low molecular weight molecules. A polymer is degradable if it
can undergo chain scissions, resulting in a decrease of its molecular weight, under
specic conditions (temperature, pH, humidity, etc.). A polymer is biodegradable if
it has the ability to be degraded by biological entities (enzymes, microorganisms,
etc.). More denitions of the specic terminology for biorelated polymers were
recently given by Vert et al. (2012).
In the design of nanoparticles used as drug delivery systems, one important step
consists in the synthesis of (co)polymers having specic properties which can be
transferred to the nanoparticles. While biocompatibility of a (co)polymer is a
property directly linked to its chemical composition and structure, the degradation
property (Table 2) can be engineered to be adjusted to the selected application,
notably in terms of degradation rate, and controlled by various environmental
(water, pH, temperature), internal (enzymes, reactive oxygen species, etc.) and/or
external (light, magnetic/electronic elds, etc.) stimuli (Deshayes and Kasko 2013).
Consequently, it seems obvious that obtaining a single ideal polymer family is a
utopia. Instead, multiple polymer families are developed and researchers can use
them to design the best (co)polymer which matches the specications of the
Table 2 Example of degradable polymers used to design nanoparticles and information about
their degradation
Polymer family Degradation Factors modulating Degradation Ref.
process degradation timea
Accelerating Slow down
Polysaccharide Enzymatic Hydrophilic Hydrophobic Not de Medeiros Modolon
(glucoamylase monomer monomer specied et al. 2012
from
Aspergillus
niger)
Poly(esters)
Natural PMLA Enzymatic Hydrophilic Hydrophobic 12 days Portilla-Arias et al.
(lipase) monomer monomer 20 weeks 2008a, Portilla-Arias
Hydrolysis Temperature et al. 2008b,
Acidic or Lanz-Landzuri et al.
basic pH 2011, Lanz-Landzuri
et al. 2012
Synthetic Hydrolysis Insert Insert Few days to Stolnik et al. 1996,
PMLA hydrophilic hydrophobic several Martinez Barbosa
monomers monomers months et al. 2004, Wang et al.
2006, Nottelet et al.
2010, Schott et al.
2013
Hy-PEI-g-PCL- Hydrolysis Increase Decrease Not Liu et al. 2010
b-PEG Enzymatic hydrophilic hydrophobic specied
(lipase from block content block content
Candida
Antartica)
3-arm Hydrolysis Decrease the Increase the At least Shen et al. 2007
PEG-PCL length of PCL length of PCL 6 months
block block
Poly(HEMA-g- Hydrolysis in Decrease Increase 57 h for the Ferrari et al. 2013
CL-b-PEG) culture hydrophobic hydrophobic more
medium block length block length hydrophilic
At least 1
month for
the more
hydrophobic
PLA Hydrolysis Nanoparticle Insert From Belbella et al. 1996,
Enzymatic preparation optically pure 12 days to Guinchedi et al. 1998,
(lipases) Insert lactide units 2 years Zweers et al. 2004,
glycolide or Duan et al. 2007,
units or hydrophobic Santander-Ortega et al.
racemic units 2010, Mohammad and
lactide units Reineke 2013
Other Enzymatic Increase Decrease Not Herzog et al. 2006
polyesters (lipases) hydrophilicity hydrophilicity specied
(continued)
Table 2 (continued)
Polymer family Degradation Factors modulating Degradation Ref.
process degradation timea
Accelerating Slow down
Polyamide
Natural PGGA Hydrolysis Increase Increase alkyl 2045 days Portilla-Arias et al.
carboxylic groups 2009
acid groups content
Others polymers
PEI-PEG Fragmentable 2h Zhao et al. 2011
Reducing
agent
PDMAEMA Fragmentable 48 h under Zhang et al. 2012
Hydrolysis basic
Enzymatic conditions
(lipase)
Triblock Fragmentable 6h Yang et al. 2013
HPMA-Dox Enzymatic
conjugate (papain)
Polypeptide Hydrolysis Increase Decrease Not van Dijk et al. 2010
with LCST hydrophilicity hydrophilicity specied
PLG-NCA Enzymatic 12 h (13 % Cheng et al. 2011
based (proteinase K) decrease in
polypeptide molecular
weight)
Poly(carbonates)
PTMC-b-PBLG Enzymatic Change the 2 days Le Hellaye et al. 2008
(Pseudomonas PBLG to PCL (70 %)
lipase)
a
Time of degradation was given as an indication. It greatly depends on the initial molecular weight of the polymer
and of the type of formulation
considered application (Ulery et al. 2011). The polymers can be from natural
(chitosan, polysaccharides, etc.) or synthetic (polyesters, polycarbonates, poly
(amino acids), etc.) origins. To adjust their properties, one can either use chemical
modications of the preformed natural or synthetic polymers or
polymerize/copolymerize specic monomers specically synthesized to provide the
desired properties to the materials and therefore to the nanoparticles.
1.1 Chemically Modied Natural Polymers
Naturally available polymers do not necessarily answer to all the requirements of

materials which can be used to prepare nanoparticles. Consequently, one has to
chemically modify them in order to adjust their physicochemical properties
(hydrophilic/hydrophobic balance, degradation rate functional groups, etc.) with the
goal to obtain well-dened degradable nanoparticles for site-specic drug delivery.
Theoretically, all the reactions known in organic chemistry might be applied to
modify polymers. However, it is important to keep in mind that the main and side
chains of polymers have an influence on the reactions leading to a modication of

the reactivities usually observed in organic chemistry. Moreover, because of the
steric hindrance of the polymer chain and/or of the side groups, the chemical
reactions, known to be total in organic chemistry, might be only partial when they
are applied to polymers.
Besides such reactivity considerations, one has to pay a special attention to:
First, the purication procedures applied to the modied polymers are of crucial
importance for obtaining very pure materials without any trace of catalyst, side
products, and/or solvents to ensure the absence of toxic effects resulting from
the presence of such molecules even in extremely low concentration. One
efcient method, in my opinion, is dialysis which allows the elimination of low
molecular weight molecules from the polymers solution. However, such
technique can be quite long, at least 24 h, to be efcient. Therefore, the dialysis
conditions (solvent, temperature, etc.) have to be carefully selected in order to
minimize the polymer degradation which may occur during this process.
Second, the characterization of the modied polymers, especially in terms of
changes in molecular weights and polymolecularity values upon modication
and purication procedures. Such changes can dramatically modify the com-
portment of the modied polymers leading to non well-dened nanoparticles
incompatible with applications in the biomedical eld.
Third, the nature of the groups introduced on the natural polymers as well as the
nature link between these groups and the polymer chains because, upon
degradation, non-biocompatible low molecular might molecules weight can be
released and lead to dramatic toxic effects.
Among all the organic chemistry reactions, the click chemistry is a more and
more used technique in polymer science to modify preformed natural or synthetic
macromolecules (Lutz 2007). The main click chemistry reaction which is com-
monly used for polymer modication is the copper-catalyzed Huisgen 1,3-dipolar
cycloaddition of azides and terminal alkynes as shown by Scheme 1 (Lutz 2007).
This technique seems to be quite simple to set up and can be performed in
various solvents as well as in the presence of other functional groups. Despite the
evident interest of such technique, one major drawback lies in the presence of
copper catalyst as such that, even at low concentrations, may lead to unwanted
toxicity in the case of biomedical applications.
Halila et al. used the click chemistry for the preparation of linear amphiphilic block
co-oligomers (BCO) constituted by fully biocompatible and biodegradable saccha-
ride blocks. These blocks were synthesized through a Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAc) of an azide maltoheptaose derivative (hydrophilic block) and
Scheme 1 Principle of the copper-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and

terminal alkynes
an alkyne peracetylated maltoheptaose (hydrophobic block) (de Medeiros Modolon

et al. 2012). Both blocks were prepared by chemical modication of maltoheptaose
which was obtained by ring opening of -cyclodextrin (Scheme 2). All the inter-
mediates and nal BCO were puried by flash chromatography technique which
allows the elimination of side products, catalysts, and starting materials (de Medeiros
Modolon et al. 2012). This purication procedure can be used in this case because
only oligomers are prepared. For higher molecular weight polymers, purication by
flash chromatography is usually impossible and other techniques have to be used
(precipitation, centrifugation, dialysis).
The strategy used by Halila et al. to prepare amphiphilic oligomers is quite
elegant in the sense that hydrophilic and hydrophobic blocks are synthesized sep-
arately thus avoiding solubility problems often encountered during amphiphilic
molecules synthesis. As mentioned above, one major drawback is the use of
copper during the click reaction. Nevertheless, this click chemistry technique is an
attractive method to prepare block copolymers and/or to introduce functional
groups or biologically active molecules on preformed polymers, especially since
researchers have developed more biocompatible Copper(I) catalyst (Soriano del
Amo et al. 2010).
Scheme 2 Synthesis of amphiphilic block oligomers based on saccharide blocks (Lutz 2007)
Starting from the synthesized amphiphilic BCO, micelles were successfully pre-
pared in an aqueous solution. Their degradation catalyzed by glucoamylase from
Aspergillus niger was evaluated in vitro at 40 C by static light scattering and dynamic
light scattering (DLS) and by reducing sugar assays. The degradation proceeded in
two steps. First, the hydrophilic shell made up by the maltoheptaose was hydrolyzed
upon the action of the glucoamylase until the substrate (maltoheptaose) became
inaccessible. This step resulted in a decrease of micelles stability leading to a
reassembly of the degraded micelles to form more stable aggregates. In the second
step, such aggregates underwent again the degradation by the glucoamylase up to
complete degradation of the BCO or until the substrate was again inaccessible, in this
later case the cycle reassembly/degradation restarted (de Medeiros Modolon et al.
2012). It is obvious that degradation rate can be modied by playing on the ratio
hydrophobic blocks/hydrophilic blocks: nanoparticles containing high ratio of
hydrophilic blocks will be degraded faster than nanoparticles containing high ratio of
hydrophobic blocks. However, formation of stable nanoparticles depends also on this
ratio: if the hydrophilic character of the block copolymers is too high, stable
nanoparticles cannot be formed. Therefore, the preparation of degradable nanopar-
ticles with controlled degradation rate needs to nd a compromise between
nanoparticles formation and degradation rate. This study opens the way to the
preparation of a vast family of fully degradable amphiphilic block oligosaccharides
thanks to the versatility of the synthetic approach elaborated by Halali et al. which will
allow introducing various protecting groups and/or biologically active carbohydrates.
Besides this click chemistry, more classical organic reactions can be used to
modify naturally available polymers to confer them the properties
(hydrophobic/hydrophilic balance, degradation rate, etc.) allowing the preparation
of degradable nanoparticles. Partial or total esterication of lateral carboxylic acid
functions is a quite efcient method to adjust both the hydrophilic/hydrophobic
balance and the degradation rate. Several techniques can be used to realize this
esterication procedure, as presented thereafter. Once again, the modication
procedure has to be selected in function of the nature of both the functional groups
present in the polymers and the molecules to be grafted. Moreover, it is important to
pay a special attention to the coupling conditions because of the degradable char-
acter of the polymers. As for click chemistry, one crucial step concerns the
purications procedures of the modied polymers which must lead to the total
elimination of catalysts and other side products.
Methylation of lateral carboxylic acid functions of the natural poly(malic acid),
PMLA, was realized using diazomethane as methylation agent as shown by
Scheme 3 (Portilla-Arias et al. 2008a, b; Lanz-Landzuri et al. 2011).
PMLA is a natural polymer which received the attention of researchers for
applications in the biomedical eld under various forms (Loyer and
Cammas-Marion 2014; Portilla-Arias et al. 2008a, b; Lanz-Landzuri et al. 2011,
2012; Ljubimova et al. 2013). This polymer has been isolated by Holler et al. at the
end of the 80s, from plasmodium extracts and from the culture medium of
Physarum polycephalum (Fisher et al. 1989). The natural PMLA has been indeed
used, either unaltered or following chemical modications, to formulate
Scheme 3 Methylation of natural PMLA (Soriano del Amo et al. 2010; Portilla-Arias et al.
2008a, b)
nanoparticles (Loyer and Cammas-Marion 2014; Portilla-Arias et al. 2008a, b;

Lanz-Landzuri et al. 2011, 2012) or as nanoplatforms called Polycens (Loyer
and Cammas-Marion 2014; Ljubimova et al. 2013). The natural PMLA used in
these studies was produced from cultures of Physarum polycephalum and had a
molecular weight of 34,000 g/mol with a polymolecularity index (Ip) of 1.08.
The methylation of this natural PMLA using diazomethane does not seem to
induce any degradation of the macromolecular chains in the sense that the
molecular weights before and after modication are similar (Portilla-Arias et al.
2008a, b; Lanz-Landzuri et al. 2011). Moreover, the purication procedure is very
simple and rapid because this reaction leads only to the formation of volatile side
product (N2): the modied polymers are puried by precipitation into an excess of
nonsolvent. However, such technique can only allow the grafting of methyl groups
and other methods have to be used to graft different alkyl groups.
The study of hydrolytical degradation of the synthesized methylated copolymers,
PMLAMe, PMLAMe75H25, PMLAMe50H50, and PMLAMe25H75, highlighted that:
(1) the hydrolysis was delayed by esterication whatever the incubation conditions;
(2) the hydrolysis of water-soluble methylated PMLA, PMLAMe25H75, and
PMLAMe50H50, was signicantly enhanced by enzymes (lipases); (3) the degra-
dation rate was accelerated with an increase of the incubation temperature; and (4).
the hydrolytic degradation of methylated PMLA took place by hydrolysis of the
methyl carboxylate side groups followed by the hydrolysis of the ester backbone
which was enhanced by the presence of initially liberated carboxylic side groups
(Portilla-Arias et al. 2008; Lanz-Landzuri et al. 2011).
Water-insoluble PMLA derivatives, PMLAMe, and PMLAMe75H25, were used to
prepare either microspheres by an emulsion/evaporation solvent method in the
presence of poly(vinyl alcohol), PVA, as an emulsier (Portilla-Arias et al. 2008a) or
nanoparticles by the precipitation-dialysis method (Portilla-Arias et al. 2008b;
Lanz-Landzuri et al. 2011). First, the degradation of PMLAMe microspheres was
slower than the corresponding lms and high hydrolysis rates were observed at high
temperature, for both acidic and basic pH and in the presence of lipase; the micro-
spheres underwent a morphological alteration evidencing both an erosion mechanism
and a degradation in bulk (Portilla-Arias et al. 2008a). The PMLAMe75H25-based
nanoparticles underwent a quite fast molecular weight decreased, from 34,000 to
5000 g/mol in 12 days when the nanoparticles were incubated in phosphate buffer
saline (PBS) under physiological conditions; higher rates in molecular weight
decreased were observed upon incubation at acid and basic pH and in the presence of
lipase; a decrease in the nanoparticle sizes and dramatic changes in their morphology
were also observed upon incubation. These results showed that the degradation of the
nanoparticles occurred rst on their surfaces with a solubilization of the degraded
chains (Portilla-Arias et al. 2008b). In parallel, the degradation rate of
PMLAMe-based nanoparticles was slower than the one observed for PMLAMe75H25-
based nanoparticles. Moreover, the presence of PVA, used as an emulsier, reduced
the hydrolysis rate suggesting that the PVA attached to the nanoparticle surfaces acted
as a hydrolysis protecting coat (Lanz-Landzuri et al. 2011). The study of the
degradation mechanism by Nuclear Magnetic Resonance (NMR) and Size Exclusion
Chromatography (SEC) showed that the degradation took place rst by the degra-
dation of the lateral ester groups as evidenced by the apparition of methanol followed
by the degradation of ester backbone with, in ne, the apparition of malic acid when
the degradation was total. For PMLAMe-based nanoparticles the degradation was
total after more than 20 weeks upon incubation in PBS at 37 C (Lanz-Landzuri et al.
2011). The study of the toxicity of the PMLAMe-based nanoparticles on various cell
lines revealed that toxicity appeared for exposure times over 12 h and may be due to
the release of methanol during the degradation process (Lanz-Landzuri et al. 2011).
This example demonstrates that the nature of the groups used to modify the
natural polymer has to be carefully selected in order to confer the expected properties
to the corresponding nanoparticles without providing any toxic effect upon degra-
dation of the modied polymers constituting the nanoparticles. The degradation
process and the nature of the molecules obtained upon degradation have to be
thoroughly determined in order to avoid the apparition of undesirable toxic effects.
A solution to the toxicity observed for methylated PMLA may consist in selecting
more biocompatible substituted groups such as amino acids. Therefore, the L-leucine
ethyl ester (Leu) or the L-phenylalanine methyl ester (Phe) were conjugated to the
carboxylic acid functions of the natural PMLA using dicyclohexylcarbodiimide
(DCC) as coupling agent (Scheme 4) (Lanz-Landzuri et al. 2012).
This coupling reaction using DCC is a well-known esterication reaction in
organic synthesis and is also applied for polymer modications. Its major drawback
lies in the difculties to eliminate the dicyclohexyl urea (DCU) formed during the
reaction. Even if most of this side product is removed by ltration, its total elim-
ination needs a dialysis for at least 24 h. However, there are at least two important
advantages in using such DCC coupling reaction; rst, it is a reaction under mild
conditions avoiding the polymer degradation; second, the substitution degree can be
easily controlled by the ratio lateral carboxylic groups/DCC/substituted groups.
Nanoparticles were prepared from natural PMLA containing 60 % of amino acid
(L-leucine ethyl ester or L-phenyl alanine methyl ester) using the
Scheme 4 DCC coupling reaction between natural PMLA and amino acids (Ljubimova et al.
2013)
precipitation-dialysis method. The hydrolytic degradation was carried out under

physiological conditions by dissolving the copolyesters in PBS at pH 7.4 at 37 C.
The degradation rates were decreased with an increase in the amidation degree for
both Leu and Phe derivatives. The hydrolysis started with the cleavage of the amino
acid ester groups with the release of the corresponding alcohol and continued with
the degradation of the main chain at the ester linkages between nonamidated units
with the generation of oligomeric fragments and malic acid. Finally, free malic acid
and L-leucine or L-phenylalanine were released upon total degradation.
Cytotoxicity assays realized with two kinds of nanoparticles on various cancer cell
lines showed a concentration-dependent cytotoxicity but only for polymer con-
centrations above 125 g/ml (Lanz-Landzuri et al. 2012).
Alkylation of polyacids can be also realized by reaction of alkyl bromide with
lateral carboxylic acid functions under basic condition. However, such reaction can
be only applied to degradable polymers which are not sensitive to basic conditions.
Consequently, before choosing a method to modify natural (or synthetic) polymers,
it is crucial to evaluate the impact of modication conditions (pH, temperature, etc.)
on the polymers in terms of potential chain degradation. Poly(amides) being less
sensitive to degradation than poly(esters), this alkylation technique was applied to
poly (-glutamic acid), PGGA.
The natural PGGA, produced by Bacillus subtilis with a D/L enantiomeric ratio
of 59:41 and a weight average molecular weight of 300,000 g/mol, was partially
alkylated by the reaction of PGGA with excess of corresponding alkyl bromide
under basic conditions (Scheme 5) (Portilla-Arias et al. 2009). This procedure
allows a ne control of the substitution degree by adjusting the reaction time.
However, to nd the good reaction time to achieve the desired esterication degree,
one needs to realize several experiments because the adapted reaction time is
determined by a trial-to-error method, method which is time and products
Scheme 5 Coupling reaction between PGGA and alkyl bromide (Fisher et al. 1989)
consuming. The purication procedure is quite simple and fast: precipitation of the
modied polymer which was recovered after centrifugation.
The hydrophobicity and wetting properties of the modied PGGA decreased
with the increase of alkyl content and size of the alkyl groups (Portilla-Arias et al.
2009). The nanoparticles based on the synthesized PGGARxHy were prepared by
the precipitation-dialysis method leading to spherical nanoparticles with average
diameters ranging between 200 and 300 nm depending on the alkyl groups
(Portilla-Arias et al. 2009). The hydrolysis of the prepared nanoparticles under
physiological conditions (PBS at pH 7.4 and 37 C) was followed by measuring the
molecular weight changes by SEC. All the nanoparticles were degraded following
an almost linear prole. They are fully degraded in a period of time that ranges from
20 to 45 days. The degradation rate of copolymers was observed to increase with
the content in carboxylic groups and to decrease with the length of the alkyl ester
group (Portilla-Arias et al. 2009). The degradation might occur by the total or
partial hydrolysis of the carboxylate side groups followed by cleavage of the main
chain amide bonds (Portilla-Arias et al. 2009).
This example demonstrated that by modifying the nature
(hydrophilicity/hydrophobicity, structures, etc.) of the lateral groups introduced by
chemical modication of naturally occurring polymers, one can tune the degrada-
tion rate of the corresponding nanoparticles and adapt it to the selected application
and rate of drug release.
1.2 Use of Synthetic Polymers
Chemical modications are also applied to synthetic polymers to confer them

adapted physicochemical properties and/or to render then fragmentable in order to
be able to prepare well-dened degradable biocompatible nanoparticles. Besides
chemical modications, another way to design specic (co)polymers having the
desired properties consists in the (co)polymerization of specic monomers elabo-
rated to answer to specications of degradable nanoparticles. All the available
polymerization techniques can be used, the more adapted one being selected in
function of both the (co)monomers nature and the functional groups present on the
monomers (Table 3).
A lot of polymers families were developed, and continue to be developed,

trying to answer to the following question: Are researchers able to synthesize an
ideal polymer allowing the design of degradable and biocompatible nanocarrier for
site-specic sustainable drug release?
Despite undeniable progress in this area since the Magic Bullet of Ehrlich
(1906), this ideal nanocarrier has not yet been elaborated and may never be
designed. Instead, specic nanocarriers may be prepared for a given application and
a biologically active molecule. To prepare such specic nanocarriers answering to
dened specications, one can either use chemically modied preformed synthetic
polymers or (co)polymerize one or more functional monomers.
Table 3 General polymerization procedures used to prepare the presented polymers

Polymerization method Conditions Ref.
Radical polymerization Schlenk tube or flask Zhang et al. (2012), van Dijk et al.
Anhydrous organic (2010)
solvent
Nitrogen or Argon
atmosphere
70 C (depending on the
initiator)
Reversible addition Schlenk tube or flask Yang et al. (2013)
fragmentation chain Water or organic solvent See also Chiefari et al. (1998),
transfer (RAFT) Nitrogen or Argon Ratcliffe et al. (2014) for general
polymerization atmosphere RAFT procedures
4560 C (depending on
the RAFT initiator)
Ring-opening Schlenk tube or flask Cheng et al. (2011), Le Hellaye
polymerization Nitrogen or Argon et al. (2008), Liu et al. (2010), Ryu
atmosphere et al. (2001), Shen et al. (2007),
Vacuum for PLA and Ferrari et al. (2013), Nampoothiri
PLGA synthesis et al. (2010), Zweers et al. (2004),
Glovebox for the Cammas et al. (1996)
synthesis of PTMC
copolymers
Bulk or anhydrous
organic solvent
Room temperature to
130 C (depending on
the selected monomers)
Polycondensation Flask Herzog et al. (2006)
Bulk See also Witt et al. (1994) for
Reduced pressure general procedure
Acid catalyzed
130 C
In order to treat acquired or inherited diseases, gene therapy using nonviral

vectors has been developed to overcome the drawbacks of the therapy using viral
gene vectors. Among the developed polymers, the poly(ethylene imine), PEI, is one
of the most promising cationic polymer (Zhao et al. 2011). However, both trans-
fection efciency and cytotoxicity of PEI are strongly correlated to its molecular
weight (Zhao et al. 2011). To decrease the cytotoxicity while keeping the high
transfection efciency of high molecular weight PEI, a strategy consists in intro-
ducing within the PEI chains some degradable bonds which, upon degradation,
might allow the obtaining of lower molecular weight PEI thus decreasing the
cytotoxicity while keeping high transfection efciency. This strategy was selected by
Zhao et al. (2011) who prepared a fragmentable poly(ethylene glycol)-PEI thanks to
click chemistry. For that, low molecular weight (poly(ethylene glycol), PEG, chains
containing both a disulde bound and azide terminal groups and an alkyne func-
tionalized PEI were coupled using Huisgen 1,3-dipolar cycloaddition (Scheme 6).
The obtained copolymer was submitted to degradation studies and was involved
in DNA complexation assays. In the presence of a reducing agent, the dithiothreitol,
the disulde bounds were cleaved within 2 h leading to PEG-PEI copolymers of
low molecular weights (Zhao et al. 2011). It is important to note that such
copolymers are not really degradable but only fragmentable in that sense the
degradation phenomenon concerns only the cleavage of the sensitive bonds leading
to copolymers with lower molecular weight than the ones of the initial copolymers.
Such strategy could be of interest when low molecular weight (co)polymers are less
toxic than the parent one and can be excreted from the body. In general, these (co)
polymers are used to produce nanoparticles by complexation with nucleic acid.
Thus, the DNA condensation ability of these PEG-PEI copolymers as well as their
cytotoxicity and transfection efcient were evaluated. The level of toxicity of these
PEG-PEI copolymers was comparable to that of the low molecular weight
(2000 g/mol) PEI which was considered as nontoxic. The transfection efciency of
these PEG-PEI/DNA complexes was higher than the one obtained with low
molecular weight PEI and free DNA plasmid but lower than the one observed for
high molecular weight PEI (25,000 g/mol) (Zhao et al. 2011). This strategy may be
of interest since the copolymer structure can be modied by changing the PEG
chain length and/or the PEGylation degree in order to reach high transfection
efciency while keeping the cytotoxicity at a low level.
Using a different synthesis strategy, Zhang et al. developed a degradable poly
(dimethylaminoethyl methacrylate), PDMAEMA, based copolymer to prepare gene
delivery systems (Zhang et al. 2012). Indeed, although PDMAEMA has been
shown to have transfection efciency similar to the one of high molecular weight
PEI, the key problems remain in its nonbiodegradability nature, its toxicity, and the
need to improve its transfection efciency (Zhang et al. 2012). To fulll the
requirements of high transfection efciencies, low toxicity and degradability, Zhang
et al. synthesized, by free radical copolymerization of
5,6-benzo-2-methylene-1,3-dioxepane (BMDO) and N,N-dimethylaminoethyl
methacrylate (DMAEMA) in the presence of poly(ethylene glycol) macro azo
Scheme 6 Click chemistry reaction between PEG and PEI (Zhao et al. 2011)
initiator, a poly[PEG-co-(BMDO-co-DMAEMA)] copolymer (Scheme 7) and

quaternized it with ethylbromide (Zhang et al. 2012).
Scheme 7 Synthesis of poly[PEG-co-(BMDO-co-DMAEMA)] (Zhang et al. 2012)

The presence of PEG blocks improved the water solubility of this macro-
molecular material. The hydrolytic degradation of such copolymers, which con-
tained ester bounds in their backbone, was studied under basic (pH 14) and
physiological (pH 7.4) conditions and the presence of enzymes (lipase from
Pseudomonas cepacia). For nonquaternized copolymers, NMR studies showed that
93 % of the ester bonds were hydrolyzed after 48 h of incubation under basic
conditions. The degradation of quaternized copolymers followed by SEC showed
that, after 24 h of basic hydrolysis, the copolymers were completely degraded into
low molecular weight molecules and PEG. Similar behaviors were observed after
longer period of incubation for degradation assays under physiological and enzy-
matic conditions (Zhang et al. 2012). Finally, the toxicity of all the prepared
copolymers was lower than the one observed for PEI. Surprisingly, the unquater-
nized copolymers showed very encouraging results in the capability to achieve
plasmid DNA transfection (Zhang et al. 2012).
With the aim to transform a nondegradable polymer into a fragmentable one by
introducing hydrolysable groups within its backbone, Yang et al. synthesized, via the
Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization, an
amphiphilic triblock (hydroxypropyl methacrylamide) copolymer-Doxorubicin conju-
gate with the nal goal to prepare the corresponding nanoparticles (Yang et al. 2013).
A well-dened poly(2-hydroxypropyl methacrylate), PHPMA, was rst synthesized by
RAFT polymerization of (2-hydroxypropyl methacrylate), HPMA, using a bifunctional
peptide 2CTA (GFLGKGFG peptide) as the RAFT chain transfer agent. In a second step,
owing to the living character of the terminal dithiobenzoate groups present at both ends of
PHPMA, this prepolymer was used as macro chain transfer agent during the RAFT
polymerization of N-methacryloyl-glycylphenylalanylleucylglycyl-doxorubicin
(MA-GFLG-Dox) thus leading to the expected amphiphilic triblock HPMA
copolymer-Dox conjugate (Scheme 8) (Yang et al. 2013).
The authors highlighted that this two-step RAFT polymerization procedure
might allow a large-scale synthesis of the selected copolymer. Until now, methods
to modulate degradation of nanoparticles in which drug was physically (hy-
drophobic and/or electrostatic interactions) entrapped were exposed. The release
rate of the drug was controlled by its diffusion through the nanocarrier as well as by
the degradation rate of the (co)polymer constituted the nanoparticles. This encap-
sulation method is quite convenient because no chemical modication of the drug is
needed thus ensuring the release of the active form of the drug. However, a very fast
drug release, also called burst effect (Huang and Brazel 2001), is always observed
within the rst hours of incubation probably resulting from the release of the
adsorbed drug at the nanoparticles surface. To have a better control of the drug
release and to avoid this burst effect, a solution consists in coupling the drug to the
polymer via degradable links such as ester linkages if sustained release over several
weeks or more is desired or anhydride linkages if faster release is wanted; several
degradable linkages can be used, some of them being sensitive to stimulus such as a
change in the environmental pH (Deshayes and Kasko 2013). In the study of Yang
et al., the drug, Dox, was linked to a monomer via a degradable peptidic link before
the copolymerization reaction. This technique leads to a better control of the
Scheme 8 Synthesis of the amphiphilic triblock (hydroxypropyl methacrylate) copolymer-Dox

conjugate by the RAFT polymerization (Yang et al. 2013)
amount of the incorporated drug with the good molecular structure by adjusting the
comonomers ratio. Moreover, it is always easier to purify monomers than poly-
mers. The prepared amphiphilic triblock copolymer self-assembled into spherical
nanoparticles in PBS at pH 7.4 with a neutral surface charge and an average
diameter of around 100 nm (Yang et al. 2013). The degradation of the triblock
HPMA copolymer-Dox conjugate was evaluated in the presence of papain at pH6
and 37 C. After 6 h of incubation, the triblock copolymer was degraded into
smaller copolymers with an average molecular weight of 44,000 g/mol, molecular
weight lower than the renal threshold (Fox et al. 2009). Yang et al. attributed this
decrease in molecular weight to the enzymatic degradation of the peptide
GFGKGLFG located within the copolymer backbone. The results of in vitro and
in vivo assays conrmed the potential of such PHPMA derivatives to be used to
design nanoparticulate drug delivery systems (Yang et al. 2013).
These examples demonstrated that it is possible to render a polymer excretable

from the body by introducing in its backbone hydrolyzable groups which allow
reducing the polymer molecular weight under the renal threshold (Fox et al. 2009)
while keeping the capacity of the considered polymer material to form efcient drug
delivery system.
However, even if this strategy seems to give interesting results and can be
considered as an efcient way to improve biocompatibility of polymer nanovectors,
the use of fully (bio)degradable (co)polymers might be a more elegant method to
ensure the total elimination of the polymer materials from the body after drug
delivery.
Various monomers leading to degradable (co)polymers are available. Among
them, amino acids are of interest in the sense that such molecules might allow
obtaining of fully degradable (co)polymers which, upon degradation, might lead to
the formation of biocompatible amino acids. In addition, some poly(amino acids)
show a lower critical solution temperature (LCST) . At the LCST, the water sol-
ubility of the considered polymer changes: above the LCST it becomes
hydrophobic. Usually, the changes in polymer solubility are happening upon a
sharp variation of temperature and are reversible. The value of the LCST of a
thermosensitive (co)polymer can be tuned by playing on the hydrophobic (or
hydrophilic) character of the (co)polymer: an increase of the hydrophilicity of the
macromolecular material leads to an increase of the LCST (Takei et al. 1993;
Taylor and Cerankowski 1992). The poly(isopropylacrylamide), PIPAAN, is the
best known thermosensitive polymer (Takei et al. 1993; Schild 1992; Heskins et al.
1968). The obtaining of (bio)degradable (co)polymers with adjustable LCST is of
great interest in the drug delivery eld because one can control the drug release
upon sharp variation of temperature.
In this context, van Dijk et al. have synthesized a series of monomers based on
methyl, ethyl, and isopropyl esters of N-(methacryloyl)-serine and threonine
(Scheme 9) (van Dijk et al. 2010).
These monomers were then polymerized by radical polymerization with 2,2-
azobis(isobutyronitrile), AIBN, as initiator. Polymers, with an average molecular
weight ranging from 6600 to 23,800 g/mol and a LCST comprised between 1.5 and
more than 100 C depending on the hydrophobicity of the monomer, were thus
obtained (van Dijk et al. 2010). In order to be able to prepare thermosensitive
nanoparticles, the N-(methacryloyl)-threonine (Thr) ethyl ester (OEt) was poly-
merized with (m-PEG5000)2-ABCPA, (4-4-azobis(4-cyanopentanoic acid),
ABCPA), as macro initiator leading to the obtaining of the expected amphiphilic
block copolymer poly(N-(methacryloyl)-Thr-OEt)-b-PEG (Scheme 9) (van Dijk
et al. 2010). Van Dijk et al. evaluated the degradation behavior of both the
monomers and the polymers under simulated physiological conditions (sodium
phosphate buffer at pH 7.4 and 37 C). The hydrolysis rates of monomers depended
on the nature of the alkyl substituent. Whatever the amino acid residue, the
hydrolysis rates of the studied polymers were slower than the one observed for the
corresponding monomers as a result of the hydrophobic nature of the polymer main
chain (van Dijk et al. 2010). Van Dijk et al. prepared thermosensitive microparticles
Scheme 9 Synthesis of poly(N-(methacryloyl)-Thr-OEt)-b-PEG (van Dijk et al. 2010)
by dissolving the poly(N-(methacryloyl)-Thr-OEt)-b-PEG in aqueous buffer above

the LCST of this copolymer measured to be at 21 C. When microparticles were
incubated under physiological conditions, the authors observed a decrease in par-
ticle amount which might result from the hydrolysis of main chain ester bonds
inducing an increase of the overall hydrophilicity leading to the destabilization of
the microparticles (van Dijk et al. 2010).
Because the obtaining of temperature-sensitive degradable nanoparticles can be
of interest to control the drug delivery, Chen et al. (2011) have synthesized a series
of temperature sensitive polypeptides. The copolymers (Scheme 10) were prepared
in two steps: ring-opening polymerization of -propargyl-L-glutamate N-
Scheme 10 Synthesis of thermosensitive polypeptides (Cheng et al. 2011)

carboxyanhydride (PLG-NCA) followed by click reaction between the pendant

alkyne groups and 1-(2-methoxyethoxy)-2-azidoethane or 1-(2-(2-methoxyethoxy)-
ethoxy)-2-azidoethane (Cheng et al. 2011).
The obtained graft copolymer exhibited LCST varying between 22 and 74 C as
a function of the molecular weight of the polypeptide backbone, the length of the
oligo(ethylene glycol) side chain, the polymer, and salt concentrations. Using these
copolymers, nanoparticles, with average diameter ranging from 30 to 60 nm, were
prepared by the nanoprecipitation/dialysis method (see chapter 2 from Miladia et al.
): a block copolymer solution was added dropwise to water and the DMF was
removed by dialysis against water for 24 h. The in vitro enzymatic degradation of
the copolymers was performed at 37 C in PBS (pH 7.4) containing proteinase K:
after 12 h of incubation, a 13 % decrease in the molecular weight of the copolymer
was observed (Cheng et al. 2011). However, neither the study of the degradation
nor the evaluation of the corresponding nanoparticles degradation was conducted.
Therefore, despite interesting results obtained concerning nanoparticles formation,
in vitro cytotoxicity assays, and in vitro Dox release experiments, more details on
the degradation mechanism and rate of these nanoparticles are still needed before
concluding on the interest of such nanoparticles.
However, these studies evidenced that by carefully selecting both the nature of
the repeating units and of the side groups, one can control the properties of the
considered copolymers in terms of temperature sensitivity and rate of degradation.
Associating polypeptides and polycarbonates would result in unique copolymers
with tunable amphiphilic and degradation properties leading to the design of
nanoparticles with adjusted properties. In this context, Le Hellaye et al. have pre-
pared poly(trimethylene carbonate)-b-poly(-benzyl L-glutamate), PTMC-b-PBLG,
by sequential ring-opening polymerization of TMC and BLG (Scheme 11) (Le
Hellaye et al. 2008).
Their properties, notably degradation rates and nanoparticles formation, were
compared to those of poly(-caprolactone)-b-PBLG, PCL-b-PBLG, which was also
obtained by sequential ring-opening polymerization of CL and BLG (Le Hellaye
Scheme 11 Sequential ring-opening polymerization of TMC and BLG (Le Hellaye et al. 2008)
et al. 2008). Since homopolymers, PTMC and PBLG, and copolymers, PTMC-b-
PBLG and PCL-b-PBLG, had well characterized structures and properties, the
corresponding nanoparticles were prepared by the nanoprecipitation method (see
chapter 2 from Miladia et al.): spherical nanoparticles with average diameters
ranging from 60 to 320 nm, depending on the nature of the (co)polymers used, were
obtained (Le Hellaye et al. 2008). The enzymatic degradation by pseudomonas
lipase was monitored for all the prepared nanoparticles by light scattering through
the variation of the scattered intensity over the degradation time (Le Hellaye et al.
2008). The PTMC and PCL-based nanoparticles were degraded after 2448 h of
incubation as no more signal could be detected after this period of time. The
PTMC-based nanoparticles were very rapidly completely destructurized while the
PBLG-based nanoparticles were stable under the degradation conditions. The PCL-
b-PBLG-based nanoparticles had a degradation prole similar to the one observed
for PBLG-based nanoparticles. The PTMC-b-PBLG-based nanoparticles were
degraded faster with an intensity decrease of 70 % after 2 days of incubation with
nanoparticles diameter remaining constant (Le Hellaye et al. 2008). Such results
are quite interesting. Indeed, based on the very different degradation behaviors
between the different homopolymers and copolymers, it could be possible to mix
the three monomer units in order to adjust the degradation rate and time of the
resulting nanoparticles to the desired value.
Among all the (bio)degradable polymers, the poly(esters) family is one of the
most studied (co)polymer classes. Degradation of poly(esters) is usually the result
of the hydrolysis of ester bonds, accelerated by a decrease or an increase of the pH,
and/or the presence of enzymes such as lipases. The degradation rate may be tuned
by modulating the access, by water and/or enzymes, to the ester bonds obtained by
changing the hydrophilic/hydrophobic balance of the (co)polymer and/or using
steric hindrance around the ester bonds.
Herzog et al. investigated the enzymatic degradation of nanoparticles constituted
by various polyesters obtained by polycondensation in bulk under reduced pressure
between a dicarboxylic acid and a diol in the presence of an acid catalyst, their
average molecular weights ranging from 16,000 to 54,000 g/mol with an Ip around
2 (Table 4) (Herzog et al. 2006).
The polyesters-based nanoparticles were prepared by the nanoprecipitation
technique without adding stabilizing agent in the aqueous phase. These nanopar-
ticles with diameters ranging from 50 to 250 nm were then involved in enzymatic
degradation experiments with lipases from Candida Cylindracia and Pseudomanas
species in order to determine both the mechanism and the kinetic of such enzymatic
hydrolysis (Herzog et al. 2006). The enzymatic hydrolysis took place on the surface
of the polyesters-based nanoparticles with a Langmuir type of enzyme adsorption
on the polymer. The nanoparticles are submitted to a uniform surface erosion
process with a constant decrease of the average diameter of the nanoparticles
(Herzog et al. 2006).
As a member of poly(esters) family, poly(-caprolactone), PCL, is known since a
long time as a biodegradable polymer with very good mechanical properties and
very slow (bio)degradation rates (Albertsson et al. 1998; Hgland et al. 2007). In
Table 4 Monomer composition of the various polyesters used by Herzog et al. (2006)
Monomers Average molecular Polymolecularity index (Ip)
Diol Dicarboxylic acid weight (Mw)g/mol
1,3-Propanediol Adipic acid 20,800 1.9
1,3-Propanediol Sebacic acid 15,400 2.0
1,3-Propanediol Dodecanic acid 16,200 2.2
1,4-Butanediol Succinic acid 41,000 1.7
1,4-Butanediol Adipic acid 54,500 2.0
1,4-Butanediol Suberic acid 39,700 2.0
1,4-Butanediol Sebacic acid 52,200 2.2
1,4-Butanediol Dodecanic acid 33,700 1.6
1,5-Pentanediol Adipic acid 40,400 1.9
1,5-Pentanediol Pimelic acid 37,600 2.5
1,6-Hexanediol Succinic acid 19,700 2.3
1,6-Hexanediol Adipic acid 28,000 2.3
-Caprolactone 50,000 nd
1,4-Butanediol Isophtalic acid 23,500 1.8
1,5-Pentanediol Terephthalic acid 22,200 2.1
order to accelerate the degradation of polymer materials based on PCL, copolymers

containing hydrophilic parts, such as PEG, were designed (Sawhney and Hubbel
1990). Because of its interesting properties, PCL was used for the design of block
copolymers for application as (bio)degradable drug delivery systems (Liu et al.
2010; Ryu et al. 2001; Shen et al. 2007; Ferrari et al. 2013). Usually, PCL is
obtained by ring-opening polymerization of -caprolactone (CL) in the presence of
a molecule of interest containing a hydroxyl group as initiator and stannous oc-
tanoate (SnOct2) as catalyst (Scheme 12).
The molecular weight of the PCL segment can be controlled by the ratio
monomer/initiator.
In this context, Liu et al. developed triblocks copolymers containing hyper-
branched poly(ethylene imine), Hy-PEI, PEG, and PCL for gene delivery. These
copolymers were obtained in three steps (Scheme 13): ring-opening polymerization
of CL in the presence of -methoxy, -hydroxy PEG as initiator and stannous
octanoate as catalyst, modication of the hydroxyl end groups using acryloyl
chloride, and conjugation of the acrylated mPEG-b-PCL to PEI through a Michael
addition under mild conditions (Liu et al. 2010). The resulting block copolymers
were not puried but the authors noted that dialysis might be realized if necessary.
Such purication step has to be conducted since it can remain some stannous
octanoate traces, product known to be toxic even at low concentration, and other
side products which can lead to unwanted toxic effects.
Scheme 12 Ring-opening polymerization of CL
Scheme 13 Synthesis of Hy-PEI-g-PCL-b-PEG (Liu et al. 2010)
Such Hy-PEI-g-PCL-b-PEG copolymers are of interest for the design of gene

delivery systems because of the versatility of molecular modications, via the
variation of copolymer compositions, allowing adjusting solubility, reducing
cytotoxicity, increase polyplexes stability and enhancing transfection. Moreover,
the circulating half-time of polyplexes based on Hy-PEI-g-PCL-b-PEG might be
enhanced as a result of slow PCL degradability and PEG hydrophilicity (Liu et al.
2010). Therefore, to conrm the interest of such triblock copolymers as gene car-
riers, the in vitro degradation behavior of amphiphilic triblock copolymers Hy-PEI-
g-PCL-b-PEG having long PCL segments under various conditions (water at 37 C,
buffers with different ionic strength at pH 5.2, 7.4 and 9 at 37 C, PBS buffer at pH
7.4 and 37 C with immobilized lipase from Candida Antartica) was investigated.
The Hy-PEI-g-PCL-b-PEG block copolymers self-assembled in aqueous medium

through hydrophobic interactions to form coreshell micelles with a PCL inner core
surrounded by a hydrophilic corona of PEG and Hy-PEI. The water can freely cross
the PEG corona to be in contact with the surface region of PCL core leading to a
swelling of the micelles: PEG and Hy-PEI blocks are then partially detached from
the micelles surfaces as a result of the random cleavage of ester bonds. Because of
a possible aminolysis effect, PEG chains and PCL segments might be reconnected
to form amide-containing degradation products. After a prolonged degradation, the
PCL core shrinked until complete degradation by random cleavage of ester and
amide bonds. Moreover, the degradation was shown to be faster under basic con-
ditions and in the presence of lipase (Liu et al. 2010). This study highlighted that
the copolymer compositions (PEG and PCL lengths, Hy-PEI molecular weight) had
an influence on the degradation of Hy-PEI-g-PCL-b-PEG copolymers. Because of
the presence of PEG block, the swelling of the micellar structure promoted the
degradation. The degradation time varied from few days to more than 50 days in
function of: (1) copolymer composition, faster degradation rate being observed for
copolymers with shorter Hy-PEI and PCL segments and longer mPEG chain;
(2) the pH of the degradation medium, faster degradation rate being observed under
basic conditions; and (3) the presence of lipases, very fast degradation being
observed with the higher lipase concentration (Liu et al. 2010). Such polymer
material might be of interest for in vivo gene delivery but several in vitro assays,
such as cytotoxicity and gene transfer efciency, have to be realized.
Besides this study, several researchers worked on the design of PCL/PEG
copolymers (Fig. 1) to obtain partially degradable nanoparticles (Ryu et al. 2001;
Shen et al. 2007; Ferrari et al. 2013).
Ruy et al. synthesized triblock copolymers by ring-opening polymerization of
CL with dihydroxy PEG as initiator without any catalyst. This triblock copolymer
formed spherical coreshell-type nanoparticles with an average diameter of 20
60 nm. Despites the presence of hydrophilic PEG block, the molecular weight of
the PCL block decreased only very slowly showing a very slow degradation of
micelles inner core constituted by the PCL block. However, the degradation
mechanism was not explored (Ryu et al. 2001).
Star shaped, 3-arm, PEG-PCL block copolymers were synthesized via
ring-opening polymerization of CL in the presence of 3-arm PEG as an initiator
with stannous octanoate as catalyst. The spherical coreshell-shaped nanoparticles
were obtained by the emulsication/solvent evaporation technique (see Chap. 4
from Mendoza-Muoz et al.) with an average diameter around 30 nm. Their
degradation behavior was evaluated upon incubation in PBS at pH 7.4 and 37 C
over a period of 6 months by dynamic light scattering (variation of the nanopar-
ticles diameter), SEC (evolution of the copolymer molecular weight), and proton
NMR (changes in chemical composition of the copolymers). The diameter of the
nanoparticles slightly varied at the beginning until it suddenly decreased up to a
nonmeasurable value and this decrease was much more rapid for copolymers with
shorter PCL chains. The molecular weight of the copolymer decreased and
broadened during the hydrolytic degradation process, these changed being faster for
Fig. 1 Structures of the PCL derivatives: PCL-PEG-PCL, 3-arm PEG-PCL, and Poly
(HEMA-g-PCL-PEG)
the copolymer with shorter PCL chain length. Finally, the composition of the 3-arm
PEG-PCL block copolymers changed during the degradation process. Therefore,
the cleavage of the ester bonds occurred rst within the PCL blocks (fluctuation in
the nanoparticle sizes) followed by the cleavage of ester bonds between PEG and
PCL oligomers (Shen et al. 2007). Such study showed that it is possible to adjust
degradation rate by modifying the length of the hydrophobic block: the longer the
hydrophobic block is, the slower the degradation rate is.
Brushed nanoparticles having tunable degradation kinetics were prepared from
PCL-b-PEG block copolymers (Ferrari et al. 2013). PCL blocks with controlled
molecular weight were synthesized by ring-opening polymerization of CL in the
presence of HEMA. This macromonomers were then copolymerized with
HEMA-PEG through a semi batch emulsion polymerization process leading to the
formation of nanoparticles constituted by a comb-like polymer with PHEMA
backbone brushed with PCL and PEG chains in the absence of surfactant (Ferrari
et al. 2013). This technique consists in loading the hydrophilic macromonomer,
HEMA-PEG, into the reactor containing water and potassium persulfate, while the
more hydrophobic macromonomer, HEMA-PCL, was fed into the reactor during
the process at a determined injection rate (2 ml/h). The obtained nanoparticles had
an average diameter ranging from 90 to 250 nm. It is important to note that
well-dened nanoparticles were prepared without adding any surfactant which is a
crucial point for applications in the biomedical eld since the presence of surfactant
may lead to biocompatible problems. These nanoparticles were submitted to

degradation studies in the medium used for 4T1 cell line growing. The nanopar-
ticles showed a shrinking of their diameter during the initial stage of degradation
process as a result of the increase in ionic strength of the medium. After few hours,
the nanoparticle diameters started to increase due to the loss of CL units which
increase the nanoparticles hydrophilicity and their swelling in water. Finally, as
expected, nanoparticles with longer hydrophobic PCL blocks had a longer degra-
dation time. The nal degradation time was evaluated to be of 57 h for the most
hydrophilic copolymer, poly(HEMA-g-CL2-b-PEG19), 96 h for the poly(HEMA-g-
CL3-b-PEG19) and more than 1 month for the hydrophobic poly(HEMA-CL5). At
the ultimate stage of PCL segment degradation, it remains only nanoparticles based
on poly(HEMA-g-PEG19) which might be eliminated through kidney as a result of
their high hydrophilicity and small diameter (Ferrari et al. 2013).
Therefore, one can adjust the degradation time and rate of nanoparticles by
playing on both the length of the hydrophobic PCL block and on the structure of the
PCL-based copolymers.
Another way to modulate degradation rate consists in changing the nature of the
hydrophobic polyester block. By selecting a polyester more sensitive to degrada-
tion, one may be able to accelerate the degradation rate. For example, polymers
based on lactic acid (LA) are known to be degraded faster than PCL-based polymer
materials. The PLA-based polymer materials (Fig. 2) were developed either for the
preparation of biodegradable plastics or for the design of sustained release system
(Nampoothiri et al. 2010).
PLAs with controlled molecular weights are commonly prepared by
ring-opening polymerization of L, D, or DL-lactide in the presence of stannous
octanoate or tin (II) chloride (Nampoothiri et al. 2010). One great advantage of such
Fig. 2 Structures of the PGA, PLA, PLGA, and PEG-PLGA polymers

degradable polymers is that several PLA-based materials are commercially avail-

able. For example, Belbella et al. prepared nanospheres from commercially avail-
able poly(D,L-lactides) of two molecular weights (25,000 and 95,000 g/mol) by the
nanoprecipitation technique (Belbella et al. 1996). The nanospheres degradation
was studied at various pH and temperature. The slower degradation was observed at
neutral pH. Following the decrease of the molecular weight of the polymer, it was
concluded that PLA degraded through different mechanisms depending on the pH
of the medium. Degradation resulted from a random scission at acidic pH, while it
consisted on a sequential cleavage in alkaline medium. At 37 C, the degradation
rate was also influenced by the enantiomeric ratio of the D and L lactide residues
composing the polymer. The fastest degradation occurred with nanoparticles
composed of DL-PLA at an enantiomeric ratio of 0.3 at acidic pH (pH 2.2.) and at
37 C. In these conditions, the half-life of the polymer considering the molecular
weight was 12 days and that of the nanoparticles considering the residual solid
mass of nanoparticles was 42 days (Belbella et al. 1996).
The degradation rate of nanoparticles based on PLA can be also modulated by
introducing glycolic acid (GA) units in the PLA polymers. Such PLGA copolymers
are also commercially available. (Bio)degradable microspheres were therefore
prepared from poly(DL-lactides), PDLLA, and poly(DL-lactide-co-glycolide)
50/50, PLGA, by solvent evaporation and spray drying methods (Guinchedi et al.
1998). The microparticles obtained by spray drying technique showed smaller
diameters, smoother and more regular surfaces than the ones observed for the
microparticles obtained by solvent evaporation method. To dene the degradation
patterns of PDLLA and PLGA microspheres, Giunchedi et al. have developed a
rapid HPLC method allowing the simultaneous determination of the end products of
degradation (glycolic and lactic acids). These authors showed that the preparation
method played an important role in the degradation behavior of the corresponding
microspheres: the ones prepared by the spray drying method were characterized by
a higher monomer release rate than the microspheres prepared by solvent evapo-
ration technique (Guinchedi et al. 1998). On the other hand, the PGLA micro-
spheres were degraded faster than PDLLA microspheres as a result of the higher
hydrophilicity of the PGLA copolymer. The glycolic acid was released faster than
lactic acid. Finally, higher molecular weight PDLLA microspheres were degraded
more slowly than lower molecular weight PDLLA microspheres (Guinchedi et al.
1998).
The same degradation behaviors were observed by Zweers et al. with
nanoparticles prepared, by the salting out method, from PDLLA and PLGA syn-
thesized by ring-opening polymerization of DL-lactide and/or glycolide in the
presence of hexanol as initiator and stannous octanoate as catalyst (Zweers et al.
2004). The PDLLA nanoparticles were gradually degraded over a period of 2 years
with a constant size demonstrating no particle aggregation, while the degradation of
PLGA nanoparticles was almost complete within 10 weeks with also a constant
size. In parallel, a PEG-b-PLGA block copolymer was synthesized by ring-opening
copolymerization of lactide and glycolide in the presence of -methoxy, -hydroxy
PEG as initiator and stannous octanoate as catalyst. Corresponding nanoparticles
were prepared by the salting out method and degradation studies showed that the
ester bond between PEG and PLGA blocks was preferentially cleaved leading to a
fast decrease in the overall molecular weight and particle aggregation with a total
degradation within 8 weeks (Zweers et al. 2004).
A similar degradation behavior was also reported for nanoparticles composed of
PEG-PLGA-PEG triblock copolymers (Duan et al. 2007). Obviously, the degra-
dation rate of PLA-based nanoparticles was accelerated by the hydrophilicity of the
copolymers forming the nanoparticles.
The degradation process observed for PDLLA, PLA, PGA, and PLGA-based
nanoparticles was the following: water uptake, swelling, local pH drop inside the
particles, ester hydrolysis, and diffusion of oligomeric degradation products (gly-
colic and lactic acids) (Santander-Ortega et al. 2010). Because the rst step in such
nanoparticle degradation was shown to be water uptake, Santander-Ortega et al.
have prepared PLGA nanoparticles blended with poloxamer or poloxamine poly-
mers with the objective to obtain a better control of the nanoparticle degradation
rate and stability. The presence of these polymers in the PLGA nanoparticle for-
mulation signicantly improved their colloidal stability. The nanoparticles obtained
with the more hydrophobic poloxamine polymer had a signicantly modied
degradation rate (Santander-Ortega et al. 2010). However, the presence of such
surfactant may lead to unwanted toxic side effects if nanoparticles are used for
in vivo applications.
Consequently, it seems better to play on the repeating units nature
(hydrophilicity/hydrophobicity, enantiomeric excess, etc.) than to add surfactant to
tune the degradation rate in order to avoid biocompatible problems resulting from
the presence of this surfactant. It can be concluded also that the hydrophobicity of
the copolymer constituted the nanoparticles, the method of nanoparticle preparation
and the nature of the repeating units might have a great influence on the degradation
rate of the corresponding nanoparticles. All the parameters have to be taken into
account for nanoparticle preparation.
Commercially available PLGA with a molecular weight of 44,000 g/mol was
used to prepare nanoparticles with diameters of 200 nm (addition of an acetone
solution of PLGA to an aqueous solution containing PVA followed by centrifu-
gation) and 500 nm (addition of a dicholomethane solution of PLGA to an aqueous
solution containing PVA followed by sonication and centrifugation) (Mohammad
and Reineke 2013). Their in vivo degradation was evaluated in parallel to their
in vitro degradation behavior after incubation in PBS at pH 7.4 and at 37 C
followed by SEC. In vitro degradation data showed that the larger PLGA
nanoparticles (500 nm) had a faster degradation rate than the smaller one, difference
of which can be explained by an accumulation of oligomers inside the larger
nanoparticles due to longer diffusion time out of the nanoparticles resulting in an
increase acidication and therefore an increase of the polyester degradation
(Mohammad and Reineke 2013). For in vivo studies, the degradation behavior of
both types of nanoparticles was determined in spleen and liver because the
biodistribution studies showed an important accumulation of the nanoparticles in
these tissues. The degradation in liver was much higher than in spleen whatever the
considered nanoparticles. The larger PLGA nanoparticles had a similar degradation

rate in liver than the one observed in vitro while in spleen their degradation rate was
slightly slower than in vitro. On the other hand, smaller PLGA nanoparticles had a
degradation rate in liver and in spleen nearly two times faster than the one observed
in vitro. Finally, this study showed that more than 1/3 of the administrated
nanoparticles remain not degraded after 1 week (Mohammad and Reineke 2013).
The differences may be explained by the presence of enzymes in vivo which can
also participate to the degradation of the poly(esters). Consequently, if studying
degradation in vitro is an important step allowing determining degradation process
and mechanism, the in vivo degradation kinetics and mechanism could be totally
different because of the presence of enzymes and other factors which might dra-
matically influence the nanoparticles degradation rate and mechanism.
In the 80th, poly(-malic acid), PMLA, a poly(ester) with carboxylic acid lateral
groups, was synthesized with the goal to use it exclusively for biomedical appli-
cations (Vert and Lenz 1981). As shown by Scheme 14, the PMLA and its
derivatives are usually obtained by anionic ring-opening polymerization of a
-substituted -lactone in the presence of a carboxylate as initiator (Cammas et al.
1996). The molecular weight of the polymer is controlled by the ratio
monomer/initiator. The monomer is synthesized, in four steps, from aspartic acid
(Cammas et al. 1996).
Scheme 14 Synthetic route to PMLA

Malic acid is the ultimate stage of PMLA degradation (Vert and Lenz 1981). The
structures of all the derivatives which will be presented thereafter are given in
Fig. 3.
Stolnik et al. (1996) prepared and characterized nanospheres constituted by
benzyl esters of PMLA. Anionic ring-opening polymerization of benzyl malolac-
tonate allowed to obtain the poly(benzyl malate), PMLABe, which was then
Fig. 3 Structures of the PMLA derivatives

partially deprotected by catalytic hydrogenolysis to lead to the selected poly(benzyl

malate-co- malic acid), PMLABe90H10 and PMLABe80H20 (Stolnik et al. 1996).
The corresponding nanospheres, prepared by the emulsication/solvent evaporation
method (see Chap. 4 from Mendoza-Muoz et al.), had a spherical shape with
average diameters ranging from 90 to 130 nm (Stolnik et al. 1996). The degradation
of nanospheres was realized in unbuffered water at 37 C and followed by SEC on
freeze-dried nanosphere samples over a period of 5 months. The chromatography
proles for all the degradation times appeared monomodal with broadening of the
molecular distribution with incubation time (Stolnik et al. 1996). The degradation
process resulted in the formation of low or intermediate molecular weight frag-
ments, and the molecular weight decreased with time almost linearly (Stolnik et al.
1996). After 5 months of incubation, it appeared that the polymer molecular
weights decreased more than 70 % of the initial molecular weights for all the
studied nanospheres (Stolnik et al. 1996). The benzyl esters of PMLA constituted
the nanospheres which were degraded in aqueous medium by hydrolytic scission of
the ester bonds between monomeric units leading to a decrease in the molecular
weight of the copolymers with time. Moreover, the presence of benzyl ester
functions reduced the hydrolysis of the main chain ester bonds as the result of their
hydrophobic character. Therefore, for these kind of poly(esters) it seems also
possible to adjust the degradation rate by introducing hydrophobic groups on the
lateral carboxylic acid functions.
Few years later, Martinez Barbosa et al. realized an in vitro degradation prole
study with nanoparticles constituted by PMLABe, poly(hexyl malate), PMLAHe,
and poly(hexyl malate-co-malic acid), PMLAHe90H10 (Martinez Barbosa et al.
2004). The homopolymers were synthesized by anionic ring-opening polymeriza-
tion of either MLABe or hexyl malolactonate, MLAHe. The PMLAHe90H10 was
obtained by catalytic hydrogenolysis of the corresponding PMLAHe90Be10 syn-
thesized by anionic ring-opening copolymerization of MLAHe (90 mol%) and
MLABe (10 mol%). Corresponding nanoparticles were prepared by the nanopre-
cipitation technique (Martinez Barbosa et al. 2004). Nanoparticles, with average
diameters ranging from 100 to 160 nm, were incubated in aqueous buffer solutions
at pH 1.2 and pH 7.5 and 37 C. The degradation was followed by DLS (evolution
of nanoparticle diameters), SEC (changes in polymer molecular weights), and by
proton NMR (identication of degradation products). DLS studies were not pos-
sible because nanoparticles precipitated quite rapidly, probably as a result of dra-
matic changes of the nanoparticle morphologies (Martinez Barbosa et al. 2004).
SEC and proton NMR analyses suggested that the degradation occurred by statis-
tical hydrolysis of the main chain ester bonds leading to the formation of polymers
with lower molecular weights whatever the nature of the PMLA derivatives con-
sidered (Martinez Barbosa et al. 2004). Moreover, this study conrmed that the
degradation rates of such nanoparticles are related to the hydrophilic character of
the polymers which constitute them. Indeed, it was shown that the molecular weight
decreased more rapidly for PMLAHe90H10 than for PMLAHe and PMLABe
(Martinez Barbosa et al. 2004). Such observation could be of interest because one
can modulate the degradation rates by introducing carboxylic groups and/or

hydrophobic moieties on the lateral chains of the PMLA derivatives.
This conclusion was supported by the work realized by Wang et al. on
nanoparticles constituted by the amphiphilic diblock copolymer PEG-b-poly
(DL-lactide-co-RS--malic acid), copolymer obtained by ring-opening polymer-
ization of lactide and MLABe in the presence of monohydroxy PEG as
macroinitiator (Wang et al. 2006). The authors studied the degradation behavior of
the formed nanoparticles and concluded that the hydrophilicity/hydrophobicity
balance played an important role in the degradation of such copolymer because they
observed that the degradation rate increased with increasing hydrophilic malic acid
content (Wang et al. 2006).
Fully degradable micelles were prepared by Nottelet et al. starting from PLMA-
b-PLA copolymers and other PMLA-based amphiphilic copolymers (Nottelet et al.
2010). Besides the usual characterization of the prepared nanoparticles, the study of
their degradation behavior (Nottelet et al. 2010) led to the same conclusions than
the ones formulated above: the degradation of nanoparticles based on PMLA
derivatives was dependent on the global hydrophilicity of the copolymer and
proceeded through statistical hydrolysis of the main chain ester bonds.
Very recently, Schott et al. published the results obtained with randomly
hydrophobized poly(dimethyl malic acid) in terms of nanoparticle preparation, drug
encapsulation, and degradation behavior (Schott et al. 2013). The amphiphilic
copolymers were obtained by anionic ring-opening polymerization of benzyl
dimethylmalolactonate and either hexyl or decyl dimethylmalolactonate followed
by the catalytic hydrogenolysis of the benzyl lateral groups (Schott et al. 2013). The
copolymer degradation studies were conducted by incubation in PBS at pH 7.4 and
37 C. The degradation of these amphiphilic polymers occurred by random
hydrolysis of main chain ester bonds leading to the formation of copolymers with
lower molecular weights than the ones of the initial copolymers (Schott et al. 2013).
The hydrolytic process was slower for such alkylated poly(dimethylmalic acid)
derivatives because the polymer backbone was probably less accessible to water as
a result of the presence of both hydrophobic side groups (hexyl or decyl) and two
methyl groups in the backbone (Schott et al. 2013).
The degradation of nanoparticles based on PMLA derivatives proceeds through
a statistical hydrolysis of the ester bond of the (co)polymer backbone leading to (co)
polymers of lower molecular weight. Moreover, it is obvious that the degradation
rate can be controlled by the global hydrophilicity or hydrophobicity of the (co)
polymers.
2 Conclusion
Several methods are available to control the elimination of the (co)polymers con-
stituted nanoparticles. They can be fragmentable meaning that degradable bonds
and/or units are introduced in the main chain or between two blocks leading to a
general decrease of the molecular weight of the initial polymer materials which can
be, if the resulting molecular weight is lower than the renal threshold, excreted from
the body. However, one has to pay attention to the nonimmunogenic and nontoxic
character of the fragmented (co)polymer generated during the cleavage of the parent
species.
A more elegant manner is to design fully degradable (co)polymer which, upon
hydrolysis and/or enzymatic degradation, can be degraded into low molecular
weight assimilable molecules. In this case, one has to pay attention to the non-
toxicity of the resulting low molecular weight molecules resulting from the (co)
polymer degradation.
The degradation mechanisms are often related to the nature of the degradable
bounds present within the (co)polymer backbone and/or side chains. Moreover, the
degradation rate can be controlled by mixing various monomer units within a
polymer material, each unit bringing a specic property such as hydrophilicity or
hydrophobicity, targeting, colloidal stability, drug encapsulation efciency, etc.
Therefore, one can consider the monomers as building blocks with specic
properties which can be mixed to obtain the expected macromolecular materials
having properties adapted to the application.
The obtaining of bioassimilable and/or (bio)degradable macromolecular materi-
als can be realized either by chemical modications of natural or synthetic polymers
or by (co)polymerization of various monomers. In the latter case, several (co)
polymerization procedures can be used such as anionic ring-opening polymerization
of cyclic monomers, classical or RAFT radical (co)polymerization of monomers
bearing the good functions for radical polymerization. Each (co)polymerization
procedure has advantages and drawbacks, and has to be selected in function of the
nature of the monomers, especially in function of the reactive groups they presented.
The mechanism and degradation rate depend on the nature of both the repeating
units and the links between the monomer units and/or the polymer and the drugs.
They can be also modulated by the methods used to prepare nanoparticles.
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Chapter 13
Associating Drugs with Polymer
Nanoparticles: A Challenge
Christelle Zandanel and Christine Charrueau
Abstract Conditions to achieve drug association with polymer nanoparticles are

examined in this chapter. The different types of interactions and modes of associ-
ation were considered using examples taken among 12 drugs that were associated
with different types of nanoparticles using different approaches. The drugs were
selected to represent the various properties of active pharmaceutical ingredient
(API) varying from their lipophilicity and hydrophilicity and their low-or
high-molecular weights. Strategies developed to enhance performance of drug
loading are discussed in relation with the different methods used to associate drugs
with polymer nanoparticles.
Keywords Active pharmaceutical ingredient

Adsorption Drug loading
Association
Covalent bonding
Electrostatic interactions Entrapment

Hydrophobic interactions Hydrophobic drugs Hydrophilic drugs Polymer

nanoparticles Small molecules Macromolecules
1 Introduction
Developing strategies to administer drug using nanoparticles has been widely

studied and reported in the literature for drugs that presented delivery problems.
Delivery of active pharmaceutical ingredients (API) by nanoparticles offers many
possibilities to enhance their therapeutic potential while the formulations are suit-
able to be administered by systemic routes or used in topical applications.
Depending on the nature of drugs and their applications, nanoparticles can be used:
C. Zandanel (&)
Institut Galien Paris Sud, UMR CNRS 8612, Univ. Paris-Sud, Universit Paris Saclay, 5 Rue
J.B. Clment, 92296 Chatenay-Malabry Cedex, France
e-mail: christelle.zandanel@gmail.com
C. Charrueau
Facult de Pharmacie de LUniversit Paris Descartes, Unit de Technologies Chimiques et
Biologiques Pour La Sant UTCBS, CNRS UMR8258 Inserm U1022, Paris, France

DOI 10.1007/978-3-319-41421-8_13
382 C. Zandanel and C. Charrueau
to improve the bioavailability of drugs that are poorly absorbed helping the drug
to go across biological barriers or preventing premature degradation in bio-
logical environments.
to reduce severe side effects of anticancer, anti-inflammatory, antifungal,
antiviral, or immunosuppressive drugs thanks to a better control of their
biodistribution enhancing the delivery to target sites and diverting distribution
from sites, where the API is highly toxic.
Nanoparticles are intentionally produced particles with dimension characteristic
ranging from 1 to 100 nm and that have properties that are not shared by
non-nanoscale size particles of the same composition (Auffan et al. 2009). As
underlined by the authors, the second part of the denition is more relevant to
characterize nanoparticles from the bulk as the term of nanoparticles is often
generalized to all nano-objects with a size in the range of 101000 nm.
Following European Medicines Agency (EMA), nanotechnology is dened as
the production and application of structure, devices and systems by controlling the
shape and size of materials at nanometric scale [] from the atomic level at around
0.2 nm up to around 100 nm scale (reference EMEA/CHMP/79769/2006).
Considering polymer nanoparticles that are engineered to improve drug delivery,
different types can be distinguished depending on their structures. These include
nanocapsules (NC), polymer nanospheres (P-NPs), core-shell nanospheres (CS-NPs),
mesoporous nanoparticles (M-NPs), magnetic nanoparticles (Mag-NPs), nanogels
(NG), nanoassemblies including polymer micelles, polymersomes, and polyelectrolyte
complexes (PEC). All these types of nanoparticles are engineered by specic methods
of preparations that are using processes that may be more or less stressful for the API
being associated with the nanoparticles. Preparation of drug loaded nanoparticles is
expected to provide nanoparticles having a high drug payload, while the drug molecule
will not be degraded during association with the nanoparticles.
This chapter describes strategies that were proposed to associate drugs with
polymer nanoparticles. Different aspects of the association were taken into con-
sideration as encapsulation depends on many parameters including solubility
properties of the drug in various solvents, hence its hydrophilic or hydrophobic
character, stability in different media (biological or preparation media), the occur-
rence as a salt or prodrug of the molecule and the type of nanoparticle intended to
be used. Twelve APIs were selected to examine the different situations that were
found so far with molecules that were associated with different types of nanopar-
ticles to improve their therapeutic potential overcoming delivery problems identi-
ed to be at the root of their low in vivo activity. This choice includes a series of
API with low-and high-molecular weight and characterized by various water sol-
ubility and partition coefcients between octanol and water (Table 1).
Octanol/water partition coefcient and water solubility are generally well
described in the literature for molecules that were developed as drugs. The
octanol/water partition coefcient was used as a quantitative parameter to represent
the lipophilicity and hydrophilicity of a molecule and considered over time to draw
prediction of in vivo fate of biologically active substances including their
Table 1 Chemical structure and properties of APIs taken as examples selected to discuss association of drugs with nanoparticles
13
Drug Structures M Solubility in watera Log Pa NPs used for

(g/mol) encapsulation
Acyclovir 225.20 2.5 g/L (37 C) 1.56 P-NPs
CS-NPs
NG
Amphotericin B 924.08 0.75 g/L (28 C) 0.8 P-NPs
PEC
Ciprofloxacin 331.34 30 g/L (20 C) 0.28 P-NPs

CS-NPs
PEC, NG
Cisplatin 300.05 2,53 g/L (25 C) 2.19 P-NPs
CS-NPs
PEC
Cyclosporine 1202.61 0.0095 g/Lb 4.1b P-NPs
M-NPs
NC
PEC
Dexamethasone 392.46 0.089 g/L (25 C) 1.83 P-NPs
CS-NPs
Associating Drugs with Polymer Nanoparticles: A Challenge
M-NPs
Self-assemblies
PEC
Doxorubicin 543.21 1.18 g/Lb 1.27 M-NPs
Self-assemblies
(continued)
383
Table 1 (continued)
384
Drug Structures M Solubility in watera Log Pa NPs used for

(g/mol) encapsulation
Doxorubicin 579.98 10 g/Lc Non CS-NPs
hydrochloride determined M-NPs
Mag-NPs
NC
PEC
Ibuprofen 206.28 0.021 g/L (25 C) 3.97 P-NPs
CS-NPs
Mag-NPs
Insulin (porcine) 5795.6 Slightly soluble 0.22 P-NPs

At pH 23 solubility on the order of 1 NC
10 g/L Self-assemblies
PEC, NG
Paclitaxel 853.91 0.0056 g/Lb 3 P-NPs
CS-NPs
Mag-NPs
Self-assemblies
Sirolumus (Rapamycine) 914.17 0.0017 g/Lb 4.85b P-NPs
M-NPs
Mag-NPs
These compounds were cited in Fig. 1. Works reporting their association with nanoparticles are summarized in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13
a
Source: http://www.drugbank.ca/drugs (expect for doxorubicin hydrochloride); bpredicted properties; chttp://www.chemicalbookcom/
ChemicalProductProperty_EN_CB0110633.htm
C. Zandanel and C. Charrueau
13 Associating Drugs with Polymer Nanoparticles: A Challenge 385
bioavailability and toxicity (Sangster 1989). Here, it was included in the table
together with the water solubility as indicators of the lipophilic/hydrophilic nature
of the molecule and of their solubility in aqueous media and eventually in organic
media that are important to know for their association with nanoparticles. From the
table, several molecules, such as acyclovir and doxorubicin. HCl appear frankly
hydrophilic (high water solubility, low octanol/water partition coefcient). Others
including cyclosporine or sirolimus are frankly hydrophobic or lipophilic (poor
water solubility, high values of the octanol/water partition coefcient). It is note-
worthy that a third category of compounds can be identied based on their low
water solubility and low absolute value of their octanol/water partition coefcient.
These compounds that are generally poorly soluble in water are also characterized
by a low solubility in organic solvents. From their molecular structure, an
amphiphilic character may be found (amphotericin B for instance) while for others,
solubility in aqueous media may greatly depends on the pH due to their capacity to
form an ion-pair (doxorubicine and dexamethasone). Compounds listed in Table 1
were associated with various types of nanoparticles. They were designed to be used
for different therapeutic purposes consistently with the activity of the drug. The
Fig. 1 summarizes the different therapeutic activities that were associated with
Peptides/protein based treatments

Metabolic disease
Drugs highly sensitive (diabetes: insulin)
to degradation
Sens/antisense therapies
(oral, IV, pulmonary, transdermal, nasal)

Cancer Viral infections
Local treatment (dermal, ocular, vaginal)
(siRNA) (siRNA)
Drugs with severe side effects due Systemic treatment

Chemotherapies to a lack of delivery specificity Chemotherapies
Viral infections Hydrosoluble Viral infections
(acyclovir) Low bioavailability due to a (acyclovir)
poor capacity to diffuse Cancer
accross biological membrans (cisplatin, doxorubicin-HCl)
API (Drug)
Cancer
Low bioavailability (paclitaxel, doxorubicin)
Bacterial infections Poorly Fungal infections
(ciprofloxacin) (amphotericin B)
water soluble
Inflammatory disorders Inflammatory disorders
(dexamethasone) (Ibuprofen)
Graft rejection management Graft rejection management
(sirolimus) (cyclosporin, sirolimus)
Drugs with severe side effects due
Chemotherapies to a lack of delivery specificity Chemotherapies
Fig. 1 API associated with nanoparticles to improve their therapeutic efcacy. The center of the
scheme point out the natures of APIs and problems linked to their in vivo use as the free
molecules. Examples of API are given together with their therapeutic interest and route of
administration of the nanoparticle formulations
nanoparticles highlighting routes of administrations that were considered to apply

treatments and motivations to design polymer nanoparticle formulations.
2 Preparation of Nanoparticles for Drug Delivery
Methods of preparation of polymer nanoparticles to be used as drug delivery sys-

tems are described in the rst part of the present book (see Chaps. 1 and 5 from
Vauthier, 2 from Miladia et al., 3 from Tang and Prudhomme, 4 from
Alcala-Alcala et al., 6 from Ponchel and Cauchois). Reviews on the subjects are
also available in the literature (Mandal et al. 2013; Pardeshi et al. 2012; Tang et al.
2012; Vauthier and Bouchemal 2009). A brief summary reminding the structure of
the different nanoparticles and their methods of preparation was found useful here
for the understanding of the topic of the present chapter.
2.1 Components of Nanoparticles and Type of Interactions

Promoting Drug Association
The nanoparticles can be divided into four groups depending on their structure and
methods of fabrications (Fig. 2a, b):
the nanospheres (NPs) are composed by a solid polymer or inorganic core
surrounded or not by an hydrophilic shell including the polymer nanoparticles
(P-NPs), the coreshell nanoparticles (CS-NPs), the mesoporous nanoparticles
(M-NPs) or magnetic nanoparticles (Mag-NPs) or inorganic nanoparticles
(I-NPs)
the nanocapsules (NCs) are composed by a solid shell and a water or oil
reservoir as a core
the self-assembled NPs prepared by spontaneous association due to electrostatic
bonds as polyelectrolyte complexes (PEC) or nanogels (NG) or hydrophobic
interactions and stacking (nanoassemblies)
the nanolipids including solid lipid nanoparticles (SLNs) or lipid nanoparticles
(LNs) composed of lipids.
Association of hydrophilic or hydrophobic drugs depends on the structure and
the composition of these nanoparticles.
Association of drugs with nanoparticles is mostly achieved by non-covalent
interactions preserving integrity of the chemical structure of the drug molecules that
can be seen as an advantage. It can be achieved by entrapment or by
adsorption/diffusion. While entrapment is mainly found with hydrophobic
(a) Preformed polymers Monomers, macromers
CS-NP
EMULSION RT or 40C
CS-NP
CeIV P-NP
(with copolymers)
O
w
NC
O
RT NC
P-NP NANOPRECIPITATION
RT
SOL-GEL PROCESS basic media
M-NP
(b)
Micelle polymersome
Amphiphilic
polymers
Polyelectrolytes with Gelling

opposite charges SELF-ASSEMBLING polymers
PEC NG
(c) SURFACE
FUNCTIONALIZATION
Fig. 2 Structures of polymer nanoparticles produced by the different methods. a Methods based
on emulsications, b methods based on polymer self-assembling processes, c surface function-
alization of particles after preparation by other methods
molecules, the two types of process are used to associate hydrophilic drugs with
nanoparticles especially API issued from biotechnologies including nucleic acids
and proteins and peptides (Vrignaud et al. 2011). The advantage of encapsulation by
entrapment is the higher protection of drug from a premature degradation in bio-
logical media. This was, for example, the case with insulin that was encapsulated in
nanocapsules (Zhang et al. 2012) and with oligonucleotides encapsulated in
nanocapsules as well (Lambert et al. 2000). Adsorption of hydrophilic drugs on
nanoparticle surface was considered with drugs that are unstable in the conditions
used to achieve the preparation of the nanoparticles (see Sect. 2.2). However, this
mode of association applied with an hydrophilic molecule may lead to a rapid release
from the nanoparticles as it was reported considering a small molecules like dox-
orubicin hydrochloride (Alhareth et al. 2012). The adsorption is more stable con-
sidering macromolecular drugs such as small interfering RNA (siRNA) (de
Martimprey et al. 2010).
Interactions of interest that are used to associate drugs with nanoparticles include
electrostatic forces that occur between compounds of opposite charges. Formation
of electrostatic bonds are one of the main driving forces for the preparation of
PECs, NG, and the most common method used for the encapsulation of hydrophilic
macromolecules.. A huge number of examples are reported in the literature on the
association of siRNA with nanoparticles mainly by adsorption (Vauthier et al.
2013). A high encapsulation efciency (>90 %) of azidothymidine (AZT-TP) into
poly(isobutylcyanoacrylate) nanocapsules was possible by the addition of poly
(ethyleneimine) (PEI) to avoid a low encapsulation efciency (EE) and a rapid
release of the encapsulated molecule (Hillaireau et al. 2006). The electrostatic
interactions are also used to adsorb small molecules as doxorubicin hydrochloride
at the surface of preformed nanoparticles (Yang et al. 2000).
Van der Waals or hydrophobic interactions that also include stacking are
other types of attractive interactions that allow drugs to associate with nanoparti-
cles. Entrapment of sirolimus in the P-NPs, insulin in nanocapsules (Zhang et al.
2012), and cyclosporine in SLNs during the preparation of the nanoparticles were
achieved thanks to these types of interactions. The uploading of mesoporous
nanoparticles with ibuprofen through a diffusion mechanism was also achieved
thanks to Van der Waals interactions. Complexation of API with cyclodextrin is
also generally based on hydrophobic interactions (Ageros et al. 2011).
Beside non-covalent interactions, the literature provides examples of association
of drugs with nanoparticles achieved by covalent binding. For instance, siRNAs
were attached on nanoparticle surface via disulde linker (Giljohann et al. 2009).
Another approach consists in designing prodrugs by attaching the API to another
compound that is often a component of the nanoparticles. This approach was used
for instance with doxorubicin, paclitaxel, cisplatin, or acyclovir. In general, labile
linkers were used to link the API with the nanoparticles. It can also serve as a mean
to control the release of the parent drug (see Sect. 4).
More rarely, coordination bonds were used to associate drugs with nanoparticles.
Insulin was incorporated into poly(-glutamic acid) and chitosan-based nanoparti-
cles thanks to coordinating bond involving Zn2+ ions (Sung et al. 2012). This mode
of association is also commonly found with cisplatin due to its chemical structure
and the ease of formation of coordination linkage with polyanions including poly
(-glutamic acid) (Oberoi et al. 2013).
2.2 Methods of Preparation of Nanoparticle and Drug

Stability
Methods of preparation determine the structure and characteristics of the produced

nanoparticles. The Fig. 2 summarizes the different structures of nanoparticles
produced by different procedures and from different media.
Preparation methods can be divided into four groups:
Methods based on polymerization (Chap. 5 from Vauthier).
Methods based on nanoprecipitation of polymers induced by removal of sol-
vent (Chap. 2 from Maladi et al. and Chap. 3 Tang and Prudhomme).
Methods based on self-association of molecules or macromolecules. One part
of methods of this category promotes the formation of highly structured
nanoparticles including micelles and polymersomes due to the amphiphilic
characteristics of components used to form nanoparticles. The other methods are
forming nanogels and polyelectrolyte complexes. NG form by gelation while
PEC results from the association of polyelectrolytes with opposite charges.
Methods based on solgel process are specically used to produce MNPs.
Each method has its own requirements in terms of physicochemical and engi-
neering conditions. Obviously, APIs that are associated during preparation of
nanoparticles must remain stable in those conditions. Experiments dedicated to
verify the stability of the drug in conditions used to produce the nanoparticles may
be required if a loss of activity during preparation is suspected. These may include a
stability study in conditions simulating the physicochemical environment of the
preparation or the use of specic engineering procedures including ultrasound and
high pressure homogenization for instance. For example, methods based on the
formation of a simple oil-in-water emulsion have been widely used to encapsulate
hydrophobic drugs. Preparation of hydrophilic drug loaded nanoparticles requires
the formation of a double emulsion in which the solvent phase is comprised
between the two aqueous phases forming a water-in-oil-in-water emulsion. Success
of the encapsulation with these techniques greatly depends on solubility properties
and partition coefcient of the API between the different phases of the emulsions.
The drug may be difcult to associate with the nanoparticles if it is too soluble in
the continuous phase of the emulsion (Lee et al. 2011). In general, engineering
methods used to produce emulsion with characteristics that suits with the prepa-
ration of nanoparticles may be damageable for the API but also for polymers due to
the strong homogenization processes that are needed. Risks are caused by the high
shear that can induce the breakage of polymer chains into chains of lower molecular
weight than the parent polymer. This effect may cause signicant shift on the drug
releasing property of the nanoparticles that often depends on the molecular weight
of the polymer that form the matrix of the delivery system. The second type of risk
is caused by the local elevation of temperature in the preparation medium during the
course of the homogenization process that can be damageable for thermosensitive
compounds. The increase of the temperature during the process can be detrimental
for the activity of peptides like insulin for instance (Vrignaud et al. 2011). With
methods of polymerization, a modication of the chemical structure of the API may
occur especially in the course of the production of nanoparticles by polymerization
of alkylcyanoacrylates. Alkylcyanoacrylate monomers can react with molecules
that have nucleophilic groups in their structure hence any API with this chemical
characteristic may be modied during the polymerization process. Although many
drugs have been associated with poly(alkylcyanoacrylate) nanoparticles prepared
by polymerization, only a very few number were found modied and have lost their
biological activity (Chap. 5 from Vauthier).
Spontaneous association is another way to promote association of drugs with
nanoparticles during preparation. It is generally preferred with hydrophilic APIs due
to their faculty to form salts, hence to develop electrostatic interactions with
oppositively charged components entering in the composition of the nanoparticle.
In general, these methods are preferred with API produced from biotechnologies,
such as peptides, proteins, and nucleic acids because they are achieved in gentle
both physicochemical and engineering conditions.
In few examples, the uploading with drug is independent of the method of
preparation of the nanoparticles as it is performed on already prepared nanoparti-
cles. Association is achieved by surface adsorption or diffusion. Based on a dif-
fusion mechanism, MNPs and Mag-NPs can associate either hydrophilic
compounds, such as doxorubicin hydrochloride or hydrophobic molecules, such as
ibuprofen.
3 Associating Drugs with Nanoparticles via Non-covalent

Binding
Association of drugs with nanocarriers is one challenge that needs to be completed

designing nanoparticles to be used to enhance in vivo delivery methods of the
molecule with the aim to increase its therapeutic efcacy. Strategies developed to
achieve association of drug with nanoparticles are often based on a compromise
taken into consideration the composition of the nanoparticles and their specica-
tions that depend on the nal application. The choice is mainly guided by
physicochemical properties of the molecule that it is desired to associate with the
nanocarrier. Among generally known physicochemical parameters, two are par-
ticularly important, the partition coefcient (log P) and the water solubility of the
molecule. The partition coefcient is a ratio between concentrations of the molecule
found in each phase of a water/octanol mixture after phase separation. Hydrophobic

molecules that are less soluble in water are characterized by log P > 0. In contrast,
hydrophilic molecules that are highly soluble in water are expected to show a
negative value of log P (log P < 0). Presence of chemical groups like carboxylic
acid or amines that can be easily transformed into carboxylates or ammonium can
be used to modulate the solubility of the drug molecules and is also interesting to
promote attractive interactions with components of nanoparticles and to favor the
association thereby increasing the drug loading based on non-covalent linkage.
Two parameters are used to express the performance of the association of a drug
with a nanoparticle. The encapsulation efciency (EE %) given in Eq. 1 and the
drug loading (DL %) that is calculated from Eq. 2 (Lehtovaara et al. 2012).
Total drug Free drug

EE % 100 1
Total drug
Total mass of drug Free mass of drug

DL % 100 2
Total mass of NPs
In the literature, the EE is also called the drug loading efcacy (LE). It corre-
sponds to the yield of association of the drug with the nanoparticles. The DL is also
called the drug loaded content (LC) and it denes the drug content or payload of the
nanoparticles with the drug (Jger et al. 2012).
Details about the association of the different API with nanoparticles are sum-
marized in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.
It is generally assumed that the drug load is homogenously spread over the
whole population of nanoparticles composing the dispersion. However, as pointed
out in the work of Kim et al. (2011a) the situation may be quite different from this
ideal case. Indeed, in dispersions composed of various populations of nanoparticles,
DL can vary in the different populations hence the efcacy and safety prole of the
nanoparticle formulation of the drug. Underestimation of the heterogeneity of the
drug distribution in nanoparticles may come from the determination of the
nanoparticle size characteristics of dispersions that are generally only evaluated by
dynamic light scattering methods. This well implanted method that is recognized by
health authorities is however suitable to evaluate size and size distribution of dis-
persions containing one population of nanoparticles. Evaluated by other size
measurement methods, populations with different sizes may be revealed as it was
the case for the dispersions analyzed in the work of Kim et al. (2011a, b). To
circumvent this problem, it was recently recommended to evaluate size character-
istics of unknown dispersions by at least two methods including one based on a
single particle measurement or with a method that applies a separation by size prior
to size measurements (Varenne et al. 2016). The drug load found in the different
populations of nanoparticles of the dispersion varied from 4 to 25 % (Kim et al.
2011a). This work that also revealed a difference of interactions of proteins with the
nanoparticles contained in the different populations indicates that they may show
different in vivo fate after administration in the body. Consequently, safety issues
Table 2 Overview of doxorubicin encapsulation
392
Type of Doxorubicin Process Initiation Monomers/polymers Encapsulation Administration References

nanoparticles form Type DL EE
(%) (%)
CS-NPs HCl Emulsion Anionic PIBCA-PIHCA Entrapment NA 95 IV De Verdire et al.
polymerization (1997)
HCl Radical PIBCA Adsorption 3.7 74 IV Alhareth et al.
(2011)
M-NPs HCl Solgel IPA-MAA Adsorption >20 IV Chen et al.
(2013)
HCl NaOH PEG Adsorption 14 NA IV Gu et al. (2012)
HCl NH4OH COOH Entrapment 4 22 IV He et al. (2011)
CTAB Chitosan Entrapment 4 85 IV Yuan et al.
93 (2012)
HCl Co-condensation PEG Adsorption 24 62 Xie et al. (2013)
Mag-NPs HCl LbL/co-precipitation Carboxylchitosan/Fe Adsorption NA 44 Anirudhan and
95 Sandeep (2012)
HCl Adsorption 10 90 IV Pilapong et al.
(2013)
HCl PLGA/Fe Entrapment 2 35 IV Li et al. (2011)
27 85
HCl W/O/W emulsion PLGA-PEG/Fe Entrapment NA 69 IV Akbarzadeh et al.
75 (2012)
NPs HCl Reduction Xanthan Adsorption NA 71 IV Pooja et al.
(2014)
NC HCl Nanoprecipitation from HPCD-PLA Complex with 90.6 Wang et al.
W/O/W emulsion cyclodextrin (2011a)
(continued)
Table 2 (continued)
13
Type of Doxorubicin Process Initiation Monomers/polymers Encapsulation Administration References

nanoparticles form Type DL EE
(%) (%)
Self-assemblies Self-association Curdlan-PEG Entrapment 1040 20 IV Lehtovaara et al.
25 (2012)
Dextran Adsorption 25 NA IV Li et al. (2013a)
boronate
PEI Entrapment 32 NA IV Theodossiou
et al. (2013)
PEC HCl Self-association Polypeptide Entrapment 22 98 IV Lv et al. (2013)
HCl Carboxylmethyl Entrapment 6 54 IV Guo et al.
chitosan 16 70 (2013b)
HCl Alginate Entrapment IV Guo et al.
(2013a)
NG HCl Gelation Calcium Entrapment 87 IV Nmati et al.
alginate + poly (1996)
(lysine)
393
394
Table 3 Overview of paclitaxel encapsulation

Polymerization Monomers/polymers Encapsulation Administration References
Process Initiation Type DL EE (%)
(%)
P-NPs Nanoprecipitation PBS/PBDL Entrapment 67 95 IV Jger et al. (2012)
Emulsion/solvent evaporation MPEGPTMC Entrapment 6 94 IV Jiang et al. (2011)
Solid-state reaction PDM Entrapment 58 IV Lee et al. (2011)
O/W/O emulsion Chitosan/HTCC Entrapment 3538 8783 oral Lv et al. (2011)
O/W emulsion Chitosan-PLGA Entrapment 66 Parveen and
Sahoo (2011)
O/W emulsion PEG/Folic-PLGA Entrapment 1.3 oral Roger et al.
(2012)
O/W emulsion-crosslinking Chitosan/PLGA Entrapment 8.5 94 IV Xu et al. (2012)
Interfacial polymerization PBCA Entrapement 1 99 IV Ren et al. (2011)
CS-NPs Nanoprecipitation Pullunam-acetate Entrapment 613 IV Lee et al. (2012)
Emulsion-precipitation PLGA Entrapment 9496 IV Narayanan et al.
(2014)
Coaxial tri-capillary PLGA-PEG Entrapment 4150 7276 IV Cao et al. (2014)
electrospray-template removal process
Mag-NPs LbL/co-precipitation Fe/poly Adsorption 30 % IV Hua et al. (2010)
[aniline-co-sodium
N-(1-one-butyric
acid) aniline]
Self-assemblies Self-association Serum albumin Entrapment >20 % 100 % IV Ding et al. (2014)
13
Table 4 Overview of ibuprofen encapsulation

Process Initiation Type DL (%) EE (%)
P-NPs Precipitation SDS/TEA/SLS/PVP-tween 80 Entrapment Oral Mansouri et al. (2011)
Nanoprecipitation Eudragit Entrapment 4.5 50 Oral Galindo-Rodrguez et al. (2005)
Emulsication-diffusion 7.7 86
Emulsication-salting out 8.8 88
Precipitation PLGA Entrapment nd Nd Oral Bonelli et al. (2012)
Emulscation-evaporation HEMA/Gelatin Entrapment nd Nd Oral Haroun et al. (2014)
Cubic NPs Fragmentation Phytantriol-poloxamer 407 Entrapment 8.3 >85 Oral Dian et al. (2013)
CS-NPs Co-precipitation DEAE-dextran Entrapment 822 5672 Oral Jiang et al. (2005)
M-NPs Solgel NaOH Chitosan Adsorption 22 Oral Chen et al. (2012)
Solgel Ammoniac Adsorption 2124 Oral Xu et al. (2009)
Mag-NPs MNPs-Fe Adsorption Oral Xing et al. (2012)

395
396
Table 5 Overview of amphotericin B encapsulation

Amphotericin Polymerization Monomers/polymers Encapsulation Administration References
B form Process Initiation Type DL EE
(%) (%)
P-NPs Nanoprecipitation PLGA Entrapment 54 IV Van de Ven et al.
63 (2012)
Solvent PLGA Entrapment 42 Verma et al.
displacement (2011)
Deoxycholate Interfacial Polysorbate Entrapment 56 IV Xu et al. (2011)
polymerization
Desolvation method Gelatin Entrapment 49 IV Nahar et al.
(2008)
PEC Sulfate Self-association CS/chondroitin Entrapment 111 90 IV Ribeiro et al.
sulfate 92 (2014)
Dialysis PLA-g-Chitosan Entrapment 18 70 Topical Zhou et al. (2013)
22 81
LNPs Emulsion solvent Lipid/PEG Entrapement 76 IV Jung et al. (2009)
evparotation
13
Table 6 Overview of cyclosporine encapsulation

P-NPs Emulsiondiffusion PLA/PEG Entrapment 1030 4350 Oral Ankola et al. (2010a)
evaporation
High homogeneization PLGA/PLA Entrapment 69 8994 Lee et al. (2002)
Emulsion-evaporation PLGA Entrapment 1096 Rahman et al. (2010)
Emulsion-evaporation PLGA/chitosan Entrapment 85105 Topical Hermans et al. (2012)
Cubic NPs Fragmentation Glyceryl monooleate-poloxamer Entrapment 85 Oral Lai et al. (2010)
M-NPs Solgel Adsorption Lodha et al. (2012)
Mag-NPs Peral milling Zr/poloxamer 99 IV Nakarani et al. (2010), Oliveira et al. (2012)
NCs Emulsion diffusion PLA Entrapment 4.59.4 7292 Oral Park et al. (2013)
Emulsion (o/w) PLGA/Eudragit/PVA Entrapment 8395 Topical Aksungur et al. (2011)
PEC Self-association PEI-cetyl Entrapment 20 Oral Cheng et al. (2006)
SLNs High homogeneization Compritol, Tween, Poloxamer Entrapment 95 Oral Karavana et al. (2012)
High homogeneization Compritol/Poloxamer Entrapment 95 Topical Gke et al. (2009)
397
398
Table 7 Overview of sirolimus encapsulation

P-NPs Nanoprecipitation PLA/chitosan Entrapment 329 689 Topical Yuan et al. (2008)
Emulsion-salting out PEO-PLGA Entrapment 0.10.4 2533 Topical Zweers et al. (2006)
Supercritical antisolvent PVP Entrapment nd Nd Oral Kim et al. (2011b)
process
Crosslinked micelles PEG-PLA-Polyester Entrapment nd Nd IV Woo et al. (2012)
Emulsication-evaporation PLGA Entrapment 1.6 80 IV Acharya and Sahoo (2011)
w/o/w emulsion PLGA Entrapment 0.05 81 IV Haddadi et al. (2008)
M-NPs Emulsion PFC Adsorption Topical Cyrus et al. (2008)
Mag-NPs Co-precipiation Emulsion FE-oleic acid Entrapment 5.66.2 8296 Oliveira et al. (2012)
solvent evaporation
13
Table 8 Overview of dexamethasone encapsulation

Dexamethasone Polymerization Monomers/polymers Encapsulation Administration References
form Process Initiation Type DL EE
(%) (%)
P-NPs Solvent displacement PLGA entrapment 59 IV Ali et al. (2013)
89
Emulsion-Salting out PEO-PLGA entrapment 229 11 topical Zweers et al. (2006)
100
CS-NPs Interfacial KPS PA-co-AA entrapment 28 25 IV Fratoddi et al. (2012)
emulsion 87 88
M-NPs Supercritical CO2-assisted Poly(-caprolactam) adsorption <1.5 local de Matos et al. (2013)
foaming/mixing
Self-assemblies Self-association PEG dentritic entrapment nasal Kenyon et al. (2013)
PEC Phosphate Self-association Quaternary ammonium entrapment oral Uccello-Barretta et al.

disodium salt chitosan (2014)
399
400
Table 9 Overview of ciprofloxacin encapsulation

Ciprofloxacin Polymerization Monomers/polymers Encapsulation Administration References
(%) (%)
P-NPs HCl Double emulsion PLGA Entrapment 3569 Mobarak et al. (2014)
solvent diffusion
CS-NPs Free base Solvent diffusion Pullulan PCL Entrapment 3540 3540 IV Shady et al. (2013)
PEC HCl Self-association Dextran sulfate Entrapment 80 Cheow and Hadinoto
(2012)
NG Self-association Carboxylchitosan Entrapment 3985 Zhao et al. (2013)
LNs HCl w/o/w emulsion PLGA/phosphatidylcholine Entrapment 4 Cheow and Hadinoto
(2011)
13
Table 10 Overview of acyclovir encapsulation

Acyclovir form Polymerization Monomers/polymers Encapsulation Administration References
P-NPs Nanoprecipitation PLA Entrapment 0.53.1 2.313.3 Patel et al. (2014)
Nanoprecipitation PLA Entrapment 1443 IV Kamel et al. (2009)
CS-NPs Emulsion PBCA Entrapment 18 Zhang et al. (1998)
polymerization
NG Ionotropic gelation Chitosan-TPP Entrapment 6 14 topical Hasanovic et al. (2009)
401
402
Table 11 Overview of insulin encapsulation

. Insulin Polymerization Monomers/polymers Encapsulation Administration References
(%) (%)
P-NPs Nanoprecipitation PLGA Entrapment 6 Oral Barichello et al.
12 (1999)
Radical APS Poly(N-isopropylacrylamide)- Adsorption 2 Oral Leobandung et al.
polymerization PEG (2002)
NCs Humalog Interfacial Anionic PIBCA Entrapment 60 Oral Cournarie et al.
Umulin polymerization (2004), Damg et al.
(1988)
w/o/w emulsion solvent PLGA-Chitosan Entrapment 1.3 53 Oral Zhang et al. (2012)
evaporation
w/o/w emulsion solvent PLGA Entrapment 80 Oral Reix et al. (2012)
evaporation
Self-assemblies Self-association via Chitosan/PLGA/Zn Entrapment 15 75 Oral Sung et al. (2012)
electrostatic and
coordination bonds
Self-association PEG-graft-PLA Adsorption > Oral Wang et al. (2011b)
60
PEC Self-association Chitosan/TMC/carboxylChitosan Entrapment >90 Oral Chaudhury and Das
(2010)
NG Self-association Pectin-calcium Entrapment 32 Oral Cheng and Lim
96 (2004)
13
Table 12 Overview of siRNA encapsulation

siRNA form Polymerization Monomers/polymers Encapsulation Administration References
Process Initiation Type DL EE (%)
(%)
P-NPs DOTAP Emulsion solvent diffusion PLGA-Chitosan Entrapment 2844 IV Tahara et al. (2010)
complexe
Nanoprecipitation Chitosan-PLG Entrapment IV (Wang et al. 2010)
PEG-PLE-PEI
Coacervation method Chitosan-glutamate/PG Entrapment 6080 IV Lee et al. (2009)
Coacervation method BSA-PEG-PLL Entrapment IV Yogasundaram et al.
(2012), Tzeng et al.
(2011)
CS-NPs Phosphodiester Emulsion Ce4+ PIBCA-PIHCA/Chitosan Adsorption 120/280 IV de Martimprey et al.
polymerization siRNA/NPs (2010)
M-NPs Sol gel-coating PEG/PEI Adsorption IV Xia et al. (2009)
Mag-NPs Co-precipitation coating Fe/PEI Adsorption IV Li et al. (2013b)
NCs Phosphodiester Emulsion PIBCA Entrapment >95 IV Toub et al. (2006)
PEC Self-association Chitosan-PEI Entrapment 3598 IV Saengkrit et al. (2012)

Ca Entrapment 90 IV Giger et al. (2013)
PEG alendronate
NG Self-association Chitosan/TPP Entrapment 7094 IV Csaba et al. (2009)
PEG-Chitosan Entrapment IV Malhotra et al. (2013)
MTC/TPP Entrapment Oral He et al. (2013)
Nanoprecipitation BHEM-chol/PEG-PLA Entrapment 95 IV Yang et al. (2012)
403
404
Table 13 Overview of cisplatin encapsulation

Process Initiation Type DL EE
(%) (%)
P-NPs W/O/W emulsion PLGA Entrapment 0.1 3 IV Alam et al. (2014)
Mini-emulsion PBCA Entrapment 5 25 IV Ebrahimi Shahmabadi et al.
(2014)
Coacervation crosslikinkg Albumin Entrapment 30 IV Das et al. (2011)
80
W/O/W emulsion PEG-PLGA-PLL Entrapment 4 80 IV Wang et al. (2013)
Mag-NPs Co-precipitation + coating PLA Adsorption 80 IV Devi and Prakash (2013)
PEC Self-association PAA-MMA Entrapment 210 IV Lee et al. (2013)
LNs Reverse microemulsion Lipid coated Entrapment 80 IV Guo et al. (2013c, 2014)
precipitaiton Ca/PEG
and drug delivery efcacy may differ depending on the nanoparticle population that
is concerned. In terms of regulatory perspectives, the characterization of nanopar-
ticles that is already challenging may be complicated in the case of complex dis-
persions but this will be needed to assess the safety of the product that will be
administered to patients.
Different polymers were employed for the encapsulation of both hydrophobic
and hydrophilic drugs. Among all, poly(lactide-co-glycolide) (PLGA), poly(lactide)
(PLA), and poly(alkylcyanoacrylate) (PACA) have generated the largest interest for
the design of nanoparticles consistently with their good biocompatibility when
administered in vivo. Examples of low-molecular weight APIs associated with
nanoparticles made of PLA and PLGA included ibuprofen, paclitaxel, amphotericin
B, sirolimus, ciprofloxacin have been reported. Nanoparticles composed with
PLGA were also proposed for the delivery of antigenic proteins that are biological
macromolecules. Degradability of polymers including PLGA and PLA depends on
the lactic/glycolic acid residue ratio and on their molecular weight (Belbella et al.
1996; Danhier et al. 2012). The lipophilicity of the polymer can also be tuned by
varying the amount of glycolic acid residue in the copolymer making possible the
association of a wide range of molecules (Danhier et al. 2012). Nanoparticles
composed of PACA associated small molecular weight APIs, such as doxorubicin,
paclitaxel, and macromolecules including siRNA and peptides like insulin have
been described. Other nanoparticles made of polyacrylic-based polymers were
applied with doxorubicin, ibuprofen, and sirolimus.
The paper of Ma and Mumper is interesting to mention to nd more examples
about polymers that were used to encapsulate paclitaxel in nanoparticles (Ma and
Mumper 2013).
3.1 Association of Hydrophobic Drugs with Nanoparticles
Clearly, beside their low solubility in aqueous media, a high number of

hydrophobic drugs are encapsulated into different nanoparticles with variable EE
and DL either for the systemic or local route of administration. Their encapsulation
was performed in P-NPs (sirolimus, ibuprofen), NCs and self-assemblies
(paclitaxel).
DL is generally low (<25 %) independently of the drugs, the type of polymers
used to prepare the nanoparticles and the process of preparation.
Except for MNPs for which the association of the drug occurs by impregnation
or adsorption on already prepared nanoparticles, the encapsulation of hydrophobic
drugs is generally performed by entrapment during formation of the nanoparticles.
Methods by which the nanoparticles are produced include those based on the
formation of an emulsion, methods of nanoprecipitation or co-precipitation and
methods generating self-assembling nanoparticles.
3.1.1 Association of Drugs with Nanoparticles Prepared by Methods

Based on an Emulsication Process
Nanoparticles prepared by methods based on an emulsication process were used to

encapsulate the different hydrophobic drugs selected as examples in this review. In
these methods, the drug is incorporated with the polymer in the dispersed phase of
the emulsion that is generally prepared with an organic solvent considering
hydrophobic drugs. Then the solvent is extracted either slowly by evaporation or
extremely rapidly by an extraction caused by dilution. The EE and DL are generally
influenced by the solubility property of the drug molecule and the partition of the
drug between the different phases that are composing the emulsion. They can also
be influenced by the speed at which the nanoparticles are formed. In the case of the
production of CS-NP, it was shown that the EE and DL of cyclosporine A in PLGA
nanoparticles increased with the decrease of shell/oil core ratio (Park et al. 2013). It
is noteworthy that in general, formulation of drug loaded nanoparticles is still
mostly based on empirical approaches. However, few works have reported the use
of rational approaches based on experimental design. In general, these approaches
are helpful to identify relevant parameters that are influencing the DL and EE,
hence the performance of the method of preparing drug loaded nanoparticles. For
instance, an experimental design approach was applied to associate cyclosporine
with P-NPs based on PLGA (50/50)/chitosan. The use of a homogenization process
during the preparation of the emulsion after sonication led to a decrease of EE 105
89 % around independently of the presence or not of chitosan in the composition of
the emulsion (Hermans et al. 2012). In another work, influences of the concen-
tration in PLGA (50/50), drug, emulsier, the stirring rate, the nature of organic
phase, and the organic/water ratio were investigated on the EE. The increase of
PLGA and emulsier concentration caused a decrease of EE. In contrast, EE was
enhanced increasing the drug concentration. The stirring rate and type of organic
solvent had no influence on the EE. Another parameter of the process that con-
sidered the dilution of the organic phase into aqueous phase decreased the EE
(Rahman et al. 2010). With dexamethasone, similar conclusions were drawn
regarding the influence of the concentration of PLGA and of the drug on the EE
(Ali et al. 2013). The increase of drug concentration increased EE while an increase
of PLGA concentration led to an opposite effect reducing the EE.
Similar approach applied considering the encapsulation of paclitaxel in PLGA
(50/50) nanoparticles (Yerlikaya et al. 2013). In that case, the increase of the
amount of PLGA was favorable to increase the EE while an increase in the sur-
factant concentration produced the reverse effect.
The impact of polymer, of its molecular weight and of the drug/polymer ratio
was studied for the encapsulation of ciprofloxacin hydrochloride in NPs prepared
by double emulsion solvent diffusion (Elkheshen 2013). EE was not affected by the
type of polymer that included PLGA 50/50 and PLA but it was influenced by the
drug/polymer concentration ratio and by the molecular weight of the polymer. The
authors of this work have suggested an interesting interpretation of their results

based on the effect of the modication of the composition of the different phases
involved in the method on the viscosity. In turn, this change in viscosity can
influence the rate of diffusion of solvent during the solvent diffusion process that is
used to induce the formation of the nanoparticles. It was assumed that an increase of
the viscosity may slow down the precipitation that occurs during nanoparticle
formation hence reduces the EE (Elkheshen 2013).
The work of Zweers et al. (2006) highlighted the influence of the concentration
of the drug in the dispersed phase of the emulsion on the DL considering two drugs,
dexamethasone and sirolimus while the nanoparticles were prepared by the method
of salting out. In both cases, the DL was improved by increasing the amount of drug
dissolved in the emulsion droplets. With dexamethasone, the DL was increased
from 2 to 29 % while the drug concentration expressed as a weight % of the drug
relative to the total weight of drug and polymer used during preparation of the
nanoparticles varied from 17 to 19 %. With sirolimus, DL varied from 0.1 to 0.4 %
upon an increase of the drug concentration from 0.3 to 1 %.
In summary, association of drugs with PLA and PLGA nanoparticles prepared
by methods based on an emulsication process depends on the type of surfactant
and the amount of PLGA. These parameters are influencing the EE. Generally,
increasing the amount of PLGA or of molecular weight of polymer led to a decrease
of EE. Parameters relevant to the process such as the homogenization have no
influence on the EE considering the rate and only a low influence considering the
duration. The solubility and concentration of the drugs in the organic phase that
constitute the dispersed phase of the emulsion influence the DL but no correlation
could be established with the molecule partition coefcient given by their Log P.
3.1.2 Association of Drugs with Nanoparticles Prepared

by Nanoprecipitation or Co-precipitation
Preparing nanoparticles by nanoprecipitation, the drug and the polymer are usually
dissolved in an organic solvent which is miscible with water. The nanoparticles are
obtained by precipitation of the polymer while the organic solvent diffuses in water
during mixing the two phases. The drug is assumed to associate with the
nanoparticles thanks to a co-precipitation with the polymer or to interactions with
the polymer.
Considering the association of sirolimus with nanoparticles prepared by nano-
precipitation, the EE was low (around 6 %) in chitosan NPs. PLA was a better
polymer to associate sirolimus to nanoparticles prepared by nanoprecipitation.
The DL and EE reach maximum values of 29 and 75 %, respectively (Yuan et al.
2008). With amphotericin B, EE was influenced by the initial amount of drug in the
organic phase and was independent from the type and nature of the organic phase
type (Dimethylsulfoxide (DMSO) or mixture of DMSO/acetone) (Van de Ven et al.
2012). With ibuprofen it was shown that the DL decreased with the pH of the
medium and the EE increased with the ratio polymer/ibuprofen for diethy-
laminoethyl (DEAE)-dextran nanoparticles prepared by co-precipitation (Jiang
et al. 2005) probably due to the hydrophilicity of DEAE-dextran polymer.
No real trends can be deduced for nanoprecipitation technique except the fact
that EE and DL depend on the initial concentration of drugs.
3.1.3 Association of Hydrophobic Drugs with Nanoparticles Obtained

from Self-Assembled-Based Systems
Hydrophobic molecules can be associated with nanoparticles that are formed by

self-assembling (Torchilin 2007; Jhaveri and Torchilin 2014). Amphiphilic poly-
mers that occur as block or graft copolymers or hydrophobized polysaccharides
spontaneously form nanoparticles in aqueous environment. When the amphiphilic
polymer is enough soluble in water, formation of the self-assembling structure is
straightforward from an aqueous solution above a minimal concentration of the
polymer. However, in many cases, the solubility of these copolymers is very low in
water. Then, the copolymer is rst solubilized in ethanol or acetone that both are
solvents miscible with water. By mixing the copolymer solution with water, the
self-assembling structure forms immediately thanks to the aggregation of the
hydrophobic parts of the macromolecules. that are insoluble in water. Generally, the
nanoparticles formed take the structure of micelles that are composed of a
hydrophobic core which is surrounded by the hydrophilic parts of the macro-
molecules that are soluble in aqueous media and insure the colloidal stability.
In general, such nanoparticles are suitable for hydrophobic API and were
developed as carriers for poorly soluble anticancer drugs (Lee et al. 2012). The
hydrophobic drugs associate in the hydrophobic domains of the nanoparticles that
generally composed the core of the micelles. Incorporation of the drug depends on
the partition coefcient between the hydrophobic domains and the surrounding
medium. This is greatly favored when the two compounds have a high chemical
compatibility that can be estimated from the calculation of their FloryHuggins
interaction parameter including their solubility parameters (Lavasanifar et al. 2002).
3.1.4 Association of Drugs with SLNs and LNs
Few examples of SLNs and LNs based on the association of polymers and lipids
were reported in the literature. For instance, with nanoparticles based on
self-assembled structures of lipid dextran, the encapsulation of API was driven by
the length of lipid and the log P of the API (Abeylath and Amiji 2011). To retain the
hydrophobic drugs after freeze-drying process, dextran chains were linked together
via click chemistry.
3.2 Association of Hydrophilic Drugs with Nanoparticles
Most low molecular weight hydrophilic drugs molecules associated with

nanoparticles are salts soluble in aqueous media (doxorubicin, acyclovir and cis-
platin). Other hydrophilic drugs are macromolecules including peptides such as
insulin, proteins or siRNA. Their association with nanoparticles can be achieved
either by adsorption on already prepared nanoparticles or by entrapment where the
API is engulfed within the nanoparticles. It is noteworthy that most of the
macromolecular hydrophilic APIs are fragile molecules. Specic care should be
taken to preserve their biological activity during association with nanoparticles.
3.2.1 Association of Hydrophilic Drug by Adsorption

on Nanoparticles
Association of API by adsorption on already prepared nanoparticles is considered

when the activity of the API is lost during preparation of the drug carrier. This
approach was often used with API composed of biological molecules including
peptides and nucleic acids that cannot resist to the stringent conditions that are used
to prepare nanoparticles and that are deleterious for the activity of the molecules.
This can be found in case of methods requiring a strong agitation or the use of
homogenization methods that may break down the molecules. Methods in which
the temperature is elevated during the process can also be deleterious for these
molecules as well as methods that are using organic phases or extreme pH con-
ditions that may cause the denaturation of peptides and proteins.
In general, electrostatic interactions are privileged to associate the API by
adsorption on already prepared nanoparticles. This approach was described with
nucleic acids that were associated with nanoparticles by surface adsorption. In most
cases, the nanoparticle surface is covered with a polycation that has an opposite
charge compared with the nucleic acid. For instance, siRNA was adsorbed on
nanoparticles made of poly(alkylcyanoacrylate) thanks to the presence of chitosan
on the nanoparticles surface (de Martimprey et al. 2010). In another example,
plasmid DNA was associated with nanoparticles by surface adsorption on
chitosan-coated PLGA nanoparticles (Hillaireau et al. 2006; Tahara et al. 2010).
The use of polycation is important to obtain a stable association of the nucleic acid
with the nanoparticles and to insure a good protection of the nucleic acid in bio-
logical media after in vivo administration and prior to the delivery to the target
cells. Nanoparticles coated with simple cationic surfactant lack to insure a stable
association of oligonucleotides with nanoparticles by surface adsorption. The
nucleic acid remains accessible to nucleases in this case (Lambert et al. 2000).
Considering mesoporous nanoparticles, the API is generally uploaded by
adsorption. In one example of adsorption of siRNA on mesoporous nanoparticles,
the mesoporous nanoparticles were further coated with PEI to hinder premature
release of the adsorbed siRNA (Li et al. 2013b).
With low-molecular weight API as well, a few conditions used for the prepa-
ration of nanoparticles can be deleterious for the biological activity of the molecule.
Specic attention is required with methods using stringent conditions of pH, tem-
perature, polymerization reactions, and involving oxidant reagents, such as CeIV
ions. For instance, doxorubicin hydrochloride that is the most commonly used form
of the drug to be associated with nanoparticles is susceptible to oxidation by cerium
ions used in preparations of poly(alkylcyanoacrylate) nanoparticles coated with
dextran by radical polymerization. While, it was not possible to associate the
molecule with the corresponding nanoparticles during their preparation by the
method of radical polymerization, it could be successfully associated with the
nanoparticle after preparation by surface adsorption (Alhareth et al. 2012). After an
hour of contact between the nanoparticles and the drug, the DL reached 3.7 % and
the EE was 74 %. These performances were comparable to those reported by
entrapment (EE = 79 %, DL = 4.2 %) in nanoparticles of the same composition
but prepared by anionic polymerization. In this latter case, as the preparation does
not require the use of cerium IV, the API can be associated with the nanoparticles
during the preparation. Doxorubicin hydrochloride was also associated with PLGA
nanoparticles thanks to the approach based on surface adsorption. In general, PLGA
nanoparticles are developed as carrier for hydrophobic drugs and are not applied
with hydrophilic molecules. The EE of doxorubicin with PLGA nanoparticles could
be increased by coating the nanoparticles with PEG and thanks to the hydrophilicity
of the nanoparticle surface. The EE increased with the presence of PEG on the
nanoparticle surface and with the molecular weight of the PEG moiety of the
PLGA-PEG used to prepare the magnetic nanoparticles (Li et al. 2011; Akbarzadeh
et al. 2012). EE efciencies of 6978 % were reported with these formulations after
3 days of contact of doxorubicin with the nanoparticles. It is noteworthy that this
method can be time demanding to upload the nanoparticles with the API.
3.2.2 Association of Hydrophilic Drugs by Entrapment

in Self-Assembling Nanoparticles Including PEC and Nanogels
The majority of encapsulation of hydrophilic drugs is performed in PEC and in

nanogels obtained by ionic gelation due to the facility to form stable nanoparticles
by electrostatic interactions. Nanogels and PEC can be considered as
self-assembling nanoparticles as they form spontaneously thanks to the occurrence
of electrostatic interactions between the different components that are needed for
their formation. In general, these nanoparticles form in aqueous media at a low
temperature and do not require strong agitation. Their methods of preparation are
suitable to preserve integrity of peptides and proteins while they are extremely
fragile API. EE is generally higher than 95 % for optimized formulations. In some
cases, the DL can be quite interesting as the API serves as component of the
nanoparticles.
With such nanoparticles, the pH is a crucial parameter that influences the pro-
tonation of compounds. In turn, it also influences the establishment of attractive
electrostatic interactions that are needed to form PEC. As relevant examples, EE

varied with the pH influencing the degree of protonation of carboxylchitosan and of
doxorubicin (Anirudhan and Sandeep 2012). A similar effect was described in the
case of the encapsulation of insulin in nanogels of pectincalcium which formation
depends on the protonation of pectin that is influenced by the pH (Cheng and Lim
2004). In this latter system, the molecular weight of pectin was another parameter
that was found to influence the formation of PEC with insulin and thereby the EE
(Cheng and Lim 2004). The increase of the lipophilicity of insulin by the formation
of a complex with phosphatidylcholine can also be used to increase the EE in
PLGA nanoparticles (Cui et al. 2006). EE was more affected by the ratio
PLGA/phosphatidylcholine than by the nature of the organic phase or the molecular
weight of PLGA.
Antigenic peptides were also associated to PEC-based nanoparticles. In this case,
the nanoparticles were obtained by assembling two polyelectrolytes of opposite
charges including for example chitosan and dextran sulfate. The peptide of interest
was entrapped in the PEC in the same time of its formation. The DL is generally
low (a few percent) but interesting results were reported considering these
nanoparticles as new formulations of vaccines (Weber et al. 2010; Delair 2011).
Formation of PEC is widely used to design carriers for nucleic acids. The
method of association is based on ion-pair formation between positive charges of
the polycation and negative charges found on the drug molecules that include
different types of nucleic acids, i.e., genes incorporated in plasmids, oligonu-
cleotides, siRNA, miRNA. They form PEC in which the drug is a constituent of the
nanoparticles allowing accessing quite high DL. The assembling occurs through
many ion-pairs making possible the formation of rather stable nanostructures. Many
parameters were identied having an influence on the efcacy of association of the
nucleic acid with nanoparticles occurring as PEC. While the nucleic acid compose
the negatively charge partner of the PEC, a polycation that is very often used is
chitosan that consists in a polysaccharide composed of N-acetyl glucosamine and
glucosamine residues. The positive charge is due to the protonation of the glu-
cosamine residues. Considering association of siRNA in PEC complex with chi-
tosan, the different parameters that were found to influence association included the
molecular weight of chitosan, its deacetylation degree, the nature of counterions
and its eventual chemical modications (Vauthier et al. 2013).
3.2.3 Association of Hydrophilic Drugs by Encapsulation

in Nanocapsules
The most obvious nanoparticles that could be considered to encapsulate hydrophilic

API are nanocapsules that are composed of an aqueous core in which the molecule
can be solubilized. Such nanocapsules were suggested as delivery systems for AZT
which is a nucleotide used in treatments of acquired immunodeciency syndrome
(AIDS) and for siRNA and other oligonucleotides. Oligonucleotides and siRNA
were associated with the nanocapsules being encapsulated into the aqueous core of
the nanocapsules during preparation of the carrier (Lambert et al. 2000; Toub et al.
2006). In contrast, low EE were reported with AZT which molecular weight is
much lower compared with that of oligonucleotides. The difculty to encapsulate
low molecular weight compounds in water containing nanocapsules was circum-
vented by co-encapsulation of a macromolecule with which the API could form
complexes. In the case of AZT, appropriate macromolecules were PEI and chitosan
that contain positively charged amines and can form ion-pairs with the negatively
charge phosphate groups of the nucleotide (Hillaireau et al. 2006).
Some nanocapsules that were originally designed to associate hydrophobic drugs
were eventually found suitable considering a few hydrophilic drugs. This was the
case of the PIBCA nanocapsules obtained by interfacial polymerization of the
corresponding monomer in an oil-in-water emulsion obtained by the diffusion
method (Al Khouri Fallouh et al. 1986) (Chap. 5 from Vauthier). These
nanocapsules that include an oily core surrounded by a polymer envelope were
found suitable to encapsulate a few peptides provided that their molecular weight
was above a certain threshold. Thus, insulin and calcitonin were efciently asso-
ciated in the nanocapsules. For instance, EE above 90 % were reported with insulin
(Mw 6000 g/mol) (Aboubakar et al. 1999). In contrast, a peptide of low molecular
weight like glutathione was not retained within the nanocapsules (Gate et al. 2001).
Several parameters were identied to be critical to succeed in encapsulating pep-
tides in these nanocapsules. The EE of insulin depended on the pH of the aqueous
insulin solution that was added in the organic phase composed of a large amount of
ethanol, a small amount of oil and including the monomer. It was also influenced by
the origin of the monomer which polymerization may be influenced by the type and
concentration of polymerization inhibitors added to achieve its stability during
storage. In contrast, the source of insulin (Umulin, Humalog) did not influence
the EE (Cournarie et al. 2004).
3.2.4 Association of Hydrophilic Drugs by Entrapment in Other

Nanoparticles
A very few number of examples of hydrophilic API could be associated with

nanoparticles prepared by nanoprecipitation in the course of their preparation
although this method of preparation of nanoparticles is more appropriate to promote
association of hydrophobic molecules. Suitable hydrophilic molecules are anthra-
cyclines, such as doxorubicin hydrochloride. During nanoprecipitation, the salt was
converted to the base form making possible encapsulation of anthracycline with EE
up to 85 %. Insulin was associated with PEG-graft-PLA copolymer with EE above
60 %. The association was achieved during the preparation of the nanoparticles by
nanoprecipitation with the insulin added in the aqueous phase. It is noteworthy that
the biological activity of insulin was preserved (Wang et al. 2011b). Considering
other molecules, low EE were reported indicating that the method of nanoprecip-
itation is not really suitable to associate hydrophilic drugs during the preparation of
the nanoparticles by this method (Barichello et al. 1999).
A molecule like cisplatin is an interesting example of compound that is char-
acterized by a poor solubility. It has a low solubility in water and its solubility in
organic solvents is low as well. As most of the success of association of API with
nanoparticles depends on solubility properties of the molecule, these types of
molecules present a real challenge to be associated with nanoparticles. Success of
the association of cisplatin with LN was achieved with EE above 80 % considering
the preparation of the nanoparticles by reverse microemulsion polymerization [Guo
et al. 2014]. Another approach took advantage of the association of cisplatin with
poly(glutamic acid) by formation of complexes of coordination while poly(glutamic
acid) was included in the composition of the nanoparticles (de Miguel et al. 2014).
Association of hydrophilic drugs with lipid nanoparticles can be achieved in
particles composed of lipids having a hydrophilic moiety. For instance, acyclovir
monophosphate was associated with lipid nanoparticles composed of dioleoyl
phosphatidic acid (Yao et al. 2013), insulin could be associated with nanoparticles
composed of phospholipids (Cui et al. 2006) and siRNA with lipid nanoparticles
including amino lipid or (N,N-bis(2-hydroxyethyl)-N-methyl-N-
(2-cholesteryloxycarbonyl aminoethyl) ammonium bromide in their composition
(Yang et al. 2012; Zhang et al. 2012).
3.3 Summary
From this part of the chapter, it appeared that there are different ways that can be
used to associate drugs with nanoparticles promoting simple interactions between
the drugs and polymers composing the nanoparticles. It is noteworthy that even
after optimization of the formulation where high EE can be reached, the DL that can
be obtained by these methods remained generally low of a few percent and is not
always given in the literature. This means that the amount of API associated with
the nanoparticles represents a low mass of the drug carrier. This is a drawback of
those methods that in contrast fully preserve the molecular structure of the API.
However, it can be a major limitation if the dose of API required to have a ther-
apeutic effect cannot be obtained at the expenses of the administration of a rea-
sonable amount of the formulation. In this case, efforts of formulating the API in
nanoparticles by these methods are useless. There is one exception that concerns the
incorporation of nucleic acids and therapeutic protein in PEC when they are
included in the formulation as a full partner of the complex. In that case, the DL can
be signicantly high as the amount of API associated with the nanoparticles can
represent about half of the composition of the nanoparticles.
4 Strategies Developed to Improve Efcacy of Drug

Association with Nanomedecine
As shown in the previous part of this chapter, associating drugs with polymer
nanoparticles with high DL and EE is not an easy task whatever the
hydrophilicity/lipophilicity of the molecules and using methods based on simple
entrapment or adsorption. Different strategies were suggested to improve association of
APIs with nanoparticles. These include the use of cyclodextrins and the chemical
modication of the drug molecule to design molecular and nanoparticular prodrugs. As
it will be pointed out, some of the developed approaches incidentally have an influence
on the releasing properties of the nanodevices that was interesting to keep and further
develop. Although mentioned in this chapter, releasing properties were the subject of
an extensive discussion in the Chap. 14 proposed by Charrueau and Zandanel.
4.1 Improving Drug Loading Forming Inclusion Complexes

with Cyclodextrins
The solubility of hydrophobic drugs is a major obstacle to achieve high DL and EE

in nanoparticles. In general, forming inclusion complexes between hydrophobic
poorly soluble compound and cyclodextrins enhanced greatly the solubility of the
molecule. As solubility is one of the main factors that hampered drug association
with nanoparticles, complexation with cyclodextrins was a strategy followed to
improve the DL and EE of API that were difcult to associate with nanoparticles.
For example, the DL of poly(alkylcyanoacrylate) nanoparticles in progesterone was
increased by a factor 50 when nanoparticles were prepared by anionic emulsion poly-
merization incorporating an inclusion complex of the API with
hydroxypropyl--cyclodextrine (HPCD) compared with the API alone (da Silveira
et al. 1998). Similarly, the solubility of cyclosporine can also be modied by the
formation of an inclusion complex with HPCD. Incorporation of such a complex into
PLGA nanoparticles could be achieved preparing nanoparticles by the
emulsication-solvent evaporation method with 88 % of EE (Aksungur et al. 2011).
Release was modied due to the presence of the cyclodextrine by increasing the solu-
bility of cyclosporine and the formation of pore inside the matrix (Hermans et al. 2010).
The encapsulation of paclitaxel was also modied by the incorporation of
HPCD into poly(anhydride) nanoparticles prepared by nanoprecipitation (solvent
displacement method) and developed for oral delivery. The DL could be increased
by 500 fold compared with the association of the drug measured in the absence of the
cyclodextrine. The association of the complex of paclitaxel with the cyclodextrine
also enhanced the delivery potential of the new carrier as the permeability of the drug
through the intestinal membrane was increased by a factor 12 with this formulation
compared with the nanoparticles prepared in the absence of cyclodextrine (Ageros
et al. 2009). The use of cyclodextrines also improved the association of paclitaxel in
nanoparticles made of chitosan. In this case, nanoparticles were prepared by radical

polymerization of an acrylic monomer in the presence of a copolymers composed of
cyclodextrines and chitosan. The encapsulation of paclitaxel was achieved during
formation of the nanoparticles by the method of emulsication-solvent evaporation
method. The EE was only of 2 % in the absence of cyclodextrine whereas in the
presence of cyclodextrines it was increased to 14 %, the EE was improved due to a
solubility of paclitaxel 115-fold higher than that observed in solution in the absence
of cyclodextrine (Wang et al. 2012b).
NPs prepared by nanoprecipitation with amphiphilic cyclodextrines also lead to
higher EE and DL than in polymer NPs prepared in the absence of cyclodextrine
(Memiolu et al. 2003). Two examples of amphiphilic cyclodextrines were
reported in the literature for the encapsulation of Acyclovir with respectively 13 and
3560 % of EE (Cavalli et al. 2009; Perret et al. 2013).
4.2 Overcoming Low DL and EE by Chemical Modications

of the Drug Molecule
In general, association of drugs with nanoparticles is achieved by non-covalent binding

including various types of interactions based on electrostatic forces, hydrophobic or
Van der Waals interactions, formation of hydrogen bonds, formation of complex by
ion-pairing, coordination, or by inclusion in cyclodextrines. In contrast to these
strategies that preserve the chemical structure of the API molecule, other approaches are
based on the introduction of chemical modications of the drug molecule to improve the
DL and EE with nanoparticles. The rationale behind these approaches is to tune
physicochemical prole of the drug molecule to promote its association with
nanoparticles. In the same time, the chemical modication applied can also be used to
modulate the releasing prole of the drug from the nanoparticles. Following this
strategy, drugs can be modied preparing molecular prodrugs that are then associated
with nanoparticles or they can be attached via covalent linkage on preformed
nanoparticles (DSouza and Topp 2004). In general, the API is linked via a linker to a
moiety that can be considered as another molecule that will lead to the design of a
molecular prodrug or directly to nanoparticles that will lead to the design of a
nanoparticles prodrug. The general structure of the nal molecule is illustrated in Fig. 3.
The Table 14 summarizes the structure of common linkers. In most cases, linkers
are easily hydrolysable in conditions found in vivo allowing the release of the parent
drug molecules. Hydrolysis depends on the chemical nature of the hydrolysable bond
included in the linker. It can be trigger by different mechanisms depending on con-
ditions found in the surrounding environment in which the parent API will need to be
released from the nanoparticles (Table 14). Thus, the choice of the linker is made
consistently with the release expected for the API. It may be done to design stimuli
responsive nanoparticles that will release the API only under the presence of factors
causing hydrolysis of the linker (Table 14).
(a)
Parent drug Linker Moiety
Small molecule Nanoparcle

(b) Polymer or
Molecular prodrug Nanopar cle prodrug
macromolecule
(parent drug/prodrug = 1) Macromolecular prodrug (parent drug/prodrug > 1)
(parent drug/prodrug 1)
Associa on with nanopar cles by

entrapment, forma on of complex.
Self-assemble in nanopar cles
Prodrug
(c) Prodrug Parent drug
or parent drug
Fig. 3 Prodrug concept applied to increase association with nanoparticle drug carriers. a general
structure of prodrugs, b different types of moiety to design different prodrugs with various parent
drug/prodrug ratios, c expected species released from the nanoparticles
Table 14 Example of hydrolysable linkers used to modify drug molecules to be associated with
nanomedicines
Linkers Chemical structure Cleavage References
Acetal/cetal H R Acidic environment Singh et al. (2008)
R R
O O O O
Ester O Esterases Rautio et al. (2008)

R O
R=C carboxylic ester

R=O carbonate ester
R=N carbamate ester
Disulde S Glutathione Jaracz et al. (2005)
S
Hydrazone NH Acidic environment Kievit et al. (2011)

N
R R'
Silyl ether R R Acidic environment Parrott et al. (2012)

Si
O O
Triazole N Acidic environment Cutler et al. (2010)

N
N
Ester linkers are easily cleaved in vivo after nanomedicines have been admin-
istered by intravenous injections (Rautio et al. 2008). The rate of degradation
depends on the type of the ester bond and the location in the body, tissue, and cells
where the concentrations and specicity of esterases vary a lot. For instance, the
rate of degradation by liver carboxylesterase highly depends on the type of esters
(carbonate > carbamate > carboxylate) (Huang et al. 1993). Disulde bond con-
taining linkers are stable in blood but they are cleaved in cells where the concen-
tration in glutathione is increased compared with the extracellular environment. The
release of the API will be trigger by the local increase in glutathione concentration
in cells while the linkage to the nanocarrier will remain stable during transport in
the blood compartment (Jaracz et al. 2005). Modulation of the release in an acidic
environment can be performed using different silyl ethers (Parrott et al. 2012),
hydrazone (Kievit et al. 2011) or triazole linkers (Cutler et al. 2010).
Molecular prodrugs built on the model structure illustrated on Fig. 3 include
three blocks: the parent drug, the linker and a moiety that gives new physico-
chemical properties to the molecule (Fang and Al-Suwayeh 2012). The new
molecule can be considered as a prodrug consistently with the denition that is a
compound that undergoes biotransformation prior to exhibiting the pharmacologi-
cal effects (Albert 1958). Originally prodrugs were mostly natural product (Arroo
et al. 2008) but nowadays this concept is applied with synthetic drugs to improve
their in vivo delivery. In general this approach is used to increase the hydropho-
bicity of a hydrophilic molecule improving membrane permeation hence
bioavailability, to increase the hydrophilicity of a hydrophobic molecule to improve
its solubility and bioavailability, to reduce toxicity and side effects compared to that
of the unmodied drug, to increase specicity of the biodistribution by targeting the
drug to a tissue or an organ designing a prodrug including an antibody for instance.
Thus, among 10 % of the active principles approved worldwide are prodrugs
(Zawilska et al. 2013). However, improvements of drug properties obtained from
the design of prodrugs were not enough to increase proportion of prodrugs in the
drug market. Transport in the sense of delivery, solubility, and toxicity of many
molecules remains a challenge for numerous APIs. Nanoparticles were used to
encapsulate and transport prodrugs to specic sites into the body, independently of
the administration route. With molecules that are difcult to associate with
nanoparticles, the concept of designing a prodrug with properties enhancing their
association with nanoparticles is a possible option that is worth to explore.
Synthesis of prodrugs can be achieved with drug molecules that include reactive
chemical functions in their structure, such as carboxylic acids, amines, and alcohol.
Reactive chemical functions of the API considered in this review are shown in the
Fig. 4. In general, the linker was grafted on this function. Then, the moiety will be
added to give to the molecule the desired property. Prodrugs designed to promote
association of the API with nanoparticles were mainly synthesized from anticancer
drugs that are cytotoxic. It is noteworthy that their synthesis requires in general the
use of reagents and catalysts that can be toxic. These compounds may produce a
synergetic effect with anticancer drug. In contrast, they can produce detrimental
side effects limiting the range of application of the corresponding chemical reac-
tions. This may explain why the prodrug approach was not so much proposed with
anti-inflammatory or immunosuppressive drugs while considering their association
with nanoparticles.
Ibuprofen Doxorubicin
Hydrazone bond
Amide bond
Paclitaxel
Dexamethasone
Indomethacin
Cisplatine
H3N+ N+ H3
Acyclovir SiRNA
Peptidic coupling via amine functions
Fig. 4 Reactive chemical functions that were used to prepare prodrugs with several API. These
included carboxylic acids, amines and alcohol groups that were used to react with a linker prior to
further addition of a moiety to give new physicochemical characteristics
4.2.1 Designing Molecular and Macromolecular Prodrugs

with Paclitaxel Promoting Association in Nanoparticles
A series of paclitaxel prodrugs was reported in the literature. The parent molecule,
paclitaxel is hydrophobic with a poor solubility prole in either aqueous or organic
media. Depending on the chemical nature of the moiety grafted on paclitaxel, the
prodrugs were either associated with nanoparticles prepared by previously descri-
bed methods or used as the main component of the nanoparticles thanks to its
capability to self-assemble giving birth of a nanostructure (Sohn et al. 2010)
(Table 15).
Three examples of prodrugs of paclitaxel were associated within lipid
nanoparticles prepared by different methods (Ansell et al. 2008; Lundberg 2011;
Nikanjam et al. 2007) (Table 15, lower part). Moities added to paclitaxel were long
Table 15 Paclitaxel prodrugs associated with different nanoparticles

Nanoparticles Moiety added Linker Encapsulation References
Types Method of preparation in the prodrugs (DL %)
Design of a prodrug that will serve as component of the nanoparticles
Nanoassemblies Self-assembly Heparin amide 3539 Wang et al.
(2009)
Nanoassemblies Self-assembly Squalene Ester 4569 Dosio et al.
(2010)
Nanoassemblies Self-assembly Hyaluronic Ester 1015 Xin et al.
acid (2010)
Modication of the parent drug to promote its association with nanoparticles
Lipid NPs Nanoprecipitation Lipids Ester 38 Ansell
et al.
(2008)
Lipid NPs Emulsication-solvent Oleate Ester NA Lundberg
evaporation (2011)
Lipid NPs Emulsication-solvent Oleate Ester PD/FD = 4/1 Nikanjam
extraction et al.
(2007)
hydrophobic chains that further increased the hydrophobicity of the parent molecule
but improved solubility in organic solvent that increased the DL of the nanopar-
ticles compared with that obtained with the parent drug. For instance, Nikanjam
et al. (2007) have reported a DL fourth times higher for the paclitaxel oleate than
that observed with the parent paclitaxel. In the same time the DL was increased, the
release characteristics of the molecule were modied and could be tuned by chosen
the type of the moiety. Ansell et al. (2008) has demonstrated that the release of
paclitaxel from the nanoparticles depended on the length of alkyl chain that was
covalently attached to the molecule designing the corresponding prodrug.
In the second approach, paclitaxel was modied by adding moieties that pro-
moted self-assembling capability of the resulting prodrug to form nanoparticles. In
most examples, the prodrug is made to present amphiphilic properties that are
favorable to induced self-assembly under the form of well-dened nano-objects.
The amphiphilic properties are needed to promote spontaneous formation of the
nanoparticles in water without the need of adding stabilizers (Trivedi and Kompella
2010). From a toxicological point of view this is an advantage especially for the
nanoparticles designed to be administered by the intravenous route. The prodrug
then is the unique component of the nanoparticles with high DL as the DL of these
nanoparticles corresponds to the ratio between the molecular weight of the parent
drug and that of the prodrug. Depending on this ratio, the DL of the nanoparticles
can reach values much higher than 50 % (Dosio et al. 2010). As paclitaxel is mainly
a hydrophobic compound, the more obvious approach that can be followed to
obtain an amphiphilic prodrug is adding a hydrophilic moiety to the parent drug
molecule. This was actually considered in works reporting the preparation of
prodrugs of paclitaxel with heparin (Wang et al. 2009) and hyaluronic acid (Xin
et al. 2010) that formed nanoparticles by self-assembling methods. Interestingly, a
prodrug of paclitaxel that self-assembled in nanoparticles could also be designed
with a hydrophobic moiety like squalene (Dosio et al. 2010). In this case, the linker
was composed of a hydrophilic spacer composed of 3 or 11 ethyl-oxy residues that
was grafted between the paclitaxel and squalene molecules. Although the formation
of nanoparticles by self-assembling of the prodrugs was not greatly influenced by
the length of the linker, the release rate of paclitaxel was increased with the
nanoparticles obtained from the prodrug having the longest linker (Dosio et al.
2010). Besides these examples, other paclitaxel prodrugs were designed using
hydrazone bond linked to PHPMA polymer (Etrych et al. 2010) and acetal linker to
PEG-PAA polymer (Gu et al. 2013). These prodrugs also formed nanoparticles via
a self-assembling mechanism. After administration of the nanoparticles by intra-
venous injections, the linkers were quickly hydrolyzed into tumor cells allowing the
release of paclitaxel. The hydrolysis of the acetal linker occurred much faster than
that of the hydrazone linker due to a steric hindrance effect. So this indicates that the
linkage sensitive to hydrolysis must be well exposed to obtain an optimal control of
the drug release from a stimuli responsive approach.

of Doxorubicin Promoting Association in Nanoparticles
Doxorubicin is another example of API that has generated interest in designing

prodrugs to improve its DL in nanoparticles (Florent and Monneret 2008).
A prodrug composed of glycol-chitosan-doxorubicin was suggested to prepare
nanoaggregates (238342 nm) by self-assembly. Due to the hydrophobicity of the
prodrug and because of the high molecular weight of chitosan (250 kDa), the
maximum value of DL that was reached was only 5 % (Son et al. 2003).
Nevertheless, the nanoaggregates generated by self-assembly of the prodrug were
found interesting because they promoted association of a large amount of the parent
drug with the nanoparticles by simple physical entrapment. The additional load in
doxorubicin was 39 % that brought the total DL of the corresponding nanoparticles
to a value above 40 %. This example illustrates the difculty of modulating the
amphiphilic properties of doxorubicin. Other prodrugs of doxorubicin were pre-
pared using linkers that hydrolyzed in well-dened conditions. Such types of
linkers are used to control the release of the parent drug from the prodrug in a
well-controlled condition that is required to trigger the hydrolysis of the linker
hence the release of the parent drug. A rst example reports the preparation of
pH-sensitive pullulan doxorubicin conjugates via hydrazone bonds that sponta-
neously self-assembled to form coreshell nanoparticles with DL up to 30 % (Li
et al. 2014a). In another work, doxorubicin was conjugated via hydrazone bonds to
polyphophoester (Sun et al. 2014). In both cases, it was expected that the parent
doxorubicin molecule was released from the nanoparticles in an acidic environ-
ment. Another prodrug of doxorubicin was designed including a
PEG-disulde-hyaluronic acid moiety linked to the molecule via a hydrazone bond.

This conjugate formed nanoparticles by self-assembly but DL remained quite low
(3.3 %) (Xu et al. 2013). A very different approach was suggested designing
doxorubicin polymerizable acryloyl prodrug. The linker consisted of a tetrapeptide
(gly-phe-leu-glu) that can be cleaved by esterase releasing doxorubicin. The
polymerizable acryloyl prodrug was incorporated into a poly(N-(2-hydroxypropyl)-
methacrylamide) (PHPMAm) polymer by radical polymerization. Depending on the
composition of the PHPMAm copolymer generated during polymerization, it can
self-assembled in nanoparticles (Yang et al. 2013) or remains as soluble polymer
(Kopecek and Kopeckov 2010). The DL depends on the proportion of the poly-
merizable acryloyl prodrug that is incorporated into the PHPMAm copolymer.
Other prodrugs of doxorubicin were designed to associate at the surface of gold
nanoparticles thanks to coordination bond formation. The moiety containing a thiol
group to insure association with the gold nanoparticle surface via disulde bond
formation was linked with doxorubicin using hydrazone or carbamate containing
linkers. These linkers were chosen for the conditions of their hydrolysis that con-
trolled the release of doxorubicin in respectively acidic and basic environments
(Wang et al. 2011c). The hydrazone linker was easily hydrolyzed in acidic envi-
ronment while the carbamate linker showed a slower hydrolysis rate in basic media.
4.2.3 Designing Molecular and Macromolecular Prodrugs with Other

Hydrophobic Drugs Promoting Association in Nanoparticles
As a quite general method, prodrugs incorporating other hydrophobic drugs were

designed on the model of self-assembling molecules to form nanoparticles incor-
porating the corresponding API. Indomethacin was coupled to heparin via an ester
bond containing linker (Li et al. 2014b). Thanks to its amphiphilic character, the
obtained prodrug self-assembled in well characterized nanoparticles. Release of the
API was triggered from the nanoparticles in a sustained controlled manner by
esterases that cleaved the ester bond of the linker when they were present in the
medium containing the nanoparticles. Concerning acyclovir, the API was modied
to obtain a lipid derivative that self-assembled in water as nanoparticles thanks to
hydrophobic interactions of the lipid chains and hydrogen bonding between the
nucleoside structures of the drug molecule (Jin 2007). The link between acyclovir
and the lipid that consisted in a stearyl chain was composed of a succinyl-glycerol
ester bond. The release of the API from the nanoparticles depends on the presence
of esterases in the surrounding medium. So the released is also controlled by
esterases in the case of this prodrug. The work reported by Jin (2007) points out
several issues that can be found developing prodrugs that displayed amphiphilic
properties. Although this characteristic is interesting to obtain spontaneous
assembly of the prodrug molecules to form nanoparticles, it has to be demonstrated
that the assembly remains stable under the various conditions that the nanoparticles
encountered when it is administered in vivo and prior it reaches it delivery place.
Indeed, biological media contain various salts at various concentrations, proteins,
lipids that are all components that can contribute to destabilize the nanoassembly.
Also some methods of sterilization may be deleterious and it is needed to nd the
most suitable method that will fully preserve the chemical structure of the prodrug
as well as the structure of the nanoparticles. The cytotoxicity of the new molecules
may be enhanced due to the amphiphilic property that may interfere in the lipid
bilayer of the cell membranes. This effect can be easily evaluated by performing
hemolytic tests.

with Hydrophilic Drugs Promoting Their Association
with Nanoparticles
Approach that consists in designing molecular and macromolecular prodrugs to

increase association with nanoparticles was also applied with hydrophilic API.
Strategies included the modication of the drug molecule to give suitable property
to increase association with nanoparticles and design of prodrugs capable of
self-assembling as nanoparticles.
An example of the rst strategy is given considering dexamethasone that was
modied as a lipid-based prodrugs to improve association with SLNs (Kim et al.
2011a; Wang et al. 2014, 2012a). Hydrophobic moieties such as palmitate and
stearyl alcohol were grafted with an ester linkage on the dexamethasone molecule.
The prodrugs were incorporated in SLN prepared by solidication of the droplet of
a microemulsion (Kim et al. 2011a). In another example, a dipeptide composed of a
dimer of valine was grafted on dexamethasone. The solubility of the prodrug in
water was 50 times higher compared with the water solubility of the parent drug.
The terminal amino group that was included in the prodrug molecule was used to
form an ion-pair with dextran sulfate. The complex was less soluble in water but it
was soluble in organic solvents including methylene chloride and acetonitrile that
are common solvents used in preparations of nanoparticles composed of PLGA.
The complex was then associated with PLGA nanoparticles with an adapted method
from the emulsication-solvent evaporation technique. Entrapment of the prodrug
in the nanoparticles could reach 45 % while conventional equivalent methods of
preparation of nanoparticles led to entrapment of dexamethasone below 10 %
(Gaudana et al. 2011). This approach allowed a 4.5 increase of the DL of the
nanoparticles. Although the method was efcient to increase the DL of the
nanoparticles, it should be pointed out that this benet was obtained at the expense
of the application of a considerably more complex procedure which may be a
limitation to achieve further developments. Nevertheless the approach was worth to
mention as it gives another example of strategy that can be used to promote drug
association with nanoparticles preparing a molecular prodrug with a moiety that
gives the parent drug an ion-pairing property.
With cisplatin, one prodrug was synthesized adding an adamantane residue to
allow association of the prodrug with gold nanoparticles functionalized with
cyclodextrin (Shi et al. 2013). In this case, association of the prodrug containing the
drug of interest with the nanoparticles was promoted thanks to the formation of an
adamantane-cyclodextrin inclusion complex at the surface of the nanoparticles. In
another example, a hydrophobized prodrugs of cisplatin was synthesized grafting
the drug molecule on PEG-PLA polymers via a hydrazone bond. This prodrug
associated with nanoparticles by self-assembling was induced during a
precipitation-based preparation method (Aryal et al. 2010). Despite the chemical
modication applied to hydrophobize the drug molecule, the DL remained low
(1.05 %) but it was stable compared with nanoparticles prepared with the parent
cisplatin molecule which association with nanoparticles was unstable. Another
interesting feature came from the drug releasing property of this new assembly. The
release was triggered in media of acidic pH that made this system interesting in
terms of its capacity to control the release of the drug under very dene pH
conditions Another strategy consisted in grafting lipophilic chains on cisplatin that
improved the DL of PLGA-PEG-COOH nanoparticles prepared by nanoprecipita-
tion. A linear relationship was reported between the chain length of the lipophilic
moiety grafted on cisplatin and the DL. With this approach, the DL of the
nanoparticles could reach 7 % w/w as expressed in platinum (Johnstone and
Lippard 2013).
In the last two examples, drug molecules were modied to promote their
self-assembling as nanoparticles. Ibuprofen esteried with xylan via carboxylic
ester linker self-assembled into nanoparticles (Daus and Heinze 2010). The
squalenization of doxorubicin led to a prodrug that self-assembled spontaneously in
nanoparticles giving DL of 57 % (Maksimenko et al. 2014).
4.3 Designing Prodrugs with Preformed Nanoparticles
In previous examples, prodrugs designed to increase association with nanoparticles

remained a chemical conjugate on a molecular scale. Another strategy consists in
attaching the API on the nanoparticle surface via a covalent bond that can be
cleaved in well-dened conditions triggering the release of the parent drug. In this
situation, the prodrug takes the structure of a nanoparticle having several copies of
the drug molecule attached on the nanoparticle surface. It can be considered as a
nanoparticle prodrug (Fig. 3). Covalent attachment of API on nanoparticle surface
leads to the formation of a new chemical entity that requires a full registration in
terms of pharmaceutical development. Different types of linkage were used to attach
API on the surface of nanoparticles. In general, the achievement of the linkage
requires the introduction of appropriate chemical groups on the drug molecule to
achieve the formation of the chemical bond with functions available on nanoparticle
surface. The achievement of the chemical bond was privileged using Click
Chemistry to avoid complex procedure. As in the case of the molecular prodrugs, a
spacer with hydrolysable bond can be added between the API and the nanoparticle.
Examples include association of siRNA on gold nanoparticles via the formation of
disulde linkage. Stability of the association of the siRNA with the nanoparticles
and the releasing properties of the drug from the nanoparticles were modied
compared with those observed with nanoparticles loaded with the siRNA by simple
adsorption (Giljohann et al. 2009). Using a copper-catalyzed Click Chemistry to
achieve the formation of a triazole linkage, alkyne modied oligonucleotides were
grafted at a high density on the surface of iron nanoparticles functionalized with
azide groups (Cutler et al. 2010). The density of grafting ranged from 3.2 1012 to
2.3 1013 oligonucleotides/cm2 (1070 oligonucleotide chains per 10 nm
nanoparticles). In another example, doxorubicin was covalently linked to super-
paramagnetic iron oxide nanoparticles (SPION) via a hydrazone bond containing
linker and PEI that was used as polymer coating material to insure colloidal stability
of the magnetic nanoparticles (Kievit et al. 2011). The chemistry was achieved in
several steps. The DL was 1089 molecules of doxorubicin per nanoparticles. The
hydrazone bond of the linker can be cleaved in acidic environment which pH is
similar to that of tumors. Thus, the release of doxorubicin was expected to be
triggered by the acidic pH of tumors increasing site specicity delivery potential of
the designed nanoparticles. With cisplatin, crosslinked micelles were achieved
creating an amide bond between the copolymer PEG-block-poly(L-lysine) (PEG-b-
PLL) and cisplatin. The hydrophilicity of the API was increased and its release was
controlled in a mild reducing environment (Hou et al. 2013). A strategy that
combined two linkers was considered to link paclitaxel to functionalized meso-
porous nanoparticle surface. As illustrated in Fig. 5, one linker included a disulde
bond and the second linker included a carboxylic ester (Yuan et al. 2013). This
combination was interesting to use to achieve a DL that reached 13 % and because
it allowed release of intact paclitaxel when nanoparticles reached an environment
containing a high concentration of glutathione. The mechanisms of release caused
by glutathione that is illustrated on Fig. 5 with a model molecule that contained a
thiol group are based on the formation of benzothiophen thanks to the cleavage of
PTX O
2
O 2 O
PTX OH
S
HOOC S benzothiophen 4
S
S
DTT S COOH
DTT-SH
HS SH
DTT:
HO OH
Fig. 5 Release of paclitaxel by action of DTT-SH (simulation of glutathione action) [reprinted

with permission from Yuan et al. (2013) 2012 American Chemical Society]
the disulde bond that in turn triggers the cleavage of the ester bond releasing
paclitaxel. This double bond strategy can be used to associate paclitaxel with
nanoparticles while the release of the drug will be controlled by glutathione which
concentration is elevated inside cells.
5 Designing Nanomedecines for Co-administration

of Drugs
In the past few years, the challenge of pharmaceutical companies has been focused
on the discovery of new therapeutic compounds.
In drug discovery, the co-administration of two or more active compounds via
the preparation of co-drugs, hybrid drugs, and by co-encapsulation into nanopar-
ticles was investigated in the aim of promoting synergic effects of the APIs in
treatment of different diseases. Preparation of co-drugs and hybrid drugs requires
the formation of covalent bonds between APIs (Svartz 1942; Sozio et al. 2010;
Vrudhula et al. 2002; Yang et al. 1998; Meunier 2008) in contrast with the
co-encapsulation in nanoparticles that is addressed with parent APIs without prior
chemical modication of the drug molecule.
Two other difculties can be pointed out with co-drugs or hybrid drugs.
Similarly to prodrugs, the absence of functionalized groups on API molecules
hampers the achievement of the required chemical modication (Das et al. 2010).
The other issue is found when APIs to be associated in a single chemical entity are
of very different natures. For instance chemical linkage of two molecules that are
not soluble in the same solvent remains an established challenge.
In the case of co-encapsulation, parent drug molecules are not chemically
modied that can be an advantage in terms of development and registration. It can
be applied with any type of molecules as it does not require the presence of
functional groups in the chemical structure. A few examples of co-encapsulation of
Table 16 Co-encapsulation of different active principles

Drugs NPs-polymer References
Cyclosporine/sirolimus PLGA Jiang et al. (2009)
Cyclosporine/coenzyme Q10 MSNs Ankola et al. (2010b)
Cyclosporine/doxorubicin PACA Soma et al. (2000)
Paclitaxel/doxorubicin PLGA Cui et al. (2013)
Paclitaxel/doxorubicin PEG-PLGA Wang et al. (2011d)
Elacridar/doxorubicin LNs Wong et al. (2006)
SiRNA/doxorubicin MSNs Ma et al. (2014)
Plasmide/doxorubicin CaCO3 Chen et al. (2012)
Paclitaxel/siRNA SLNs Yu et al. (2012)
Paclitaxel/sorafenib Albumine Zhang et al. (2011)
drugs in nanoparticles are mentioned in Table 16. This table highlights the different
types of nanoparticles that were used to co-encapsulate a series of drugs. As it can
be seen from these examples, the co-encapsulation approach was often considered
with anticancer drugs where a synergistic effect was expected to improve efcacy of
treatments given by the molecules taken separately.
The co-encapsulation approach can also be viewed as an alternative solution to
resolve the problem of co-administration of molecules that have different natures
and that are difcult to attach by chemical methods. Indeed, several types of
nanoparticles are designed to co-encapsulate molecules of different nature including
API being hydrophilic and hydrophobic. Suitable nanoparticles consist of Janus
type nano-objects that include in a single particle hydrophobic and hydrophilic
domains (Xie et al. 2012). A method for the synthesis of such nanoparticles consists
in the injection of two-polymer solutions each containing an API in a microfluidic
nanoprecipitation system. The method was applied to co-encapsulate paclitaxel
(hydrophibic drug) and doxorubicin hydrochloride (hydrophilic drug) in PLGA
Janus type nanoparticles while these molecules display very different solubility
properties.
6 General Conclusion
Associating drugs with nanoparticles at an interesting DL may be challenging. It

greatly depends on the physicochemical characteristics of the drug molecule, of the
method of preparation of the nanoparticles and of the nature of the polymer
composing the nanoparticles. Simple entrapment methods have the advantage to
preserve the chemical nature of the molecule but are often limited in term of the DL
and stability of the association. Various strategies were suggested to improve the
drug payload of nanoparticles promoting the formation of complexes with
cyclodextrins for instance or synthesizing prodrugs. Strategies based on the syn-
thesis of prodrugs can be applied only on drug molecules that can undergo chemical
modications. Although not applicable on all types of drugs, this approach is
widely explored with molecules that are difcult to associate with nanoparticles by
entrapment. Several advantages were identied including nanoparticles recovered
by self-assembling of the prodrug thanks to a specic design of the molecule, the
achievement of high drug loading and integration of methodologies controlling the
release of the drug with a high precision based on the use of stimuli responsive
strategies. One obstacle that might compromise the development of this approach
on a large range of molecules may be the cost generated by the required full
registration process of the prodrug. As highlighted in this chapter, different prin-
ciples were identied to associate drugs with nanoparticles from the analysis of the
various strategies reported in the literature. It is noteworthy that several can be used
with the same drug to achieve association with different types of nanoparticles
designed according different specications consistently with their nal applications.
As a general rule, association of drugs with nanoparticles cannot be viewed as a
simple problem as is the complexity of the designed nanoparticles to be used as

drug carriers. It will certainly continue to be done on a case by case basis as it is one
of the various functionalities that are integrated in a single nanoparticle fullling
stringent specications to achieve the drug delivery mission for which it is
designed.
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Chapter 14
Drug Delivery by Polymer Nanoparticles:
The Challenge of Controlled Release
and Evaluation
Christine Charrueau and Christelle Zandanel
Abstract The controlled release of the drugs at the site of action is a key issue for
nanoparticulate carriers. The purpose of this chapter is to review the current
strategies used to control the release proles of polymer nanoparticles. Based on 12
representative drugs with hydrophobic or hydrophilic properties, the mechanisms
controlling the drug release are described, the different ways to tune the release
prole are analyzed, and the methods for evaluating drug release from nanoparticles
are discussed. In conclusion, based on the physicochemical properties of the drugs,
the types and characteristics of nanoformulations, and the route of administration,
promising tracks for tuning release proles can be proposed. Suggestions for
choosing the most appropriate methods for studying drug release are also presented.
Keywords Drug release

Nanoparticles
Hydrophobic drugs Hydrophilic

drugs Dissolution Dialysis Diffusion Pharmacokinetics Release evaluation
1 Introduction
The endpoint of nanomedicines and of approaches consisting in developing polymer

nanoparticulate carriers of drugs is, rst, to control the pharmacokinetics and biodis-
tribution of the drugs to improve their delivery to target tissues and cells, and second, to
control the release of the drugs at the site of action. While the rst events are developed
in other chapters, the present chapter deals with the challenge of drug release at the site
of action. To achieve that goal, the association between the drug and its nanoparticulate
C. Charrueau (&)
Facult de Pharmacie de lUniversit Paris Descartes, Unit de Technologies Chimiques et
Biologiques pour la Sant UTCBS, CNRS UMR8258 Inserm U1022, 4 avenue de
lObservatoire, 75270 Paris Cedex 06, France
e-mail: christine.charrueau@parisdescartes.fr
C. Zandanel
Institut Galien Paris Sud, UMR CNRS 8612, Univ. Paris Sud, Universit Paris-Saclay,
Chtenay-Malabry, France

DOI 10.1007/978-3-319-41421-8_14
440 C. Charrueau and C. Zandanel
polymer carrier needs to be stable during its distribution from the administration site to
the target site. The encapsulation and retention of the drug into its carrier can be
controlled by different formulation methods that are described in the Chap. 13 by
Zandanel and Charrueau. Once the target is reached, the carrier must release the drug
with the appropriate rate. Ideally, the release would be triggered by specic stimuli
allowing for on demand release. One of the main problems to overcome is the
phenomenon of uncontrolled burst release.
This chapter aims at explaining the current strategies employed to control the
drug release from polymer nanoparticles. This will be discussed from examples of
12 representative drugs which properties are described in this chapter. Main
physicochemical properties of the drugs to know prior considering their release
from polymer nanoparticle drug carriers are described in this chapter. The tables
provide with detailed summary of the different works published on the 12 model
drugs. Data given in these tables will be useful to present mechanisms controlling
the drug release from polymer nanoparticles, to explain how tuning the release
prole, and to discuss the methods for evaluating drug release from nanoparticles.
To conclude this chapter, strategies to achieve the control of drug release from
polymer nanoparticles will be proposed, depending on the properties of the drug,
requirements imposed by the route of administration and the influence of the type of
nanoparticles (nanocapsules, nanospheres) and composition. A critical analysis of
the different methods used to evaluate drug release from nanoparticles will be
suggested as a guideline to choose the most appropriate method.
2 Properties of 12 Drugs and Their Release

from Nanoparticles
Solubility and permeability are of pivotal importance in the fate of a drug after its
administration. Hence the Biopharmaceutical Classication System, or BCS, developed
to allow prediction of in vivo pharmacokinetic performance of drugs, has been based on
solubility and permeability measurements; the latter being determined as the extent of
oral absorption (Wu and Benet 2005). For that reason, in the present work, the 12
selected drugs were chosen for their large variety in solubility and permeability prop-
erties ranging from practically insoluble drugs with high lipophilicity to very soluble
drugs with high hydrophilicity, as depicted in Fig. 1. A large range of molecular weights
was also represented (for details, see the Chap. 13 by Zandanel and Charrueau).
Insulin which solubility is pH-dependent is not indicated on the Fig. 1.
In the context of nanomedicine, the initial solubility and permeability properties
of the drugs could be modulated by their encapsulation into nanoparticles, the latter
being able to promote drug absorption and delivery to cells through various
endocytosis mechanisms (see Chap. 10 by Hillaireau).
On the basis of this drug selection, the literature has been studied in order to gather
information about the release of each drug from nanoparticulate formulations. The
Tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 present for each nanoparticle type, the
14 Drug Delivery by Polymer Nanoparticles: The Challenge of 441
Positive Negative
(Lipophilicity) (Hydrophylicity)
Permeability (log P)
SiRNA
Ciprofloxacin (0.28)
Doxorubicin HCl
Cisplatin (-2.19)
Acyclovir (-1.56)
Amphotericin B (0.80)
Paclitaxel (3.00)
Dexamethasone (1.83)
Ibuprofen (3.97)
Cyclosporine A (4.10)
Sirolimus (4.85)
Doxorubicin (1.27)
Water solubility (ml/g) (European Pharmacopoeia)

Practically Very slightly Slightly Sparingly Soluble Freely soluble Very
insoluble soluble soluble soluble (10 - 30) (1 - 10) soluble
(>10,000) (1,000 - 10,000) (100 - 1,000) (30 - 100) (<1)
Fig. 1 Water solubility and permeability characteristics of the selected drugs. Lower panel the water
solubility is expressed according to the European Pharmacopoeia in descriptive terms from practically
insoluble to very soluble corresponding to water volumes necessary to dissolve 1 g of drug and
indicated in brackets (ml/g). Upper panel the permeability is expressed as logP ranging from positive
values characteristic of lipophilic drugs to negative values characteristic of hydrophilic drugs. When
documented, the logP value is indicated in brackets after each drug name
release characteristics, i.e., methods of study, mechanisms, and prole, along with the
carrier size and the administration route. The encapsulation parameters detailed in the
Chap. 13 by Zandanel and Charrueau are mentioned as well, i.e., type E for entrapment
or A for adsorption, drug loading DL, and encapsulation efciency EE.
As for methods of study, they are classied into in vitro methods including
dissolution and dialysis methods, in cellulo methods using either cellular uptake or
biological effect evaluation, ex vivo methods determining permeation across bio-
logical membranes, and nally in vivo methods studying either pharmacokinetics in
healthy animals, or a therapeutic effect in diseased animals.
Mechanisms of release are mentioned when documented. They mainly consist in
diffusion of the drug, degradation of the nanoparticles, and release triggered by
various stimuli.
Table 1 Overview of doxorubicin release from NPs
442
Encapsulation Size-administration Release References

Type DL EE route Method of study Mechanism Prole
(%) (%)
core-shell NPs E 95 186243 nm-IV Dissolution in cell Diffusion of the 100 % at 60 min for De Verdire
culture medium at drug/degradation of the PIBCA NPs et al. (1997)
37 C polymer 100 % at 400 min for
PIHCA NPs
E *301 nm for NPs PK in rats Rapid uptake of AEP-NPs by Distribution rate: faster for Alhareth
prepared by macrophages due to loop AEP-NPs and slower for et al. (2012)
RREP/*196 nm for conformation of surface RREP-NPs
NPs prepared by dextran chains Signicant increase of
AEP-IV Lower activation of the AUC0 and total
complement by RREP-NPs due clearance for RREP-NPs
to dense brush conformation of versus AEP-NPs and free
the surface dextran chains drug
MNPs A >20 160190 nm-IV Dissolution in 0.1 Diffusion At 48 h: 49 % at pH 5.0 Chen et al.
citrate buffer at pH Low release below volume and 27 % at pH 7.4 from (2013)
5.0 and 7.4 at 37 phase transition temperature MNPs; 85 % at pH 5.0 and
C (VPTT) of the polymer coating 13 % at pH 7.4 from
PK in mice (pH 7.4) and enhanced release pH-sensitive MNPs
Antitumor effect in above VPTT (pH 5.0) AUC012h increased
mice bearing S-180 1.6-fold for MNPs and
subcutaneous 1.8-fold for pH-sensitive
tumors MNPs versus free drug
Signicant decrease of the
tumor weight: drug <MNPs
<pH-sensitive MNPs
(continued)
C. Charrueau and C. Zandanel
Table 1 (continued)
14

(%) (%)
A 14 *100 nm-IV Dialysis in PBS at Decreasing interaction between At 24 h, 18 % at pH 4.4, Gu et al.
pH 4.45.46.4 negatively charged MNPs and 15 % at pH 5.4, 10 % at (2012)
and 7.4 at 37 C positively charged drug with pH 6.4, 6.7 % at pH 7.4
decreasing pH
E 4 22 *46 nm-IV Dialysis in PBS at Diffusion At 192 h: 81 % from He et al.
pH 7.4 at 37 C Compact structure of the silicon COOH-MNPs, 61 % from (2011)
matrix affected by surface PO4-MNPs, 41 % from
modications OH-MNPs, 38 % from
NH2-MNPs, 22 % from
PEGMNPs
E 4 85 30 nm (micelles) Dialysis in PBS at Faster release through creation At 48 h: 57 % for Yuan et al.
93* 3675 nm (NPs)-ns pH 7.4 at 37 C of a mesoporous structure with chitosanstearic acid (2012)
(buffer refreshed SiO2 deposition that increases micelles, 8591 % for
regularly) specic surface area of NPs chitosanstearic acid-SiO2
versus micelles NPs
A 24 62 *64 nm-intratumor Release (dialysis? Electrostatic interactions At 8 h: *40 % at pH 7.4, Xie et al.
injection dissolution?) in between the drug and carboxyl *60 % at pH 6.5, *90 % (2013)
PBS at pH 7.4 and groups decreased at acidic pH at pH 5.0
Drug Delivery by Polymer Nanoparticles: The Challenge of
acetate buffer at pH pH-dependent release and

5.0 and 6.5 at 37 diffusion
C
MagNPs A 44 *150230 nm-ns Dissolution in Diffusion and chemically At 24 h: *40 % at pH 7.4 Anirudhan
95 phosphate citrate controlled breakage of and *90 % at pH 5.0 and Sandeep
buffer solutions at hydrogen bonds and (2012)
pH 5.0 and 7.4 at electrostatic attraction between
37 C drug and NPs
(continued)
443
Table 1 (continued)
444

(%) (%)
A 10 90 *5 nm-IV Dissolution in PBS Lower electrostatic interaction At pH 4.5: 100 % at 1 h Pilapong
at pH 7.4 and between negatively charged At pH 7.4: *40 % at 5 h et al. (2013)
acetic buffer at pH NPs and positively charged Signicant increase in cell
4.5 at 37 C drug at acidic pH toxicity
Cytotoxicity pH-dependent release and
against diffusion
doxo-resistant
(K562/ADR)
human leukemia
cells
E 2 35 *100 nm-IV Dialysis in PBS Diffusion of the drug At 2 h: 99 % from free Li et al.
27 85 0.01 M at pH 7.4 at Polymer matrix degradation drug, *27 % from NPs (2011)
37 C (*80 % at 120 h)
Toxicity on human Cytotoxic effect prolonged
breast cancer by NPs
MCF-7 cells
E 69 *3060 nm-IV Dissolution in PBS Diffusion and pH-dependent 2130 % at 12 h, 6182 % Akbarzadeh
75 at pH 7.4 or acetate gelation of the matrix due to at 2 days at pH 7.4 et al. (2012)
buffer at pH 5.8 at hydrolysis of the polymer at Enhanced release at pH 5.8
37 to 40 C acidic pH
INPs A 71 *1520 nm-IV Dialysis in PBS at Faster release in SAB due to At 10 h: *98 % in SAB Pooja et al.
pH 7.4 and sodium protonation of the drug that and *84 % in PBS (2014)
acetate buffer increases hydrophilicity and
(SAB) at pH 4.5 at solubility at lower pH
37 C in dark
(continued)
Table 1 (continued)
14

(%) (%)
Self-assemblies E 10 20 *110 nm-ns Dialysis in distilled Diffusion 100 % at 24 h Lehtovaara
40 25* water at 37 C et al. (2012)
A 25 * *273 nm-ns Dissolution in PBS Hydrolysis of the polymer at At 96 h: *20 % at pH 7.4 Li et al.
0.2 M at pH 7.4 acidic pH and NPs degradation and *80 % at pH 5.0 (2013a)
and sodium acetate
buffer 0.2 M at pH
5.0 at 37 C
E 32 * Release not studied Theodossiou
et al. (2013)
PEC E 22 98 *140 nm-IV Dialysis in PBS at Decreased electrostatic At 12 h (60 h): *22 % Lv et al.
pH 7.46.85.5 at interactions between the (*24 %) at pH 7.4, (2013)
37 C cationic drug and anionic *29 % (*30 %) at pH 6.8
In vivo antitumor polymer with decreased pH and *60 % (*65 %) at
efciency in pH-triggered release pH 5.5
subcutaneous Signicantly lower tumor
non-small cell lung volume versus free drug
cancer A549
xenograft in mice
E 6 54 *160 nm-IV Dialysis in Destruction of the core-shell At 144 h: >60 % at pH 5.8, Guo et al.
16 70 PBS + 0.2 % SDS structure at pH close to the *53 % at pH 6.5 and (2013b)
at pH 5.86.57.4 isoelectric point (<5.8) of the *38 % at pH 7.4
at 37 C polymer conjugate increased
release at acidic pH
E 47 *241 nm-IV Dialysis in PBS at pH-triggered release: acidic pH At 9 days: <10 % at pH Guo et al.
pH 4.05.06.0 hydrazine bonds 7.4, *27 % at pH 6.0, (2013a)
7.4 at 37 C cleavage + increased drug *45 % at pH 5.0, *60 %
solubility + decreased at pH 4.0
(continued)
445
Table 1 (continued)
446

(%) (%)
Uptake by electrostatic forces between the signicant increase of the
hepatoma HepG2 drug and alginate drug intracellular
cells concentration with NPs
PK in mice versus free drug
Antitumor efcacy 11.8-fold increase of
in ectopic AUC0 and 3.2-fold
tumor-bearing mice increase of half-life in the
liver with NPs versus free
drug
Tumor growth inhibition
rate *79 % with NPs
versus *52 % with free
drug
*Doxorubicin base (all other studies cited use doxorubicin hydrochloride)
Table 2 Overview of paclitaxel release from NPs
14

Type DL EE route Method of Mechanism Prole
(%) (%) study
PNPs E 67 95 *40 nm-IV Dialysis in Diffusion through the 40 % at 12 h Jger et al.
water at 37 C matrix (+hydrolysis of 90 % at 120 h (2012)
the polymers)
E 6 94 *49 nm-IV Dialysis in Diffusion 38 % at 3 h Jiang et al.
1 M sodium 80 % at 24 h (2011)
salicylate at Extended elimination
37 C half-life + plasma
PK in rats AUC increased by
Antitumor 7.17-fold
effect in Median survival time
tumor-bearing signicantly increased
mice
E 58 250 nm-IV Dialysis in pH-dependent erosion At 60 h: 60 % at pH Lee et al.
0.1 M acetic of NPs 5.2 and 30 % at pH (2011)
acetate buffer 7.4
pH 5.2 or in Signicant reversal of
PBS pH 7.4 chemoresistance
Release in
MDR breast
cancer cells
E 35 87 130 nm-oral Dissolution in Dissolution of the At 9 days: 59 % from Lv et al.
38 83 PBS + 0.1 % drug and chitosan NPs and (2011)
Tween 80 at biodegradation of the 84 % from
37 C (buffer polymer quaternized chitosan
refreshed NPs
regularly)
(continued)
447
Table 2 (continued)
448

(%) (%) study
Intracellular At 24 h: *80 % with
drug uptake in chitosan NPs and
Lewis lung *40 % with
carcinoma quaternized chitosan
(LLC) cells NPs
In vivo tumor Relative tumor
growth volume decreased by
inhibition in 3
LLC
tumor-bearing
mice
E 66 355 nm-IV Dissolution in Diffusion At 20 days: 55 % Parveen
PBS + 0.1 % from PLGA NPs, and Sahoo
Tween 80 at 50 % from (2011)
37 C (buffer PLGA-chitosan NPs
refreshed and 40 % from
regularly) PLGA-chitosanPEG
PK in mice NPs
Prolonged circulation
up to 72 h
E 1.3 200300 nm-oral Intracellular Diffusion Intracellular % Roger et al.
uptake by increased by 1.5 at (2012)
Caco2 cells in 2 h and by 3.54.0 at
HBSS at 37 C 6 h with folic acid
functionalized NPs
E 8.5 94 640 nm (pH 7.5)-IV
(continued)
Table 2 (continued)
14

(%) (%) study
Dialysis in Diffusion of the drug 52 % at 12 h, 68 % at Xu et al.
PBS pH 7.4 at and swelling of the 48 h and 88 % at (2012)
37 C polymer 7 days
E 1 99 *224 nm-IV Dialysis in Mainly diffusion 53 % at 12 h and 64 Ren et al.
PBS 0.01 M (partial polymer 71 % at 96 h (2011)
pH 7.4 at 37 degradation)
C
E *204302 nm-oral Dissolution in No hydrolysis of the No release in SGF; in Ageros
SGF at pH 1.2 poly(anhydride) SIF, slow release for et al.
(2 h) followed polymer at acidic pH; 67 h, followed by (2009)
by SIF (24 h) hydrolysis at pH 7.5 faster release from
at pH Efflux phenomenon 11 h, to complete
7.5 + 1 % reduced by release at 22 h
polysorbate cyclodextrin-polymer Permeability
80, at 37 C NPs through increased by 12 times
Permeation bioadhesive with NPs versus
across rat interactions Taxol
jejunum in
Ussing
chambers in
PBS at pH 7.4
at 37 C
E 75 *177188 nm-oral Permeation Inhibition of active Permeability Zabaleta
88 across rat secretory transport by increased by 37 et al.
jejunum in pegylated NPs times with pegylated (2012)
Ussing NPs versus Taxol
(continued)
449
Table 2 (continued)
450

(%) (%) study
chambers in Reduced impact of the Relative oral
PBS at pH 7.4 efflux pump P-gp by bioavailability of 70,
at 37 C pegylated NPs 40, 16 and 9 % with
PK in rats PEG 2000, PEG
6000, PEG 10000
pegylated NPs, and
non-pegylated NPs,
respectively
core-shell NPs E 6 *125250 nm-IV Dialysis in Diffusion 3852 % at 2 days Lee et al.
13 PBS 0.01 M and 8092 % at (2012)
pH 6 days
7.4 + 0.1 % Signicant decrease
Tween 80 of the tumor growth
In vivo tumor
growth
inhibition in
HTC116
tumor-bearing
mice
E 94 190 nm-IV Dissolution in Swelling of the casein 2.5 % at 25 h, 25 % Narayanan
96 PBS pH 7.4 at shell-diffusion of the at 50 h and 28 % at et al.
37 C drug-erosion of the 10 days (2014)
PK in rats PLGA core Signicant increase in
Tmax, AUC and mean
residence time versus
drug in solution
(continued)
Table 2 (continued)
14

(%) (%) study
E 41 72 *106 nm-IV Dialysis in Diffusion At 10 days: *7.5 % Cao et al.
51 76 PBS + 0.5 % for NPs with 44 % (2014)
Tween 80 DL, *12.5 % for
NPs with 51 % DL
At 40 days: *20 %
for NPs with 44 %
DL, *32.5 % for
NPs with 51 % DL
MagNPs A 30 ns-IV Release not Hua et al.
studied (2010)
Self-assemblies E *94153 nm-IV In vivo tumor Tumor volume Caron et al.
growth reduction by 3545 % (2013)
inhibition in versus at day 30
human lung saline
carcinoma
A549
tumor-bearing
mice
451
Table 3 Overview of ibuprofen release from NPs
452

(%) (%)
PNPs E 300400 nm-oral Dissolution in puried Greater surface Dissolution rate Mansouri et al.
water at 37 C (USP) area exposed to increased by (2011)
dissolution 2.33
medium and
reduced
cristallinity
E 4.5 50 oral Release not studied Galindo-Rodrguez
7.7 86 et al. (2005)
8.8 88
E ns-oral Diffusion in distilled Diffusion *75 % at 1 h Bonelli et al. (2012)
water at 37 C followed by and 100 % at
Release in cultured cells degradation of 2 h 30 mins
MKN-45 the polymer 0.3 % at 2 h and
0.4 % at 24 h
intracellular
E 143159 nm Dissolution in pH 7.4 Without 2883 % at Haroun et al.
without phosphate crosslinking: 1 min depending (2012)
crosslinking buffer + 0.1 % Tween diffusion on the
Oral at 37 C through monomers used
12161 nm with flexible chains 8 % at 30 min
crosslinking With followed by
crosslinking: slow release
controlled within 5 h
release by
biodegradation
of the polymer
(continued)
Table 3 (continued)
14

(%) (%)
Cubic E 8.3 >85 238 nm-oral Dialysis in SGF pH 1.2 Diffusion 4 % from NPs Dian et al. (2013)
NPs and SIF pH 7.4 at 37 C versus 34 %
PK in Beagle dogs from pure drug
at 2 h at pH 1.2
56 % from NPs
versus 95 %
from pure drug
at 6 h at pH 7.4
Relative oral
bioavailability
increased to
222 % with NPs
core- E 8 56 2560 nm-oral Dialysis in PBS at pH Diffusion Burst release Jiang et al. (2005)
shell 22 72 1.0, 5.8 and 7.4 at 37 C within 2 h
NPs followed by
continuous slow
release
At 40 h: 60 % at
pH 1.0, 80 % at
pH 5.8, 90 % at
pH 7.4
MNPs A 22 *100 nm-oral Dissolution from MNPs Low diffusion *23 % at 1 h Chen and Zhu
pressed into disks through orderly and *90 % at (2012)
(3 MPa) in pH 6.8 and aggregated 24 h at both pH
7.4 at 37 C chitosan at pH for MNPs
7.4 without chitosan
(continued)
453
Table 3 (continued)
454

(%) (%)
High diffusion *20 % at 1 h at
through both pH; at 24 h
chitosan in gel 65 % at pH 6.8
state at pH 6.8 and 35 % at pH
7.4 for MNPs
with chitosan
A 21 500, 750, Static release from NPs Diffusion At 10 h in SIF: Xu et al. (2009)
24 950 nm-oral in pressed disks in SGF 84, 75, 56 % for
pH 1.2, SIF pH 7.4 and 500, 750,
SBF pH 7.4 950 nm NPs
60 %: at 11 h in
SIF, 31 h in
SBF; <12 % at
108 h in SGF
MagNPs A 500 nm-oral Dissolution of MNPs Diffusion 2.5 % Xing et al. (2012)
compressed into tablets Eudragit: 7 %
(4 MPa) coated with at 2 h in SGF,
Eudragit S100, in SGF 50 % at 24 h in
pH 1.2 for 2 h and then SIF
in SIF pH 7.4 for 24 h 10 % Eudragit:
4 % at 2 h in
SGF, 20 % at
24 h in SIF
Table 4 Overview of amphotericin B release from NPs
14

(%) (%)
PNPs E 54 *86153 nm-IV Dissolution (50 g/mL) in Release of active *7585 % following sample Van de
63 phosphate-buffered 5 % monomeric drug dilution; 92100 % at 30 min Ven et al.
glucose + 0.082 % from NPs versus Release of monomeric drug along (2012)
sodium deoxycholate at aggregates from with decreased aggregation
37 C AmBisome tenfold increase in antifungal
Monomeric drug release activity for NPs versus free drug;
(0.1 g/mL) monitored by signicant increase of antifungal
the absorbance change at activity for NPs versus
487 nm AmBisome
Antifungal assay on A. NPs about 2 times more efcacious
fumigatus than AmBisome
Efcacy in acute A.
fumigatus mouse model
E 56* *69 nm-IV PK in mice Biodegradation of Signicant increase in brain Xu et al.
Effect against murine the polymer concentration with NPs versus (2011)
cryptoccocal meningitis liposomes
Higher survival rate with NPs
versus liposomes and free drug
E 49 *213 nm-IV Dialysis in PBS + 0.5 % Diffusion of drug At 4 h: initial burst between 25 and Nahar
DMSO at pH 7.4 at 37 C and matrix erosion 39 %; at 196 h: 7490 % et al.
(2008)
PEC E 1 90 *136 nm-IV Dialysis in PBS/methanol Delayed release due At 48 h: 5758 616859 % for Ribeiro
11 92# (60/40) at room to strong interaction NPs with 117421 % DL, et al.
temperature between the drug and respectively, versus 100 % at 12 h (2014)
polymers for free drug
(continued)
455
Table 4 (continued)
456

(%) (%)
E 18 70 *200 nm-topical Dialysis in PBS + 1 % Diffusion and *3040 % at 2 h and *80 % at Zhou et al.
22 81 Tween 80 at pH 7.4 at polymer erosion 11 h from NPs versus 100 % at 4 h (2013)
37 C from free drug
Ocular PK in rabbits 1.95-fold increase of AUC,
1.50-fold increase of half-life, and
signicant increase of mean
residence time
SLN 235 nm-ns PK in rats Matrix degradation 2.15-fold higher AUC0 with NPs Patel and
versus free drug in solution in Patravale
DMSO; relative (2011)
bioavailability = 216 %
E 59 Release not studied Tan and
Billa
(2014)
LN E 76 *7395 nm-IV Antifungal activity on C. Higher afnity of Higher antifungal activity for NPs Jung et al.
albicans and A. fumigatus NPs to fungal cell than AmBisome against C. (2009)
PK in rats membranes albicans
Antifungal efcacy in A. Higher AUC024h, Cmax and t1/2 for
fumigatus-infected mice PEG NPs than AmBisome
100 % survival rate at 14 days with
PEG NPs versus 90 % with
AmBisome
* Amphotericin B deoxycholate; # Amphotericin B sulfate
Table 5 Overview of cyclosporin A release from NPs
14

(%) (%)
PNPs E 10 43 107119 nm PLGA Dialysis in Diffusion-cum-degradation At 48 h: *53 % Ankola
30 50 NPs/ 136-139 nm phosphate process from EL14 NPs et al.
EL14 NPs-oral buffer +5 % versus *25 % (2010)
Labrasol at pH 7.4 from PLGA NPs
at 37 C (buffer *7 %/day up to
completely replaced 9 days from EL14
at each sampling) NPs
PK in rats *4 %/day up to
18 days from
PLGA NPs
Relative
bioavailability to
Neoral: 126 for
EL14 NPs and
120 for PLGA
NPs
E 69 89 300400 nm-ns Dialysis in 0.1 M Diffusion At 21 days: *35 Lee et al.
94 phosphate 50 % from (2002)
buffer +0.01 % PLGA/PLA NPs,

Tween 80 at 37 C and *2040 %
from PLA NPs
E 10 *42273 nm-ns Dissolution in Diffusion burst release Burst effect from 9 Rahman
96 0.2 M phosphate Diffusion + erosion of the to 36 % et al.
buffer +0.1 % polymer sustained (correlated with (2010)
sodium lauryl release EE) followed by
sulfate (sink
(continued)
457
Table 5 (continued)
458

(%) (%)
conditions) and sustained release
0.02 % sodium during 7 days
azide (antimicrobial Dissolution
agent) at pH 7.4 at efciency from 53
37 C for 7 days to 84 %
E 85 *156 Dissolution in Diffusion At 24 h: *45 % Hermans
105 314 nm-topical PBS polysorbate in PBS, *60 % in et al.
80 (0.01 or 0.28 %) PBS + 0.01 % (2012)
at pH 7.4 at 32 C polysorbate 80
*6090 % at 1 h
in PBS + 0.28 %
polysorbate 80
Cubic E 85 *180 nm-oral Dialysis in Enhanced bioavailability <5 % at 12 h Lai et al.
NPs water + 0.2 % due to facilitated absorption Increased Cmax (2010)
sodium lauryl of NPs rather than and AUC0 for
sulfate at 37 C improved release NPs versus
PK in dogs versus Neoral; relative
Neoral oral bioavailability
*178 % for NPs
versus Neoral
MNPs A Release not studied Lodha
et al.
(2012)
MagNPs 99 *213 nm-IV Saturation solubility Increased surface area of 5.7-fold increase Nakarani
in phosphate buffer the NPs of saturation et al.
pH 7.4 at 37 C (2010)
(continued)
Table 5 (continued)
14

(%) (%)
PK in rats solubility for NPs
versus free drug
Increased half-life
and AUC
following IV
administration for
NPs versus free
drug and
Sandimmune
NCs E 4.5 72 *202220 nm-ns PK in rats Diffusion of drug and Signicantly Park et al.
9.4 92 partial matrix erosion or higher AUC with (2013)
swelling of the NCs NCs versus
SEDDS-core
solution; blood
drug concentration
maintained above
500 ng/mL for
1420 h with NCs
versus 7 h with
SEDDS-core
solution
E 83 *148219 nm Dissolution in Increased solubility of the Initial burst Aksungur
95 uncoated NPs/ simulated lachrymal drug through the formation release followed et al.
*393 nm fluid (SLF) at 32 C of amorphous structure by slow release: (2011)
Carbopol-coated from crystals during 7590 % at 24 h
NPs-oral lyophilization of the NPs
(continued)
459
Table 5 (continued)
460

(%) (%)
Diffusion in Franz 4147 % diffusion
cells in SLF at 32 at 24 h for NPs;
C no difference
Drug release in versus Restasis
rabbit tear lm A (emulsion)
Highest AUC024h
with PLGA:
EudragitRL
(75:25) NPs
PEC E *100300 nm-oral PK in rats Higher drug dissolution At 10 mg kg1: Cheng
Promotion of paracellular two- to sixfold et al.
drug transport by NPs increase in blood (2006)
level versus free
drug
At 7.5 mg kg1:
similar blood level
as after Neoral
administration
SLN E 95 *204 nm-oral Diffusion in Franz Degradation of the lipid At 24 h: not Karavana
cells across cow matrix detectable in SSF; et al.
buccal mucosa in 72 % of the dose (2012)
simulated saliva in the mucosa
fluid (SSF) at pH Signicant
6.75 and methanol improvement of
(60:40) at 37 C; ulcer healing with
NPs versus control
(continued)
Table 5 (continued)
14

(%) (%)
drug retention in the
mucosa
Efcacy on oral
ulcer model in
rabbits
E 95 *226 nm-topical Dissolution in Enzymatic degradation of *28 % at 90 min Gke
0.01 M borate/boric the lipid matrix with enzymes (no et al.
acid buffer release without (2008)
lipase/colipase at enzymes)
pH 7.4 at 37 C Permeation close
Permeation across to the limit of
rabbit cornea detection
epithelial At 24 h: *24 %
(RCE) cells in permeation across
HBSS at pH 7.4 at the cornea and
37 C *15 %
Diffusion through penetration inside
pig cornea in the cornea
Valia-Chien
chambers in Krebs
buffer
461
Table 6 Overview of sirolimus release from NPs
462

(%) (%)
PNPs E 3 6 *300 nm-topical Dialysis in NaCl Drug diffusion and At 12 h: *50 % from Yuan et al.
29 89 0.9 % at 37 C for matrix erosion cholesterol-modied (2008)
8 days Prolonged release chitosan PLA NPs versus
Immunosuppressive attributed to *85 % from NPs
effect in a rabbit hydrophobicity of without PLA
model of corneal PLA At 8 days: *90 % from
transplantation cholesterol-modied
chitosan PLA NPs
Median graft survival
time: 27 days for NPs
versus 24 days for free
drug (NS)
E 0.1 25 *163 Dialysis in Drug diffusion and *100 % at 5 h for PEO Zweers
0.4 33 193 nm-local PBS + 0.02 % polymer degradation PLGA NPs et al.
NaN3 + 1 mM SDS Linear release over (2006)
at 37 C 25 days from NPs treated
with 14 % gelatin, and
50 days from NPs treated
with 3 % gelatin
E *250 nm-oral Dissolution by the Enhanced At 30 min: *95 % from Kim et al.
USP paddle method supersaturation and NPs versus *5 % from (2011)
in degassed water at increased specic free drug
37 C surface area resulting Peak plasma
PK in rats from reduced particle concentration increased
size by 18 and AUC012h
increased by 15 versus
free drug
(continued)
Table 6 (continued)
14

(%) (%)
E *38 nm-IV PK in rats Increased aqueous threefold increase of Woo et al.
Tumor growth delay solubility of the drug absolute bioavailability (2012)
in A549 cell with NPs versus free drug
xenograft model in At 41 days: 1.5-fold
mice increase of tumor volume
with NPs versus ninefold
increase in control group
E 1.6 80 *274 nm-IV Dissolution in PBS Diffusion *18 % at 24 h and Acharya
0.01 M + 0.1 % *50 % at 15 days and Sahoo
Tween 80 at pH 7.4 (2011)
at 37 C
A *80 nm-oral Dissolution in Diffusion At 2 h: *25 % at pH 7.4 Bisht et al.
phosphate buffer at and *65 % at pH 5.0; (2008)
pH 7.4 and pH 5.0 at *100 % at 7 days
37 C Increase of AUC0 by
PK in mice 2.3 for NPs versus
Efcacy in pancreatic Rapamune
Panc198 cancer Signicant growth

xenograft in mice retardation of tumors
with NPs versus
untreated control; same
effect as Rapamune
E 0.05 81 *165500 nm-IV Dissolution in Diffusion of the drug No burst release Haddadi
phosphate buffer: and biodegradation of *3.2 % per 24 h et al.
ethanol (9:1) at pH the polymer *22 % within 7 days (2008)
7.6 at 37 C
(continued)
463
Table 6 (continued)
464

(%) (%)
MNPs A *293 nm-local Dialysis in 0.9 % Fusion of the outer *97 % drug retention at Cyrus
NaCl + 0.2 mg/ml lipid membrane of the 3 days et al.
human serum NPs with the Signicant decrease of (2008)
albumin + 0.05 % membrane of the stenosis levels with NPs
NaN3 at pH 6.0 at target cell versus controls
37 C
Effect in a rabbit
femoral artery model
of stenosis
Table 7 Overview of dexamethasone release from NPs
14

(%) (%)
PNPs E 59 *140298 nm-IV Transport study Modication of NP binding More than tenfold Ali et al. (2013)
89 across human to proteins and altered enhanced apparent
placental interactions with efflux permeability with
choriocarcinom transporters NPs versus free
BeWo b30 cells drug
Drug and NP
permeability
inversely correlated
with particle size
E 2 11 *163 Dialysis in Drug diffusion and polymer *100 % at 5 h for Zweers et al.
29 100 193 nm-local PBS +0.02 % degradation PEOPLGA NPs (2006)
NaN3 + 1 mM SDS Linear release over
at 37 C 8 days from NPs
treated with 0.5 %
gelatin or albumin,
and 17 days from
NPs treated with
14 % gelatin
core-shell NPs E 28 25 *150500 nm-IV Dissolution in PBS Strong entrapment of drug No release from Fratoddi et al.
87 88 at pH 7.4 at 37 C in polymer chains polymer NPs; (2012)
Apoptosis inhibition absence of release release after 210 h
on HeLa cells Disruption of from copolymer
nanomorphology of NPs
copolymer NPs at pH 7.4 Same cytoprotective
drug release effect for NPs as for
free drug
(continued)
465
Table 7 (continued)
466

(%) (%)
MNPs A <1.5 ns-local Dialysis in water Superposition of Fickian *721 g drug/ de Matos et al.
with NPs or disks and of case II-type transport mg sample at 7 h (2013)
(2 mm mechanisms due to release for composite NPs
thickness 5.5 mm at near-surface regions and
diameter) to poly(-caprolactone)
surface skin erosion
Self-assemblies E *1020 nm-nasal Release not studied Kenyon et al.
(2013)
PEC E -* ns-ns Interrupted dialysis Intermolecular interactions At 1 h: >70 % in Uccello-Barretta
in water at 37 C between drug and the NPs dispersion et al. (2014)
(release dened by macromolecules + diffusion medium (within the
quantitative NMR) dialysis bag);
Dynamic dialysis in *10 % in the
the same conditions release medium
At 24 h: *60
80 % in the release
medium
Retention of a
signicant drug
fraction inside the
nanostructures
* Dexamethasone phosphate disodium salt
Table 8 Overview of ciprofloxacin release from NPs
14

(%) (%) study
PEC E 80 -* 200400 nm-ns Saturation Amorphous state and 0.30 mg/ml for nanoplex Cheow
solubility in nanoscale size versus 0.14 mg/ml for drug and
PBS at 37 C increased solubility and crystals Hadinoto
Dialysis in dissolution rate At 3 h: *100 % (2012)
PBS at 37 C dissolution for nanoplex
In vitro versus *66 % for drug
minimum crystals (dissolution
inhibitory comparable to drug
concentration hydrochloride)
on 0.25 g/ml for nanoplex
P. aeruginosa versus 0.13 g/ml for
native drug
Nanogels E 39 *151 nm-ns Dialysis in Higher antibacterial 927 % at 0.5 h Zhao et al.
85 PBS at pH 7.4 activity in relation with 95 % at 24 h (2013)
at 37 C improved intracellular Internalization into cells
Cellular uptake uptake observed by fluorescence
of NPs by Antibacterial activity
SMMC-7221 increased by 2 times for
liver carcinoma NPs versus free drug

cells
Antibacterial
activity against
E. coli
(continued)
467
Table 8 (continued)
468

(%) (%) study
SLN E 28 *161 nm-ns Dialysis in Sustained release due *30 % at 1 h Shah et al.
39* PBS at pH 6.8 to reduced mobility of *80 % at 15 h (2012
at 37 C the drug in solidied
state of the binary
lipids
LP E 4 *300 nm - Dialysis in ns *20 % at 5 days Cheow
pulmonary PBS at 37 C and
Hadinoto
(2011)
*Ciprofloxacin hydrochloride
Table 9 Overview of acyclovir release from NPs
14

(%) (%)
PNPs E 14 280300 nm-IV Dialysis in PBS at pH 7.4 Surface drug release At 1 h: initial burst release Kamel
43 at 37 C followed by diffusion of 1337 % et al.
PK in rabbits Slower release by Slower exponential release (2009)
biodegradable NPs over 48 h up to 6080 %
leading to lower AUC0 increased by 16
clearance 21-fold and mean plasma
residence time increased
by 1829-fold with NPs
versus commercial
solution
No inflammation nor
phlebitis with NPs versus
commercial solution
Nanogels E 380 nm versus Ex vivo permeation study Initial fast partitioning of Biphasic diffusion prole: Hasanovic
703 nm-topical of NPs at pH 5.4 using drug close to the NP at 8 h, *20 % and et al.
Franz-type diffusion cells surface *35 % diffusion from (2009)
and porcine abdominal Slower diffusion of drug 380 and 703 nm NPs
skin; receptor phase entrapped inside the NP respectively; at 48 h, *45
0.012 M phosphate buffer Better permeation from and *70 % diffusion from
at pH 7.4 at 32 C larger NP stronger 380 nm and 703 nm NPs
interaction between NP respectively, versus almost
surface charge density no diffusion from control
and anionic components aqueous solution over 48 h
of the epithelial cell
surface
469
Table 10 Overview of insulin release from NPs
470
Encapsulation Size-administration route Release References

Type DL EE Method of study Mechanism Prole
(%) (%)
PNPs A 2 Oral Release not studied Leobandung et al.
(2002)
NCs E 60* Oral Release not studied Cournarie et al.
(2004)
E 55 *220 nmoral and PD: glycemia ns Subcutaneously: Damg et al.
subcutaneous measurement in prolongation of (1988)
diabetic and normal hypoglycemic effect
rats with NCs versus
insulin
Oral: NCs decreased
fasted glycemia by
5060 % in diabetic
rats at day 2 at 12.5
50 U/kg, and
decreased fed
glycemial by 25 % at
100 U/kg
E 98# *297 nm-administration Stability toward ns Protection of NCs Michel et al.
at various sites in the GI digestive enzymes at against proteolysis (1991)
tract pH 1.4 (pepsin) or 7.9 from pepsin,
(-chymotrypsin and -chymotrypsin and
trypsin) for 30 min at trypsin versus
37C non-encapsulated
PD: fasted glycemia insulin
measurement in Glycemia decreased
streptozotocin-induced from day 2 by 65 %
diabetic rats (ileum), 59 %
(stomach), 52 %
(continued)
Table 10 (continued)
14

(%) (%)
(duodenum and
jejunum), 34 %
(colon) versus no
effect with
non-encapsulated
insulin, and for
18 days (ileum and
jejunum), 15 days
(stomach and
duodenum), 13 days
(colon)
E 90 *250 nm-intragastric PD: glycemia ns Reduction of glycemia Aboubakar et al.
measurement in to normal level with a (1999)
streptozotocin-induced lag time period of
diabetic rats and in 2 days and a
alloxan-induced prolonged effect over
diabetic dogs 20 days
E 100 *60300 nm-intragastric Stability in ns Degradation of the Pinto-Alphandary

reconstituted intestinal NCs et al. (2003)
medium for 5 h at 37 Uptake of the NCs in
C M-cell-free epithelium
Visualization of the of the ileum;
NCs in the intestinal degradation in
tissue by fluorescence M-cell-containing
microscopy and TEM epithelium
90 min after
administration in rats
471
E *121134 nm-oral
(continued)
472

(%) (%)
Dialysis in PBS at pH Initial burst due to At 2 h: 28 % from Zhang et al.
7.4 at 37 C release of drug PLGA NPs versus (2012)
PD: glycemia localized at the NP 18 % from
measurement in surface chitosan-coated
diabetic rats Enhanced intestinal PLGA NPs
absorption due to At 20 h: 40 % for
positive charge both types of NPs
behavior of Signicant
chitosan-coated hypoglycemic effect
PLGA NPs of NPs from 4 to 12 h
post-oral
administration versus
insulin control
solution
7.6 and 10.5 %
pharmacological
availability relative to
SC injection for
PLGA NPs and
chitosan-coated
PLGA NPs,
respectively
Self-assemblies E 15 75 *250 nm-oral PD/PK in diabetic rats Adhesion to the Signicant Sung et al. (2012)
mucosa; opening of hypoglycemic effect
tight junctions; of NPs from 4 to 10 h
pH-responsive NP post-oral
disintegration at administration versus
pH > 7 in the intestine

(continued)
14

(%) (%)
insulin release; insulin control
paracellular solution
permeation of insulin Slower absorption and
and systemic 15 % relative
absorption bioavailability versus
SC injection
A > *200 nm-oral Dissolution in 0.01 M Insulin absorbed onto At 2 h: burst release Wang et al. (2011)
60 PBS at 37 C NP surface *27 %
Measurement of insulin Readsorption of At 4 h: slight drop
concentration and insulin onto the NPs At 24 h: *80 % of
bioactivity by ELISA Inner insulin bioactive insulin
#
* Humalog Umulin; Velosulin Novonordisk
473
Table 11 Overview of siRNA release from NPs
474

(%) (%)
PNPs E 28 PLGA NPs Release in PBS at pH Diffusion followed by Initial burst: *60 % for Tahara et al.
44* 317 nm-chitosan-modied 7.4 at 37 C erosion; interactions PLGA NPs and *40 % (2010)
PLGA NPs 352 nm-ns siRNA uptake by betweek negative siRNA for chitosan-modied
human lung and positive chitosan PLGA NPs; Prolonged
adenocarcinoma Enhanced electrostatic release: *90 % for PLGA
A549 cells over 4 h interactions between NPs and *70 % for
Gene silencing cationic chitosan and chitosan-modied PLGA
efciency in negatively charged cell NPs at 5 days
A549-Luc cells membranes Higher uptake of SiRNA
from chitosan-modied
PLGA NPs than from
PLGA NPs
Prolonged and signicant
effect of chitosan-modied
PLGA NPs at 5 days
versus no effect of PLGA
NPs
E 77 % No chitosan coating Gene silencing Intertwined factors of Highest silencing effect Yuan et al.
182 nm-0.17 %, chitosan efciency in human increased siRNA loading with highest chitosan (2010)
coating 543 nm-ns embryonic kidney and positive charge coating; effect comparable
HEK 293 T cells resulting from higher to lipofectamine control
chitosan coating of NPs
E PEG/PLA/chitosan NPs Transfection ns but efcacy of the Decrease of HBsAg Wang et al.
227 nmPLGA/chitosan efciency in smallest NP size expression: 55, 58, 76, and (2010)
NPs 260 nmPLGA/PEI PLC/PRF/5 human 80 % for
NPs 200 nm liver cell line as PEG/PLA/chitosan NPs,
PEG/PLA/PEI NPs in vitro model of PLGA/chitosan NPs,
165 nm-ns human hepatitis B PLGA/PEI NPs, and
(continued)
14

(%) (%)
PEG/PLA/PEI NPs,
versus more than 80 % for
lipofectamine-delivered
siRNA
E 60 175 nm-ns Cellular association ns *29 % for HEK 293FT Lee et al.
80 of NPs to human cells and *71 % for (2009)
embryonic kidney HeLa cells
cell line HEK 293FT *60 % in HEK
and to human 293FT-Luc cells and
cervical carcinoma *50 % in HeLa-Luc cells
HeLa cells
Gene silencing
efciency in HEK
293FT-Luc and
HeLa-Luc cells
E 310880 nm-ns Release in PBS at 24 kDa poly-L-lysine At 7 days: *6393 % for Yogasundaram
37 C coating forms more stable 4.2 kDa poly-L-lysine et al. (2012)
Uptake by human NPs that retard siRNA (PLL)-coated NPs; *33
breast cancer release 43 % for 24 kDa
MDA-231 cells over PLL-coated NPs

24 h 2, 18, and 62 %
siRNA-positive cells with
PEG/PLL 24 kDa NPs,
PLL and PEG/PLL
4.2 kDa NPs, and PLL
24 kDa NPs, respectively
core- A 120/280 PIBCA NPs *60 nm Uptake by RP1 cells PIBCA NPs could cross Intracytoplasmic de Martimprey
shell siRNA/NPs PIHCA NPs In vivo activity on the blood epithelium and localization of the NPs et al. (2010)
NPs *100 nm-intratumoral mice subcutaneously accumulate within the Intratumoral treatment: 64
475
and IV injected with RP1 tumor more easily thanks and 59 % tumor growth
(continued)
476

(%) (%)
cells as a model of to their smaller size than inhibition with PIBCA
papillary thyroid PIHCA NPs; their NPs and PIHCA NPs,
carcinoma negative zeta potential respectively, at 14 days;
allows them to escape IV treatment: two-times
uptake by macrophages increase of tumor size with
PIBCA NPs versus
10-times in control group
at 10 days
MNPs A *3062675 nm-ns Transfection Better uptake with longer Knocking down of GFP Xia et al.
efciency in coating polymers expression by 55 and (2009)
GFP-expressing 60 % with NPs coated
HEPA-1 cells with 10 and 25 kD
polyethyleneimine,
respectively
MagNPs A *238 nm-intratumoral Uptake by lung Probable better attachment Intracytoplasmic uptake of Li et al.
cancer cell line A549 of positively charged NPs NPs followed by siRNA (2013b)
and human cervical to negatively charged cell release
cancer cell line HeLa membranes EGFP gene silencing with
EGFP and VEGF NPs similar to that
gene silencing in obtained with
A549-EGFP and lipofectamine; VEGF gene
HeLa cells, knockdown with NPs
respectively higher to that obtained
In vivo efciency in with lipofectamine
A549 tumor-bearing Signicant reduction of
mice tumor growth with NPs
(continued)
14

(%) (%)
NCs E >95 *325 nm-intratumoral Cellular uptake by ns Cytoplasmic localization Toub et al.
NIH/3T3 EWS-Flil of the NPs (2006)
cells 43 and 80 % of tumor
Gene silencing growth inhibition with
efciency in NPs at 0.8 and 1.1 mg/kg
NIH/3T3 EWS-Flil of siRNA cumulative dose
cells 60 % inhibition of
In vivo efciency in EWS-Flil expression by
mice xenografted NPs
EWS-Flil-expressing
tumor
PEC E 35 *400500 nm-ns Cellular uptake by ns Intracellular localization Saengkrit et al.
98 human cervical of the NPs (2012)
cancer cell line SiHa Signicant decrease in
Gene silencing gene expression with NPs
efciency in SiHa
cells
Nanogels E *5 nm-IV Transfection ns Silencing of ataxin protein Malhotra et al.
efciency in an by chitosanPEGTAT (2013)
in vitro model of NPs

spinocerebellar ataxia
in mouse
neuroblastoma cells
(Neuro 2 s)
E *147 nm-oral Permeation across Electrostatic interaction of 10 and 34-fold increase He et al. (2013)
Caco-2 cells and trimethyl groups on NPs versus naked siRNA with
Caco-2 + Raji B cells and negatively charged Caco-2 and Caco-2 + Raji
in coculture (human mucosa; disulde bonding B cells, respectively
(continued)
477
478

(%) (%)
follicle-associated of cystein residues on NPs 34 and 30-fold increase
epithelia) and mucin glycoproteins versus naked siRNA with
Cellular uptake by in the mucosa Raw 264.7 cells and
mouse monocyte improvement of chitosan PECs, respectively
macrophage Raw mucoadhesion and NP Direct transport of
264.7 and murine permeation; active encapsulated siRNA to the
peritoneal exsudate targeting of NPs toward cytoplasm; no endosomal
cell macrophages cocultured cells through entrapment nor lysosomal
(PECs) mannose ligands; degradation
Intracellular caveolae-mediated Signicant silencing of
trafcking in Raw endocytosis and TNF expression in both
264.7 cells macropinocytosis; cell types
Transfection clathrin-independent Enhancement of siRNA
efciency in Raw endocytosis distribution levels in liver,
264.7 cells and PECs Systemic distribution of spleen, lung and intestine
In vivo siRNA and versus naked siRNA
biodistribution after siRNA-mediated gene 7290 % depletion of
oral gavage in mice silencing in macrophages serum TNF production
Gene silencing in with a single gavage of
healthy mice and 1.515 nM SiRNA/kg
mice with acute
hepatic injury
* DOTAP complex
Table 12 Overview of cisplatin release from NPs
14

(%) (%)
PNPs E 0.1 3 *285 nm-IV Dissolution in Diffusion *40 % at day 1; *75 % at day Alam et al.
PBS at pH 7.4 at burst release 15 (2014)
37 C Combination of Similar anticancer activity for NPs
Cytotoxicity in drug diffusion as for free drug
human lung and polymer Liver/kidney and liver/blood drug
cancer cell line degradation ratios signicantly higher with
A549 sustained release NPs than with free drug
Tissue distribution
in mice
E 4 80 *140190 nm-IV Dialysis in PBS at Diffusion of At 24 h: *3538 % from NPs Wang
37 C drug and erosion versus 86 % from free drug; at et al.
Cytotoxicity on of the polymer 126 h: *7679 % from NPs (vs. (2013)
human ovarian matrix 78 % at 8 h from free drug)
adenocarcinoma Enhanced cytotoxicity of NPs
cell line SKOV3 versus free drug
Biodistribution Higher blood and tumor
and therapeutic concentration and lower
efciency in accumulation in kidneys for NPs
SKOV3 versus free drug; tumor inhibition

cancer-bearing rate 3.33 times higher with NPs
mice than with free drug
PEC E *114163 nm-IV Dialysis in PBS ns At 12 h: burst release up to 50 %; Lee et al.
0.01 M at pH 7.4 sustained release: 90100 % at (2013)
at 37 C 100 h versus 100 % at 24 h for
Anticancer free drug
activity on CT26
(continued)
479
480

(%) (%)
colorectal Similar anticancer activity of NPs
carcinoma cells as free drug
Tumor growth Signicantly higher tumor growth
inhibition in CT26 inhibition of NPs versus free drug
tumor-bearing
mice
LP E 80 *2030 nm-IV Uptake and ns Uptake and cytotoxicity increased Guo et al.
cytotoxicity on by 6.5 and tenfold, respectively for (2013c)
human melanoma NPs versus free drug
A375M cells 24 h post-IV injection:
Biodistribution in accumulation of 10.5 % of the
mice drug in the tumor with NPs versus
Antitumor activity 1.2 % with free drug
in A375M Signicant inhibition of the tumor
xenograft-bearing growth with NPs versus free drug
mice
Finally, release prole are generally depicted as percentage of drug by time units, but
can also correspond to pharmacokinetic parameters like the area under the curve
(AUC) or the half-life values, or to cytotoxic effects or tumor growth inhibition for
anticancer drugs, for example.
3 Mechanisms of Drug Release
Generally, the drug release is governed by three different mechanisms: (i) a standard
diffusion-controlled release, (ii) release thanks to the degradation of the nanoparticles
produced from biodegradable polymers, or (iii) a triggered pathway initiated by changing
the environmental conditions such as pH or temperature. Most sustained release for-
mulations suffer a common phenomenon of burst release at an initial stage that can be
ascribed to the presence of drug at the nanoparticle surface. The drug diffusion-controlled
release depends on its effective diffusion coefcient throughout the polymer matrix,
which in turn depends on its porosity and tortuosity (Jger et al. 2009). The drug release
from degradable NPs is controlled by the bulk erosion rate (Chan et al. 2009).
Mechanisms of drug release can be studied more precisely by analyzing release proles
thanks to mathematical models. Hence, kinetic analysis of drug release can use models
like the Korsmeyer-Peppas model (Siepmann and Peppas 2001; Siepmann and Siepmann
2012), expressed as Mt/M = ktn, where Mt and M represent the cumulative amount of
drug released at time t and at innite time, respectively; k is a pseudokinetic constant that
takes into account the structural and geometric features of the NPs; and n is the release
exponent that provides information about the involved release mechanisms (de Matos
et al. 2013). For example, n values above 0.5 obtained with dexamethasone-loaded poly
(-caprolactone) (PCL)/silica NPs indicate that drug release is principally controlled by an
anomalous transport mechanism associating Fickian- and Case II-type transport mecha-
nisms. In such a case, the drug is released both from near-surface regions and from initial
poly(-caprolactone) surface erosion (de Matos et al. 2013).
For the 12 selected drugs studied, the most encountered release mechanism is the
drug diffusion, followed by the nanoparticle degradation, and by the stimuli-triggered
release. Hence, the diffusion mechanism concerns all the molecules studied except for
ciprofloxacin and for insulin. The latter can be specically released by the combination
of nanoparticle degradation and a pH-triggered release (Sung et al. 2012).
Otherwise, the degradation of nanoparticles is used as a release mechanism of
doxorubicin from self-assemblies (Li et al. 2013a), of paclitaxel from PNPs (Lee et al.
2011; Lv et al. 2011), of amphotericin B from PNPs (Xu et al. 2011) and from SLN
(Patel and Patravale 2011), and of cyclosporine A from SLN (Karavana et al. 2012;
Gke et al. 2008).
As for stimuli-triggered release, it concerns doxorubicin included in polyelec-
trolyte complexes (PC) (Lv et al. 2013; Guo et al. 2013a, b), paclitaxel encapsulated
in core-shell nanoparticles (core shell NPs) (Agueros et al. 2009), and dexam-
ethasone entrapped in core shell NPs (Fratoddi et al. 2012). In all cases, the release
is triggered by a pH change. Theoretically, stimuli-responsive NPs can use a variety
of stimuli to trigger drug release: light, temperature, ultrasounds, magnetic force,

enzymes, pH, reductive, or oxidative stress (Kang et al. 2011; Bikram and West
2008; Ferrara 2008; Hoare et al. 2011; De la Rica et al. 2012; Felber et al. 2012;
Cheng et al. 2011; Broaders et al. 2011; Mura et al. 2013). In practice, pH is one of
the most commonly used stimuli because of the changes of its value in the different
biological compartments and the cellular organelles. In addition, the neutral pH
encountered in most human tissues becomes acidic in tumor microenvironments
(Tannock and Rotin 1989; Gerweck and Seetharaman 1996). Hence, the pH value
at the tumor extracellular environment is more acidic (6.8) than that in blood (7.4),
and the pH values in the endosomes and lysosomes is even lower (<5.5). Acidic pH
states of human tumors range from 5.7 to 7.8 according to Cheng et al (2013).
pH-sensitive nanocarriers offer two main advantages: by minimizing drug loss in
blood circulation they allow reducing the side effects to normal tissues and by
triggering faster release in endosomes of tumor cells they improve therapeutic
efcacy (Lv et al. 2013).
Finally, two mechanisms can be associated to induce the drug release. The latter
results most of the time from a drug diffusion combined with nanoparticle degra-
dation. This double mechanism of release is mainly exploited in PNPs of paclitaxel
(Jger et al. 2012; Ren et al. 2011), ibuprofen (Bonelli et al. 2012), amphotericin B
(Nahar et al. 2008), cyclosporine A (Ankola et al. 2010), sirolimus (Haddadi et al.
2008), dexamethasone (Zweers et al. 2006), acyclovir (Kamel et al. 2009), siRNA
(Tahara et al. 2010), and cisplatin (Alam et al. 2014; Wang et al. 2013). Core-shell
NPs also allow for release by diffusion/degradation mechanisms of doxorubicin (De
Verdire et al. 1997), and paclitaxel (Narayanan et al. 2014); PC of amphotericin B
(Zhou et al. 2013) and NC of cyclosporine A (Park et al. 2013) as well.
More rarely, the diffusion mechanism is associated with a stimuli-responsive
process. It is the case with Mg-NPs of doxorubicin which release either pH-dependent
(Pilapong et al. 2013; Akbarzadeh et al. 2012), or chemically controlled (Anirudhan
and Sandeep 2012). It is also the case with MNPs of ibuprofen (Chen and Zhu 2012).
Among rare mechanisms of release, one can nd the fusion of the outer lipid
membrane of the NPs with the membrane of the target cell (Cyrus et al. 2008), or
the lipid-coating destabilization of cisplatin nanocapsules following cell surface
binding or endocytic uptake and resulting in membrane passage and subsequent
intracellular drug release (Burger et al. 2002).
4 Tuning the Release Proles
While tuning the release prole of nanoformulations is still rather difcult, one can
take into account the influence of parameters like the effect of the type of
nanoparticles (NPs) and its physicochemical characteristics, the modulation of the
release prole according to the active molecule properties and to the administration
route.
One critical problem is the rapid initial release or burst release, in particular in
NPs intended for parenteral administration for which slow release for several weeks
or months may be required. Burst release is attributed to the drug that is adsorbed or
weakly bound to large surface area of NPs rather than to the drug incorporated
inside NPs (Parveen and Sahoo 2008). Sustained release is the most frequently
desired prole. As an exception a fast release can be desired for drugs like
cyclosporine (Urbn-Morln et al. 2010).
a. Effect of the type of nanoparticle and its physicochemical characteristics
By analyzing the in vitro studies implemented on the 12 selected drugs in the
literature, one can observe that the nanoparticle type as well as some characteristics
of the NPs like the nanoparticle size and size distribution, polymer crosslinking and
drug loading, influence the release prole.
Among the different types of NPs studied, the most often employed to encap-
sulate the largest variety of drugs, i.e., 9 out of 12 drugs, is the polymer nanoparticle
(PNP) type. PNPs allow drug release duration as short as 30 min (Kim et al. 2011),
and as extended as 50 days for sirolimus (Zweers et al. 2006). This type of NPs
releases its load by the mechanism the most often seen in the literature, that is to say
diffusion-erosion mechanism. PLGA NPs represent the model of this type of
biphasic release characterized by an initial burst release occurring by diffusion of
the drug from polymer matrix; drug release during a later phase is mediated both by
diffusion of the drug and erosion of poly(lactide-co-glycolide) (PLGA) itself
through a process of autocatalytic hydrolysis of ester bonds. Hence, acidic mono-
mers and polymers produced by this degradation further catalyze the hydrolysis
(Rahman et al. 2010).
Triphasic release prole from PLGA or poly(ethylene oxide)-co-poly
(lactide-co-glycolide) (PEOPLGA) NPs consists in a rst phase of burst effect
caused by the release of the drug adsorbed onto the outer particle surface, then a
second phase of relatively slow release due to diffusion of the drug out of the
matrix, and nally a third phase of increased drug release caused by extensive
polymer degradation (Li et al. 2001). There is an impact of the molecular weight
and the lactide to glycolide ratio of the polymer forming the NP matrix on the drug
release rate (Tahara et al. 2010).
Mesoporous nanoparticles (MNPs) can also encapsulate numerous drugs with a
smaller range of release duration between 7 h (De Matos et al. 2013) and 192 h
(8 days) for doxorubicin (He et al. 2011). In addition, pH-responsive drug delivery
systems can be obtained using MNPs as drug carriers and chitosan as
pH-responsive functional molecule (Chen and Zhu 2012; Chen et al. 2013). Thus, a
sensitive response in a narrow pH range between 6.8 and 7.4 could be obtained with
ibuprofen-loaded MNPs enclosed in chitosan. Thanks to chitosan being in a gel
state at pH 6.8, the drug was released at 65 % after 24 h; at pH 7.4, the orderly
aggregated state of chitosan molecules retained the drug which was released only at
35 % (Chen and Zhu 2012). In addition to controllable release from size-adjustable

pH-sensitive MNPs, the functionalization of the NP surface could be achieved by
hydrophobic trimethylsilyl groups that further delayed the drug release (Xu et al.
2009). Such pH-sensitive systems are of particular interest for specically targeting
inflammatory tissues or tumor cells whose pH is more acidic than the physiological
pH of 7.4. Nonspecic reaction with normal cells could thereby be reduced while
the curative effect of the drug could take place in diseased tissues.
The next two types of NPs the most employed to encapsulate half of the 12 drugs
studied here are core-shell NPs and polyelectrolyte complexes (PEC). The release
duration of the former ranges from 1 h (De Verdire et al. 1997) to 40 h (Jiang et al.
2005); while the latter offer longer release times up to 9 days (Guo et al. 2013a).
Finally the other types of NPs are less used. Their release capacity last for
periods of time between a few hours for cubic NPs (Dian et al. 2013; Lai et al.
2010), nanocapsules (Aksungur et al. 2011; Zhang et al. 2012) and nanogels (Zhao
et al. 2013), to a few days for magnetic NPs (Li et al. 2011) and self-assemblies (Li
et al. 2013a).
Beside the type of NPs, the characteristics of the NPs can influence the release
prole of the drug, in particular the NP size. In most studies, the size of the
nanoparticles was within the optimal range for injectable formulations with diam-
eters smaller than 200 nm. The NP size is a key parameter that influences the
release rate of the drugs. A narrow size distribution plays also an important role in
colloidal stability and later in controlled drug release (Jger et al. 2012). In most
cases, the smaller the NP size, the faster the release. This phenomenon is due to the
larger surface area exposed to the release medium. It can be observed within a type
of NPs, for example MNPs (Xu et al. 2009), but no common feature can be ruled
out among NPs of different natures. Of note, even though the release kinetic can be
well controlled by the NP size, their agglomeration modies the prole toward
delayed release probably in relation to a lowered surface area available for diffusion
(Xu et al. 2009). Interestingly, the ex vivo permeation of a drug across the skin can
increase with the size of its nanocarrier. It is the case for nanogels intended for
topical administration of acyclovir which sizes of 380 and 703 nm allow for 45 and
70 % drug diffusion within 48 h, respectively (Hasanovic et al. 2009). The better
permeation from larger NP could be explained by the stronger interaction between
NP surface charge density and anionic components of the epithelial cell surface
(Hasanovic et al. 2009).
The size of the NPs also influences the efcient delivery of the drug to cells.
Hence, Ali et al. (2013) observed that NP-mediated delivery of dexamethasone
across a model of human placental trophoblast was dependent on the particle size:
the apparent permeability of 145-nm NPs was twice as high as the apparent per-
meability of 196-nm NPs.
SiRNA delivery from cationic NPs of biodegradable polymers such as PLA and
PLGA was dependent on the size and the surface charge of the NPs: the smaller the
size, the more efcient the in vitro transfection into cultured cells (Wang et al. 2010).
In addition, the surface charge of the NPs has to be positive to favor electrostatic
interactions with the negatively charged cell membranes.
Crosslinking of the polymer forming PNPs represents a mean to modulate the
release rate of the drug. Hence, ibuprofen-loaded gelatin graft copolymer NPs prepared
using styrene or 2-hydroxyethyl methacrylate (HEMA) monomers, in the presence of
potassium persulfate as an initiator and glutaraldehyde as a crosslinker, had release
proles completely different depending on crosslinking. Burst release at 1 min was 28
and 83 % for styrene and HEMA NPs without crosslinking, respectively, while the
release was only 8 % at 30 min for crosslinked styrene and HEMA NPs (Haroun et al.
2012). In the latter case, the slow release was dynamically controlled by the
biodegradation of the copolymer matrix over 5 h. Interestingly, the size of the NPs
changed with the crosslinking step, from 143 to 161 nm for HEMA NPs and from 159
to 12 nm for styrene NPs, but in such a manner that the NP size by itself could not be
linked to the release rate observed (Haroun et al. 2012). Although the results obtained
in this study are very important as proof of concept demonstrating the influence of the
crosslinking to tune releasing properties of polymer nanoparticles, the nature of some
chemicals including the crosslinker may be problematic for the development of a
suitable drug carrier to be administered to patients. Nevertheless, the concept is worth
to consider with more acceptable components entering a drug formulation.
Drug loading is another parameter that can influence the release rate. The higher
the drug loading, the higher the release rate. Hence, Cao et al. (2014) observed a
marked increase in cumulative release of paclitaxel from PLAPEG NPs with a
core-shell structure between 44 and 51 % drug loading. Doxorubicin release from
NPs with 2 to 28 % drug content increased from 70 to 90 %, respectively (Li et al.
2011). Exceptions exist: one is published by Ribeiro et al. (2014) who observed a
lower release of amphotericin B from chitosanchondroitin sulfate NPs with higher
drug loading; another exception is described by Lee et al. (2013) who noticed that
the higher drug loading, the slower the cisplatin release from NPs of poly(acrylic
acid-co-methyl methacrylate) copolymer. In that case, one can however underline
that the drug loading was not the only parameter to vary since the particle size
increased from 114 to 158 nm with drug loading increasing from 2.3 to 10.6 %
(Lee et al. 2013).
b. Modulation of the release prole according to the active molecule properties and
to the administration route
For hydrophobic drugs with very low water solubility, the challenge of NP
delivery consists either in improving their oral bioavailability or in providing
injectable formulations with a sustained release prole. The reduction of toxicity is
also an issue. This is the case for insoluble anticancer drugs like paclitaxel and
doxorubicin. The majority of the paclitaxel formulations were intended for the IV
route, except for four PNPs formulations designed for the oral route (Lv et al. 2011;
Roger et al. 2012; Ageros et al. 2009; Zabaleta et al. 2012). The release was either
relatively rapid within 22 h (Ageros et al. 2009) or prolonged up to 9 days (Lv
et al. 2011) for oral formulations. Increased oral absorption of paclitaxel could be
achieved through transcellular transport of NPs by transcytosis and could be further

improved thanks to receptors expressed on the intestinal epithelial cells like folate
receptors (Roger et al. 2012). Injectable formulations had all sustained release
proles with paclitaxel released between 24 h (Jiang et al. 2011) and 40 days (Cao
et al. 2014).
Concerning doxorubicin, a majority of studies get around the problem of poor
water solubility using doxorubicin hydrochloride which solubility is improved. For
that reason, modulation of doxorubicin release is commented below in the para-
graph devoted to drugs with good water solubility. As for the four studies that used
the base that is practically insoluble in water (Yuan et al. 2012; Lehtovaara et al.
2012; Li et al. 2013a; Theodossiou et al. 2013), the release occurred within 2496 h
but no administration route was specied.
Clinical applications of amphotericin B are limited by its cytotoxicity (Xu et al.
2011) and pronounced side effects (chills, fever, nausea, hemolytic toxicity, and
nephrotoxicity) (Nahar et al. 2008); delivery by NPs is intended to reduce these
deleterious effects and to provide sustained release proles. Except for one topical
formulation with almost total amphotericin B release at 11 h (Zhou et al. 2013), all
NPs were intended for IV administration with a range of release time between
30 min (Van de Ven et al. 2012) and over 8 days (Nahar et al. 2008).
Amphotericin B being very slightly soluble in water, some authors used more
soluble derivatives like amphotericin B deoxycholate (Xu et al. 2011) or ampho-
tericin B sulfate (Ribeiro et al. 2014). Both nanoformulations were intended for the
IV route. In the former study, the release was estimated indirectly by an in vivo
signicant increase of amphotericin B brain concentration in healthy mice and a
higher survival rate in murine cryptococcal meningitidis when delivered with PNPs
(Xu et al. 2011). The latter study with amphotericin B sulfate included in PEC
allowed for a delayed release of 5768 % at 48 h compared to 100 % at 12 h for
free drug (Ribeiro et al. 2014).
The oral absorption of drugs with poor bioavailability like cyclosporin A is
limited by their lack of dissolution within sufcient time while being at the
absorptive site. For such drugs, oral absorption could be promoted thanks to
paracellular drug transport by NPs (Cheng et al. 2006). Cyclosporine A-loaded NPs
intended for the oral route (Ankola et al. 2010; Lai et al. 2010; Aksungur et al.
2011; Cheng et al. 2006), were characterized by prolonged release proles lasting
between 12 h (Lai et al. 2010), 24 h (Aksungur et al. 2011) and 918 days (Ankola
et al. 2010). Other administration routes like the topical ocular route (Hermans et al.
2012; Gke et al. 2008) and the IV route (Nakarani et al. 2010) were envisaged.
The release proles were comprised between 90 min (Gke et al. 2008) and 24 h
(Hermans et al. 2012) for topical use. Other formulations which administration
route was not specied had sustained release up to 7 days (Rahman et al. 2010) and
21 days (Lee et al. 2012).
NPs intended for IV delivery of sirolimus were sustained release formulations
with release proles lasting up to 7 days (Haddadi et al. 2008) and 15 days
(Acharya and Sahoo 2011). Local administration at the site of arterial stenosis used
NPs with extremely extended release up to 50 days (Zweers et al. 2006). On the
opposite, oral formulations could present a fast release within 30 min (Kim et al.
2011) or a slower release within 7 days (Bisht et al. 2008). Finally, topical corneal
administration used NPs releasing sirolimus over 8 days (Yuan et al. 2008). It is
noteworthy that the obtaining of such a wide range of releasing properties among
the different studies was associated with the wide variety of properties of the NPs
used in these studies including the nature of the polymer that composed them and
their structure. Hence, all studies employed PNPs as carriers for sirolimus. While
prolonged release proles could be achieved with PLGA NPs (Haddadi et al. 2008;
Acharya and Sahoo 2011), PEO/PLGA NPs (Zweers et al. 2006), chitosan/PLA
NPs (Yuan et al. 2008) and N-isopropylacrylamide/methylmethacrylate/acrylic acid
NPs (Bisht et al. 2008), very fast release could be obtained thanks to poly
(n-vinyl-2-pyrrolidone) (PVP)/sodium lauryl sulfate (SLS) NPs (Kim et al. 2011).
As for poorly soluble ciprofloxacin, improvement of dissolution rate and solu-
bility could be achieved through encapsulation into NPs and the difculty for this
antibiotic drug to reach its site of action could be overcome by NPs as antibiotic
carriers that crossed the mucus barrier of lung chronically infected patients to reach
the biolm colonies (Cheow and Hadinoto 2011). Sustained release of ciprofloxacin
depended on the lipophilicity of the drug; hence lipophilic ciprofloxacin allowed
sustained release attributed to the interaction with the lipid coat of lipid-polymer
hybrid NPs (Cheow and Hadinoto 2011). Except for this latter formulation of NPs
intended for pulmonary administration, the administration route of the other
ciprofloxacin nanoformulations was not specied. Their release prole could be
rapid for PEC with 100 % release at 3 h (Cheow and Hadinoto 2012), or sustained
over 15 h for SLN (Shah et al. 2012), or over 24 h for nanogels (Zhao et al. 2013).
As for the LP intended for pulmonary administration, they presented an extended
release lasting over 5 days (Cheow and Hadinoto 2011). Of note, Shah et al. (2012)
used ciprofloxacin hydrochloride with higher water solubility than ciprofloxacin.
All ibuprofen-loaded NPs were formulated for the oral route of administration
with drug release lasting for 2 h 30 min (Bonelli et al. 2012) to 40 h (Jiang et al.
2005). These NP formulations principally aimed at improving the solubility and
dissolution rate of ibuprofen and at limiting side effects affecting the gastrointestinal
tract thanks to a distribution of the dose over a larger surface area of the mucosa.
Finally, nanoformulations of dexamethasone were designed for IV or local
administration with relatively short release for the former route, i.e., 210 h
(Fratoddi et al. 2012), and short release within 7 h (de Matos et al. 2013) as well as
prolonged release up to 17 days (Zweers et al. 2006) for the latter route. All authors
used dexamethasone that is practically insoluble in water but Uccello-Barretta et al.
(2014) used dexamethasone phosphate disodium salt with higher water solubility.
Hydrophilic drugs with good water solubility are characterized by a poor per-
meability across biological membranes and thereby a limited absorption. Among
such drugs, cisplatin has limited clinical use due to three major problems: severe
adverse effects including nephrotoxicity and neurotoxicity, rapid complexation to
plasma and tissue proteins resulting in inactivation of the drug, frequent occurrence
of platinum resistance (Burger et al. 2002). Injectable formulations of NPs are
intended to reduce toxicity and sustained release of cisplatin shall lower its intrinsic
toxicity (Lee et al. 2013). Among the selected studies, cisplatin nanoformulations
were all designed for IV administration with sustained release lasting between
4 days (Lee et al. 2013) and 15 days (Alam et al. 2014).
Although practically insoluble in water as a base, doxorubicin can be considered as
a hydrophilic drug as soon as its hydrochloride form is used. A majority of studies
used doxorubicin hydrochloride which water solubility is improved. All formulations
were designed for IV injection except for one formulation of MNPs designed for
intratumoral injection (Xie et al. 2013). This latter was characterized by a total and
rather fast release of doxorubicin within 8 h at pH 5.0. Among IV formulations, three
studies reported relatively fast release of a few hours up to 5 h (Pilapong et al. 2013),
6 h (De Verdire et al. 1997), or 10 h (Pooja et al. 2014). All other formulations
achieved more sustained release between 24 h (Anirudhan and Sandeep 2012; Gu
et al. 2012) and 9 days (Guo et al. 2013a). IV delivery of anticancer drugs like
doxorubicin aims at an efcient and site-specic delivery to tumor tissues optimizing
therapeutic efcacy and minimizing side effects (Guo et al. 2013b) since clinical
applications of doxorubicin that exhibits very high cytotoxicity to normal cell are
greatly limited by the tolerance of the patients (Su et al. 2013).
Concerning acyclovir, it is commercially available for IV, oral and topical
administration; however, its oral bioavailability is considered as low about 20 %
(Gide et al. 2013) and IV administration of the solution at pH 11 causes frequent
adverse reactions like phlebitis at the injection site and renal damage through
precipitation of acyclovir crystals in renal tubules (Kamel et al. 2009). In addition,
its plasma half-life is short about 2 h 30 min. NPs intended for IV injection aim at
extending acyclovir plasma half-life, hence reducing the daily dose and the side
effects (Kamel et al. 2009). NPs are also intended for topical administration in order
to improve transdermal penetration (Gide et al. 2013; Hasanovic et al. 2009).
Acyclovir nanoformulations studied here were designed either for IV injection or
topical administration and presented an initial burst release at 1 h followed by a
slower exponential release over 48 h for the former (Kamel et al. 2009), and a
biphasic diffusion prole within 48 h for the latter (Hasanovic et al. 2009).
Oral administration of insulin is the goal of the formulation of this peptide into
NPs in order to avoid administration by injections that cause discomfort to patients.
The challenge consists in overcoming the limiting factors that hamper oral delivery,
i.e., the acidic environment of the stomach, the enzymatic degradation, the low
epithelial permeability, and the rapid clearance from the gastrointestinal tract
(Zhang et al. 2012). NCs and self-assemblies were both characterized by a burst
release of insulin at 2 h followed by a sustained release over 20 h (Zhang et al.
2012) and 24 h (Wang et al. 2011).
Finally, siRNA are hydrophilic molecules with strong negative charges derived
from phosphate groups that hamper their penetration into cells without a trans-
fecting agent. Therefore cellular uptake is the most restraining factor for siRNA
therapeutics (Tahara et al. 2010). Most of the selected studies dealing with siRNA
NPs did not specify the administration route. When documented, the administration
route could be IV, intratumoral, or oral. The administration of siRNA via the oral
route is a real challenge because of enzymatic and pH-driven degradations, poor
intestinal permeation, inefcient cellular internalization and intracellular trafcking,

and stickiness to nontarget cells. It therefore requires efcient vehicles able to
overcome all these obstacles. SiRNA delivery by polymer NPs is an alternative to
delivery after complexation with cationic lipids which present certain toxicity.
When studied in vitro, the release prole of siRNA occurred in a sustained manner
over 5 days (Tahara et al. 2010) to 7 days (Yogasundaram et al. 2012).
5 Methods for Evaluating Drug Release from NPs
The evaluation of drug release from nanoparticles is a real challenge due to the
small size of the formulation. Numerous methods were published to investigate the
release of drugs including in vitro, in cellulo, ex vivo, and in vivo methods (Fig. 2).
It is noteworthy that release proles may greatly depend on the applied methods.
The different methods are presented in this part of the chapter and the choice of the
methods will be discussed with specic examples.
a. In vitro methods
These are the most direct methods to assess the release of drugs from
nanoparticles, the easiest ones and the most commonly cited in the literature. They
comprise methods of dissolution and dialysis methods.
Methods of dissolution: these methods to study dissolution of drugs encapsu-
lated in NPs are inspired by the dissolution techniques described in the European
Pharmacopoeia and the United State pharmacopoeia (USP). These methods were
adapted to be applied to nanoparticles. The most important modication consisted
in modications of the dissolution medium volume and agitation. The critical
parameters of these methods are the followings:
The nature of the medium: the simplest one is puried water, the most
commonly encountered are saline media like phosphate buffer saline (PBS),
acetate buffer, etc.; a simulated body fluid is employed by Xu et al. (2009).
Organic solvent like methanol (Ribeiro et al. 2014) are more rarely used. For
NPs intended for oral administration, SGF, and SIF (USP) are often chosen to
study the release according to the level of the GIT considered. Other specic
media like simulated lachrymal fluid (Aksungur et al. 2011) are also used.
The pH of the medium: the physiological pH of 7.4 is frequent but other pH
characteristic of different biological compartments are also used like gastric and
intestinal pH about 1.2 and 6.8, respectively, saliva pH about 6.75 (Karavana
et al. 2012), acidic pH of 5.86.8 mimicking tumor microenvironments, and
lower than 5.5 representing pH values in endosomes and lysosomes.
The temperature: at which the release is tested: most studies are implemented
at the physiological temperature of 37 C. Very few studies use lower tem-
perature, like room temperature (Ribeiro et al. 2014), eye temperature of 32 C
(Hermans et al. 2012), or higher temperature up to 40 C (Akbarzadeh et al.
2012).
In vitro
Critical parameters Results
Dissolution Dialysis
- Medium: composition, volume, pH, sink
conditions, temperature, agitation
- Drug: solubility, molar mass, pKa, state,
concentration % release vs time
- Duration
- Detection limit of measurement
- Membrane cut off for dialysis methods
In cellulo
Uptake Biological Uptake:

effect - Type of cell - Intracellular %
concentration, extraction method Biological effect:
- Duration - Mostly cytotoxicity of
- Detection limit of measurement anticancer drugs on
tumor cells
- Transfection efficiency
for siRNA NPs
Ex vivo
Permeation across biological
membranes - Type of membrane
concentration % of absorption
- Duration
In vivo
PK in healthy Therapeutic effect in In healthy animals:
animals diseased animals - Bioavailability
- Animal species
- Biodistribution
- Administration route
- Dose administered, once or repeteadly
In diseased animals:
- Nature of the disease (mostly tumor-bearing
Healing of the disease
rodents)
(mostly reduced tumor
growth for anticancer
drugs)
Fig. 2 Schematic representation of the different methods for evaluating drug release from NPs
Sink conditions: they are conditions under which the maximum concentration
of the drug in the release medium would always be less than 10 % of the
maximum solubility (Bisrat et al. 1992). Sink conditions are either maintained
by frequent replacement of fresh medium during the release (Ren et al. 2011) or
by adding surfactants like polysorbate 80 (Parveen and Sahoo 2011; Ageros
et al. 2009), sodium lauryl sulfate (SLS) (Rahman et al. 2010; Lai et al. 2010;
Urbn-Morln et al. 2010), sodium deoxycholate (Van de Ven et al. 2012) in the
release medium, or by using organic solvents such as methanol (Karavana et al.
2012), or dimethylsulfoxide (DMSO) (Nahar et al. 2008).
Duration of release: this parameter varies from a few hours to several days.
Hence, despite intended oral administration, the release is studied during
18 days (Ankola et al. 2010) or 21 days (Lee et al. 2002). Concerning the latter
study, the reason for a so long period of study relies on the fact that NPs are to
be delivered to Peyers patches where they release the drug for long periods of
time (Lee et al. 2002). Up to 50 days of sirolimus release have been achieved
with PEOPLGA nanoparticles treated with 3 % gelatin (Zweers et al. 2006). Of
note, antimicrobial agents like sodium azide (Rahman et al. 2010) may be added
to the medium for dissolution testing lasting over days.
Dark/light conditions: The use of protection against light depends on the photo
sensitivity of the drug molecule. Most studies were carried out without pro-
tection against light. One assay studying doxorubicin hydrochloride release
from xanthan gum stabilized gold NPs mentioned that dark conditions were
used (Pooja et al. 2014).
Detection limit of the measurement method: it is a real issue since the con-
centrations to be measured are often low.
Sampling interval and sampling volume: they have to be carefully chosen
depending on the type of release studied (Patel and Patravale 2011).
Methods of dialysis: these methods consist in interposing a synthetic membrane
between the NPs and the release medium thereby allowing maintaining the for-
mulation inside a dialysis bag or cell. In addition to the critical parameters cited
above, the molecular weight cut off of the membrane is of main importance because
it controls the drug diffusion through the membrane. The drug adsorption onto the
dialysis membrane is a possible source of release underestimation.
Dialysis studies are usually dynamic ones, the receiving medium being analyzed at
intervals and the released drug fraction being calculated at time t. Interrupted dialysis
studies can also be implemented. They consist in stopping the dialysis after an
established time from the start in order to determine the drug fraction contained in each
of the NP matrix, NP dispersion medium (within the dialysis bag or cell), and release
medium (external to the dialysis bag or cell). This procedure has to be repeated for
different dialysis times to allow for plotting the drug fraction in each phase versus time
(Uccello-Barretta et al. 2014). This original method is very interesting since it allows
discriminating between the actual release of the drug from the NPs (within the dialysis
bag) and its diffusion through the dialysis membrane (toward the release medium).
While drug measurements are usually done by classical methods like UV-visible
spectrophotometry or by high performance liquid chromatography (HPLC), a original
technique was also recently published by Uccello-Barretta et al. (2014). By integrating
the nuclear magnetic resonance (NMR) signal of proton H4 of dexamethasone
21-phosphate disodium salt loaded in aggregates, it was possible to analyze its release
rate. Hence, NMR relaxation measurements allowed detecting intermolecular inter-
actions between the drug and macromolecules constituting the NPs.
In this chapter, in vitro methods are the most frequently employed to study drug
release from nanoparticulate formulations. Dissolution methods are implemented
for ten out of 12 drugs studied, and concern all types of drugs whatever their molar
mass (from 206 to 1202 g/mol), their hydrophilicity of lipophilicity (logP from
2.19 to +4.85), their water solubility (from practically insoluble to soluble), and
the type of nanoformulation. Even drugs with the lowest water solubility could be
tested through adaptation of the dissolution medium composition in order to
achieve sink conditions by adding surfactants or organic solvents. Dissolution
testing could last from 30 min for sirolimus PNPs (Kim et al. 2011) to 20 days for
paclitaxel PNPs (Parveen and Sahoo 2011), depending on the intended release
prole. Dialysis methods are used for all the drugs studied, except for siRNAs
whose molar mass of about 13 kDa prevents them from crossing dialysis mem-
branes classically used. In addition, siRNAs are very different from all other drugs
studied in this chapter for they cannot cross the cellular membrane unless carried by
a nanoparticulate system. For that reason, their release from their carrier is not a key
point in their development. The length of dialysis studies varies from 2 to 6 h for
cubic nanoparticles of ibuprofen (Dian et al. 2013) to 50 days for PNPs of sirolimus
(Zweers et al. 2006).
In vitro methods are extremely useful to study the drug release from nanopar-
ticulate systems. However, one should be very careful in interpreting the results
they can provide. Hence, the choice of the method may be of crucial importance
especially for very sparingly soluble drugs. For example, Van de Ven et al. (2012)
observed that a conventional in vitro dissolution set up was not relevant for
studying the release of amphotericin B from PLGA nanoformulations since it was
only dependent on the surfactant concentration in the release medium. In addition,
part of solubilized drug precipitated during the experiments thereby leading to an
underestimation of the percentage of release. This occurred despite a concentration
up to 2 % of sodium deoxycholate used to maintain sink conditions. In such a
situation, the dialysis method was not tested because it could not prevent the drug
from precipitation; moreover, the dialysis membrane is known to delay drug
release. A test protocol published by Legrand et al. (1997) was therefore imple-
mented where monomeric amphotericin B release was monitored at a very low
concentration of 0.1 g/mL by the absorbance change at 407 nm.
In the case of cyclosporine A-loaded NPs, the release prole was also strongly
dependent on the composition of the release medium: in PBS without surfactant the
release was slow and limited to the solubility of the drug in the medium; in PBS
with 0.01 % polysorbate 80 (below the critical micellar concentration) the release
had the same prole but the total release increased from 45 to 60 %; nally in PBS
with 0.28 % polysorbate 80 (above the critical micellar concentration) allowing for
sink conditions, a burst release of 6090 % occurred within 1 h and no additional
release was observed (Hermans et al. 2012).
Beside direct in vitro method of evaluation, more indirect methods can reflect
drug release from NPs. These are methods using biological supports like cells in
culture or biological membranes, as well as in vivo methods.
b. In cellulo methods
These techniques study the uptake of drug into the cells, either by the direct
mean of intracellular measurements [for example, measurement of cyclosporine A
permeation across and penetration inside rabbit cornea epithelial cells (Gke et al.
2008), intracellular paclitaxel uptake in Lewis lung carcinoma cells (Lv et al.
2011)], or in an indirect way by detecting a biological effect induced by the drug
within the cell.
In the present chapter, in cellulo methods are mostly implemented with
nanoparticulate systems of anticancer drugs like doxorubicin, paclitaxel, and cis-
platin, or antifungal drugs like amphotericin B, and antibiotics like ciprofloxacin.
Cultured cells are especially adapted to assess the cellular uptake of these drugs and
their respective biological activity, i.e., cytotoxicity, antifungal efcacy, and
antibacterial activity. The evaluation of siRNA nanoformulations also necessitates
in cellulo methods since cellular uptake is one of the main obstacle siRNAs have to
overcome in order to reach their intracytoplasmic site of action. In addition, their
transfection efciency can be evidenced through gene silencing assessment.
The critical parameters of these methods include in particular the cell line
employed, the conditions of their culture and the composition of the culture med-
ium in which the NPs will be tested, the concentrations at which the cells will be
exposed, the duration of the release study. The extraction method of the drug from
cells, and the detection limit of the measurement method are critical as well in
uptake studies. Finally, the sensitivity and specicity of detection of a biological
effect are also of major importance.
c. Ex vivo methods
Ex vivo methods consist in studying the permeation of drugs across biological
membranes. Among the 12 drugs studied, ex vivo methods are rarely implemented
to study drug release from nanoparticles. They concern only paclitaxel, cyclos-
porine A, and acyclovir nanoformulations. Franz cells are mostly used (Aksungur
et al. 2011; Karavana et al. 2012; Hasanovic et al. 2009), then Ussing chambers
(Ageros et al. 2009; Zabaleta et al. 2012), and Valia-Chien chambers (Gkce et al.
2008). Except for paclitaxel PNPs intended for oral administration which perme-
ation has been studied across rat jejunum (Ageros et al. 2009; Zabaleta et al.
2012), other permeation studies have been performed on tissues specic for local
administration, i.e., cow buccal mucosa (Karavana et al. 2012) and pig cornea
(Gke et al. 2008) for cyclosporin A nanoparticles, or porcine abdominal skin for
acyclovir nanogels (Hasanovic et al. 2009).
d. In vivo methods
Although costly and difcult to implement, in vivo methods of evaluation are
frequently employed either to assess pharmacokinetics in heathy animals (mice,
rats, dogs) for nine out of the 12 drugs studied, or to evidence a therapeutic effect in
diseased animals for all the drugs studied except for ibuprofen, dexamethasone,
ciprofloxacin and acyclovir. While classical pharmacokinetics studies based on
bioavailability after oral or IV administration are usually implemented, other studies

are specic of the ocular route of administration (Zhou et al. 2013; Aksungur et al.
2011). Among experimental models of diseases, beside models of tumor-bearing
animals often implemented for testing nanoformulations of anticancer drugs, vari-
ous models are proposed such as aspergilloses (Van de Ven et al. 2012), crypto-
coccal meningitidis (Xu et al. 2011), oral ulcer (Karavana et al. 2012), corneal
transplantation (Yuan et al. 2008), femoral artery stenosis (Cyrus et al. 2008),
diabetes (Damg et al. 1988; Aboubakar et al. 1999; Zhang et al. 2012; Sung et al.
2012) in mice, rats, rabbits or dogs.
One common issue to pharmacokinetics and pharmacodynamics studies concern
the monitoring of the drug: is it the drug by itself, or the nanoparticles including the
drug, or a tracer, that are monitered? Is the drug still associated with the
nanoparticles along its circulation within the organism? Ideally, one would label
both the drug and its nanoparticulate carrier in a stable manner in order to follow the
fate of both in vivo.
Table 13 summarizes the advantages and limitations of the different methods for
evaluating drug release from nanoparticles.
Table 13 Advantages and limitations of methods for evaluating drug release from
nanoformulations
Advantages Limitations
In vitro methods Easy implementation Release prole dependent on the
Dissolution Adapted to a large variety of release medium composition
Dialysis drugs especially for drugs of poor water
Low cost solubility
Possible misinterpretation with
very sparingly soluble drugs
Not adapted to evaluation of siRNA
NPs
Distant from in vivo conditions
In cellulo methods Implementation relatively easy Results highly dependent on the
Cellular uptake Closer to biological conditions cell line and on the testing
Biological effect than in vitro methods conditions
detection Methods of choice for testing Necessity to extract the drug from
siRNA NPs cells in uptake studies
Moderate cost
Ex vivo methods Closer to in vivo conditions Difculty of implementation
Release across than in vitro and in cellulo Higher cost than above-cited
biological methods methods
membranes Various biological membranes
may be used representing
various administration routes
In vivo methods Closest conditions to future Difculty of implementation
Pharmacokinetics utilization in medicine High cost
in healthy animals Determination of the Necessity to extract the drug from
Therapeutic effect nanoparticle fate in a whole biological samples
in diseased animals organism
6 Conclusion
The challenge of drug release from nanoparticles remains a key point in the
development of nanomedicines. Promising tracks for tuning release proles from
nanoparticles can be based on the physicochemical properties of the drugs, the
types and characteristics of nanoformulations, and on the route of administration.
Concerning the drugs by themselves, hydrophobic ones with very low water sol-
ubility can benet from encapsulation into nanoparticles to improve their oral
bioavailability or to achieve a sustained release after injection, and/or to reduce side
effects due to toxicity; while hydrophilic ones with good water solubility but poor
permeability can see their absorption optimized through inclusion into nanoparti-
cles, and their stability improved in challenging physiological conditions. Both
types of drugs can be loaded into a large variety of nanoformulations including
notably PNPs, MNPs, core-shell NPs, PEC, NCs, cubic NPs, MagNPs,
self-assemblies, or nanogels.
As for the administration route, it will impose constraints in terms of (i) particle
size, especially for injectable formulations; (ii) stability in biological environments
like blood for nanoparticles intended for IV administration, or gastrointestinal
media for oral formulations, of specic conditions for local administrations; and
(iii) release prole.
To meet these requirements, the choice of the nanoparticle type is decisive.
Among the large panel of nanoparticles available, PNPs are versatile nanoparticles
that allow for a rapid release within a few minutes as well as extremely extended
release duration up to 50 days, via erosion-diffusion mechanisms. The diversity of
release proles can be achieved through the control of the nanoparticle size and size
distribution, the choice of the polymer composition and molecular weight, the
control of the surface charge of the nanoparticles, the crosslinking of the polymers,
and the percentage of drug loading. Such nanoparticles can be administered orally,
locally, or by injection. MNPs present the double advantage of providing con-
trollable release through the adjustment of the nanoparticle size, and/or through the
functionalization of the nanoparticle surface, and of allowing for pH-sensitive
delivery as soon as a pH-responsive agent like chitosan is associated to the
nanocarrier. MNPs are of special interest for targeting inflammatory tissues and
tumor cells characterized by an acidic pH. They can be administered by the IV, oral,
or local routes. Core-shell NPs and PC offer release durations of about 140 h for
the former, and up to 9 days for the latter; they are principally intended for IV
administration, more rarely for oral, topical or intratumoral administration. Both
types of nanoparticles can provide pH-responsive systems, as well as cubic NPs,
MagNPs, and self-assemblies.
However, the goal of tuning release proles is still difcult to achieve and
methods for studying drug release are of particular usefulness with this intention.
While in vitro methods may be implemented in a rst approach for an easy
screening among a panel of formulas, they present limitations in terms of inter-
pretation, especially for drugs of very poor solubility. If in cellulo methods may be
complementary, notably for antitumor efcacy evaluation, they are inevitable when
cellular uptake is the major barrier to be overcome, like for siRNAs. The main
interests of ex vivo methods are to be closer to in vivo conditions and to allow
absorption assessment especially for oral or local formulations. Finally, in vivo
methods are essential to determine the fate of nanoformulations in a whole
organism and to prove their therapeutic efciency.
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Chapter 15
Interaction Between Nanoparticles
and Plasma Proteins: Effects
on Nanoparticle Biodistribution
and Toxicity
Anna N. Ilinskaya and Marina A. Dobrovolskaia
Abstract Nanoparticles are increasingly used in biomedical applications as active

pharmaceutical ingredients, drug carriers, or medical devices. Nanoparticles inter-
action with plasma proteins may influence their biodistribution by promoting
interaction with and uptake by the circulating and tissue resident phagocytes.
Biodistribution to off intended target-sites may lead to decrease in therapeutic
efcacy and result in undesirable toxicities. Therefore understanding nanoparticle
physicochemical properties, which determine protein binding, and consequences of
protein corona on nanoparticle biodistribution and toxicity are important elements
of the preclinical development of nanomedicines and nanoparticle-based medical
devices. The focus of this chapter is to discuss the most recent data on nanoparticle
interactions with blood components and how particle size and surface charge dene
their compatibility with the immune system.

Keywords Bovine serum albumin (BSA) Complement activation related pseu-
doallergy (CARPA)
Complement receptor (CR)
High density lipoprotein

(HDL) Mononuclear phagocytic system (MPS) Mass spectrometry (MS) N-
Acetyltransferase 1 (NAC1)
Polyacrylamide gel electrophoresis (PAGE)

Pharmacodynamic (PD) Poly(ethylene glycol) (PEG) Pharmacokinetic (PK)
Reactive oxygen species (ROS)
Thermo-responsive diblock copolymer
nanoparticles (TDCN)
A.N. Ilinskaya M.A. Dobrovolskaia (&)

Cancer Research Technology Program, Nanotechnology Characterization Laboratory,
Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc.,
Frederick, MD 21702, USA
e-mail: marina@mail.nih.gov

DOI 10.1007/978-3-319-41421-8_15
506 A.N. Ilinskaya and M.A. Dobrovolskaia
1 Introduction
Ability of nanoparticles to improve solubility of hydrophobic drugs, change

pharmacokinetics and/or reduce toxicity made them attractive platforms for drug
delivery. In addition, some nanoparticles may be used as therapeutics per se, as
components of medical devices, or constituents of vaccine formulations. The
application of nanoparticles as drug delivery platforms hinges on avoiding elimi-
nation by the mononuclear phagocytic system (MPS).
Nanoparticles administered through systemic route immediately interact with
plasma proteins. Human plasma proteome consists of approximately 3700 proteins,
55 % of which are represented by albumins, 38 % by globulins, and 4 % by
brinogen (Jeong et al. 2009; Anderson and Anderson 2002; Monopoli et al. 2012).
Plasma protein repertoire is very dynamic in that it changes depending on diet,
physiological condition, stress; it also varies in health and disease, as well as
depends on the genetic background of individual donor. For example, quantity of
lipoproteins in plasma varies with a diet, cytokine levels and C-reactive protein
raise during infections, serum deprivation proteins and heat shock proteins ele-
vate during stress, albumin, globulin, and brinogen fluctuate in response to
environmental changes, while blood group antigens and Rhesus factor are only
present in the blood of individuals with certain genetic background. During disease,
new proteins may appear in the plasma; examples include disease specic antigens
(e.g., cancer biomarkers) and foreign proteins from pathogens. Binding of proteins
to nanoparticle surface is also a dynamic process which depends on nanoparticle
physicochemical properties (size, charge, and hydrophobicity). The mechanisms of
binding may also be different. Some studies suggested that abundant plasma pro-
teins (e.g., albumin) bind to nanoparticle surface during earlier time points but later
they get displaced by other proteins through the so-called Vromans effect (Casals
and Puntes 2012; Milani et al. 2012). However, others concluded that there is no
typical Vromans effect in nanoparticle protein interaction (Jansch et al. 2012;
Dobrovolskaia et al. 2014). The difference in conclusions between the studies is not
surprising because nanoparticles (diverse metal colloids and polymer particles),
source of proteins (plasma, serum, puried proteins), and methods for analysis
(mass spectrometry, gel electrophoresis, fluorescent correlation spectroscopy) vary
between the studies.
The interaction of the nanoparticles with certain proteins marks them for the
uptake by the MPS. Phagocyte uptake may greatly reduce the number of circulating
nanoparticles available to reach the target site and results in reduction in the drug
efcacy and/or inflammation-mediated toxicity at the organ accumulating resident
phagocytes (Dobrovolskaia and McNeil 2013; Roser et al. 1998; Demoy et al.
1999). Function of phagocytic cells was shown to correlate with clearance of
nanocarriers (Caron et al. 2013). Moreover, the variability between individual
patients in the pharmacokinetic (PK) and pharmacodynamic (PD) parameters of
nanoparticle-delivered drugs was attributed to the phagocytic function of macro-
phages and dendritic cells (Zamboni et al. 2011a, b). This nding was consistent
15 Interaction Between Nanoparticles and Plasma Proteins 507
across different species including mice, dogs, rats, and human (Caron et al. 2013).
Protein binding was also shown to affect particles ability to aggregate and interact
with other blood cells (De Paoli et al. 2014). While reports regarding the role of
nanoparticle physicochemical properties on formation of protein corona and sub-
sequent effects of the corona on particle biodistribution are consistent, opinions
regarding the role of identity of protein corona diverge. While some studies report
correlation between identity of proteins in the corona and relevant toxicity
(Monopoli et al. 2012; De Paoli et al. 2014; Lundqvist et al. 2008; Walczyk et al.
2010); others demonstrate that composition of the protein corona per se cannot be
used to predict hematological toxicities of nanoparticles (Dobrovolskaia et al. 2014;
Deng et al. 2011, 2012).
Below we will review available literature highlighting the role of nanoparticle
physicochemical properties in formation of so-called protein corona and the
influence of this corona on particle biodistribution. We will also evaluate the link
between composition of the protein corona and nanoparticle toxicity.
1.1 Protein Binding and Nanoparticle Biodistribution
Nanoparticle uptake by the phagocytic cells is determined by physicochemical

properties, such as nanoparticle size, zeta potential, hydrophobicity, and surface
functionality. In general, larger particles are taken up by macrophages more ef-
ciently than their smaller counterparts possessing the same composition and surface
properties (Fang et al. 2006a, b). Charged particles are more attractive to phago-
cytes then their neutral counterparts of the same size (Zahr et al. 2006).
Nanoparticle physicochemical properties can be manipulated to either specically
attract the immune cells when particle uptake is desirable (e.g., during antigen
delivery for vaccine applications) (Cui and Mumper 2002) or to avoid the immune
recognition when the uptake is unwanted (e.g., during drug delivery in cancer)
(Paciotti et al. 2004). For example, addition of chitosan or mannose onto
nanoparticle surface makes these particles attractive to the phagocytes and deter-
mines their uptake through mannose receptor mediated route, which improves the
immune response to nanoparticle bound antigens (Cui et al. 2003; Cuna et al.
2006). Addition of hydrophilic polymers to the nanoparticle surface is commonly
used as a tool to avoid nanoparticle recognition by the immune cells (Paciotti et al.
2004; Csaba et al. 2006a, b). Several polymers including poly(ethylene glycol)
(PEG), poloxamer, and poloxamine are the mostly often used in formulations
specically designed for medical applications. Addition of these polymers to
nanoparticle surface prolongs particle circulation in the blood and signicantly
decreases uptake by spleen and liver resident phagocytes. PEG is thought to form a
hydrodynamic shield around the particle, which prevents plasma proteins from
binding to particle surface and subsequent uptake by macrophages.
Protein binding occurs almost instantaneously as a particle enters blood stream;
the nanoparticle protein complex is then delivered throughout the body. The
physical characteristics of nanoparticle protein corona complex can be different

from native nanoparticles. For example, protein binding to anionic gold colloids
results in increase in nanoparticle hydrodynamic size and a change in their zeta
potential (Dobrovolskaia et al. 2009, 2014; Franca et al. 2011). Binding of plasma
proteins to nanoparticle surface may also promote particle aggregation and pre-
dispose larger aggregates to elimination by liver resident macrophages, while
nonaggregating particles or particles forming smaller aggregates are distributed to
other organs (Mohr et al. 2014). The proteins which help phagocytic cells to
recognize foreign particulate and promote their uptake are called opsonins. Binding
of such proteins to nanoparticle surface, also known as opsonization, leads to
nanoparticles clearance from bloodstream by the MPS (Owens and Peppas 2006).
There are several types of proteins that can act as opsonins: complement,
immunoglobulins, acute-phase proteins, laminin, bronectin, and brinogen. Of
them complement and immunoglobulins are the most potent ones, because
phagocytes express complement receptors (CR1/3) and Fc-gamma receptors which
recognize these proteins. The amount of complement and immunoglobulins on the
surface of nanoparticles directly correlates with the level of uptake by phagocytic
cells (Owens and Peppas 2006; Nagayama et al. 2007; Leroux et al. 1995; Karmali
and Simberg 2011). In addition, phagocytic cells express scavenger receptors which
recognize variety of opsonins including fetuin (Nagayama et al. 2007).
Interestingly, nanoparticle protein corona may decrease nanoparticle uptake by one
type of cells and facilitate the uptake by other cells. For example, the presence of
protein corona signicantly reduced adhesion of di-sulde stabilized poly-
(methacrylic acid) nanoporous polymer nanoparticles to monocytic cell line THP-1
cells, but increased the uptake of these nanoparticles by THP-1 line after the cells
were differentiated to attain macrophage phenotype (Yan et al. 2013).
The mechanism by which protein binding occurs is not completely understood.
However, the important components involved in this process are pretty well known.
Of the proteins present in plasma the most common ones found in nanoparticle
protein corona are the immunoglobulins, the complement as well as brinogen and
albumin (Owens and Peppas 2006). However, the complete composition of the
protein corona at any given time depends on many factors including but not limited
to protein concentration, protein repertoire and kinetic properties of individual
proteins such as equilibrium constants, on/off rates and binding afnity (Lynch et al.
2007). Therefore, the composition of the nanoparticle protein corona is dynamic
and changes through the duration of nanoparticle presence in systemic circulation.
Even though presence of certain proteins in the corona predisposes nanoparticles
for the uptake via certain receptors, e.g., fetuin was shown to target nanoparticles
for the uptake through scavenger receptor (Nagayama et al. 2007), opsonized
nanoparticles may be internalized by a macrophage via multiple routes including
both phagocytic routes and various forms of pinocytosis (Franca et al. 2011).
Following internalization nanoparticle fate depends on its composition in that
biodegradable particles are digested and cleared from the body (Chellat et al. 2005;
Ljubimova et al. 2008a, b), while nonbiodegradable particles accumulate in
phagocytic cells and stay inside MPS organs for a long time (Niidome et al. 2006).
1.2 Protein Binding and Nanoparticle Toxicity
As discussed above, proteins forming nanoparticle corona may influence

nanoparticle biodistribution, clearance route, and uptake by cells of the immune
system. In turn, some nanoparticles may affect protein function by inducing its
conformational changes and exposure of hidden epitopes. For example, Deng et al
demonstrated that brinogen binding to poly(acrylic acid)-conjugated gold
nanoparticles results in conformational changes in the protein which lead to
exposure of a hidden ligand specic to Mac-1 receptor (Deng et al. 2011). Although
binding of a protein to nanoparticle surface may result in activation of the protein
which ultimately may result in toxicity, e.g., complement binding to carbon nan-
otubes activates complement and leads to anaphylactic reactions (Salvador-Morales
et al. 2006) or brinogen binding to poly(acrylic acid)-coated particles results in
activation of Mac-1 receptor and subsequent inflammation (Deng et al. 2011),
prediction of nanoparticle toxicity solely based on the composition of protein
corona is less likely to be accurate. Indeed, the same study demonstrating change in
the function of brinogen after binding to nanoparticle surface, has also reported
that such effect was detected only with SiO2 particles, while two other metal oxides
which also bound brinogen had signicantly less effect on conformation of this
protein (Deng et al. 2011). Likewise, binding of complement and brinogen to the
surface of colloidal gold nanoparticles neither resulted in activation of these pro-
teins nor affected their normal function in response to factors known to activate
complement and coagulation (Dobrovolskaia et al. 2014).
For the purpose of this chapter, we divide studies reporting nanoparticle toxic-
ities associated with protein corona into following categories: (1) effects of
nanoparticles on proteins composing the corona, (2) effects of the protein corona on
nanoparticle toxicity, and (3) effects of nanoparticles on cells after the uptake. The
close relationship between these categories is summarized in Fig. 1.
1.2.1 Effects of Nanoparticles on Proteins
Proteins bound to the surface of nanoparticles may retain their native state or
undergo conformational changes (Deng et al. 2011; Lundqvist et al. 2004; Shang
et al. 2007, 2009; Linse et al. 2007). Such changes may result in gain or loss of
protein function, alteration of protein stability, and/or exposure of hidden epitopes.
The induction of structural changes in the protein upon binding to a surface is not
unique to nanomaterials and was frequently reported for other articial surfaces
interfacing biological fluids. However, unlike flat surfaces, the likelihood of change
in protein structure after binding to a nanoparticle surface depends on the particle
size. Large nanoparticles are more likely to affect protein structure than their smaller
counterparts of the same composition due to the higher degree of protein coverage
(Shang et al. 2007; Vertegel et al. 2004). Lower degree of protein coverage on
smaller nanoparticles is thought to be due to their higher curvature (Cedervall et al.
Fig. 1 Dual relationship between nanoparticles and proteins. Nanoparticles may affect protein
structure and/or change protein function, as well as affect protein conformation and result in
exposure of new otherwise masked epitops available for immune recognition. Proteins in turn
result in change in nanoparticle physicochemical properties, affect nanoparticle biodistribution and
clearance, and interfere with targeting
2007). In contrast, larger particles with lower curvature resemble flat surface and as
such are more prone to unfold the proteins bound to this surface (Shang et al. 2007,
2009; Vertegel et al. 2004). The larger the particle is, the more points of contact
with the protein exist, which results in stronger interaction between protein and the
particle surface. It would be interesting to understand what role the relationship
between particle size and protein size plays in protein binding to nanoparticle
surface.
Nanoparticle effect on the protein conformation and function is also determined
by the particle surface properties including presence of functional moieties and zeta
potential. Stabilization or disruption of the secondary protein structure was shown
to depend on nanoparticle surface charge, in that protein binding to nanoparticles
baring surface charge opposite to that of a protein results in higher rate of distur-
bance of the protein secondary structure (Cukalevski et al. 2011). For example,
binding of anionic proteins [apolipoprotein A-I, apolipoprotein B100 and high
density lipoprotein (HDL)] to cationic polystyrene nanoparticles results in more
prominent changes in the protein secondary structure than binding of the same
proteins to nanoparticles with neutral or anionic surface (Cukalevski et al. 2011). In
contrast, no signicant changes in the secondary structure of cationic lysozyme
were observed after its binding to cationic particles (Cukalevski et al. 2011).
Binding of bovine serum albumin (BSA) to the surface of cationic, but not anionic,
polystyrene nanoparticles caused changes in the protein secondary structure
(Fleischer and Payne 2014). Nanoparticle hydrophobicity is another important
factor dening interaction with proteins. For instance, trypsin binds with higher
afnity to the hydrophobic surface of polystyrene nanoparticles than to the
hydrophilic surface of silica nanoparticles (Koutsopoulos et al. 2007).
Conformational changes of a protein with enzymatic activity may affect protein

activity and stability (Koutsopoulos et al. 2007; Deng et al. 2014). The stronger the
interaction between nanoparticles and adsorbed proteins is, the more likely that the
proteins structure and activity will be altered (Shang et al. 2007; Koutsopoulos
et al. 2007). For example, coagulation factor XII undergoes self-activation when
absorbed on the surface of 220 nm anionic polystyrene nanoparticles but not on the
surface of smaller (24 nm) counterparts (Oslakovic et al. 2012). Furthermore,
mechanisms leading to a change in the protein enzymatic activity vary for different
nanoparticles. For example, metal nanoparticles (TiO2, SiO2, ZnO) directly bind
and inactivate N-acetyltransferase 1 (NAC1), while synthetic layered silicate
nanoparticles do so indirectly by facilitating substrate binding prior to interaction
with enzyme cofactor (Deng et al. 2014). In contrast to other nanoparticles, NAC1
binding to thermo-responsive diblock copolymer nanoparticles (TDCN), consisting
of poly(dimethylacrylamide) and poly(N-isopropylacrylamide), results in
enhancement of NAC1 enzymatic activity (Deng et al. 2014). Another possible
consequence of protein interaction with nanoparticles is changing stability of
absorbed proteins (Shang et al. 2007; Deng et al. 2014). For instance, thermody-
namic stability of ribonuclease A decreases with adsorption on the surface of silica
nanoparticles (Shang et al. 2007). In contrast, binding of NAC1 to TDCN results in
stabilization of the protein under heat denaturing condition (Deng et al. 2014).
Protein unfolding may expose hidden epitopes and cause activation of the
immune system. For instance, interaction of brinogen with both 5 and 20 nm poly
(acrylic acid)-coated gold nanoparticles resulted in unfolding of the protein and
exposure of the epitope which serves as a ligand to Mac-1 receptor. However, only
5 nm nanoparticles were capable of binding to Mac-1 on cells. Interestingly, even
though 20 nm nanoparticles caused unfolding of the protein, the unmasked epitope
was not approachable for interaction with Mac-1 because too many brinogen
molecules blocked interaction between the epitope and the receptor. Nanoparticles
which induced modications in brinogen conformation promoting its interaction
with Mac-1 resulted in production proinflammatory cytokines (Deng et al. 2011).
Furthermore, some nanoparticles may aid in cytokine release by activating
inflammasome. For instance, TiO2 and SiO2 activate the NLRP3 inflammasome in
human macrophages and keratinocytes; this process leads to activation of caspase-1
which is essential for processing of pre-IL-1b to IL-1b and secretion of mature
cytokine (Yazdi et al. 2010).
Damaged or incorrectly processed host proteins are removed by macrophages
through scavenger receptors, therefore, the presence of damaged or altered proteins
on nanoparticle surface may facilitate uptake of nanoparticles by cells through
scavenger receptors (Yan et al. 2013; Fleischer and Payne 2014; Dutta et al. 2007).
Interestingly, the condition of a protein on the nanoparticle surface denes the way
the protein will be internalized. For instance, presence of native BSA on
nanoparticle surface favors internalization through albumin receptor, while
nanoparticles with altered BSA are eliminated through scavenger receptors
(Fleischer and Payne 2014). Coating of nanoparticles with surfactants, which
prevent protein adsorption or inhibition of scavenger receptors, signicantly

reduced uptake and toxicity of nanoparticles (Dutta et al. 2007).
Another mechanism of nanoparticle toxicity related to nanoparticle interaction
with plasma protein is associated with activation of complement system. The sol-
uble products of complement cleavage (C3a, C4a and C5a) are called anaphyla-
toxins; they induce chemotaxis, activation of B-cells, and trigger inflammation and
anaphylaxis (Moghimi 2014). Activation of complement contributes to the devel-
opment of acute reaction also known as Complement Activation-Related
Pseudoallergy (CARPA), which is a common dose limiting toxicity of certain
types of nanoparticles, e.g., PEGylated liposomes (Moghimi and Farhangrazi 2013)
(Szebeni 2005). Nanoparticles characteristics, such as composition, charge, shape,
hydrophobicity, presence of repetitive motifs on the surface are important in pre-
diction of complement activation and identity of the pathways triggering such
activation (Moghimi et al. 2011). For example, incidents of hypersensitivity upon
administration of liposomal drugs varies from 3 to 45 % (Szebeni 2005). This
variability reflects the differences in liposomal structures and characteristics. For
instance, negatively charged liposomes activate complement via classical pathway,
cationic liposomes trigger the alternative pathway, and neutral liposomes are least
likely to activate the complement (Salvador-Morales and Sim 2013).
It has been demonstrated that an interface between liquid-solid or liquid-liquid
surfaces facilitates formation of amyloid brils (Linse et al. 2007; Zhu et al. 2002).
Deposition of these toxic atypical protein clusters results in development of human
amyloid diseases, such as Alzheimer disease, Parkinson disease, and diabetes
mellitus type II. The formation of amyloid clusters is a two-stage process com-
prising of the lag (or nucleation) and the elongation phases (Hellstrand et al. 2010).
Several types of nanoparticles (co-polymers, quantum dots, carbon nanotubes, and
cerium oxide nanoparticles) enhanced formation of amyloid brils in vitro by
shortening the lag phase (Linse et al. 2007). However, in another study TiO2
nanoparticles but not cerium nanoparticles were capable of shortening nucleation
process in vitro (Wu et al. 2008). These ndings suggest that certain nanoparticles
may contribute to development of pathologies through induction of change in
protein conformation, function, and increase in protein accumulation (Zaman et al.
2014). However, more studies are necessary to better understand the cause-effect
relationship.
1.2.2 Effects of Proteins on Nanoparticles
Protein adsorption on nanoparticle surface may change physicochemical properties

of the particles. Interaction with proteins may change hydrodynamic size and
charge of nanoparticles. The size alteration can be achieved through the accumu-
lation of proteins on the surface or through induction of particle aggregation. For
instance, size of 30 nm gold nanoparticles is increased after incubation with plasma
(Dobrovolskaia et al. 2009; Franca et al. 2011). Fibrinogen can connect closely
located particles causing particle aggregation (Vauthier et al. 2011). The alteration
of particles size may affect nanoparticle distribution (Wolfram et al. 2014). Since
particle size is important determinant of nanoparticle clearance route, increase in
size as a result of nanoparticle protein corona formation may change accumulation
of nanoparticles in different organs. For example, particles smaller than 58 nm are
removed from circulation by kidneys; liver resident macrophages eliminate particles
with larger size (50200 nm), while red pulp macrophages in spleen capture par-
ticles with size 200300 nm; in all of these cases conformation of polymer on
particle surface is important factor contributing to the clearance through certain
MPS organs, while particles larger than 300 nm regardless of polymer coating are
cleared by all organs of the MPS (Wolfram et al. 2014; Nel et al. 2009;
Dobrovolskaia and McNeil 2013; Caron et al. 2013).
The presence of some proteins on the surface of nanoparticles can facilitate
rerouting of nanoparticles to different organs. For example, apolipoprotein on the
surface of nanoparticles was shown to facilitate nanoparticle transfer through
bloodbrain barrier probably through the interaction with low density lipoprotein
receptors on the brain capillary endothelial cells (Goppert and Muller 2005;
Michaelis et al. 2006; Kreuter et al. 2002). Intuitively, spontaneous binding of
apolipoprotein to a nanoparticle may have detrimental consequences if such
nanoparticle is not intended for delivery into the brain. Protein corona can interfere
with nanoparticle targeting. For instance, SiO2 grafted with targeting molecules for
transferrin receptors lost capacity to interact with transferrin receptor after incu-
bation with serum, possibly due to the protein corona shielding the targeting ligand
on the surface of nanoparticles (Salvati et al. 2013).
1.2.3 Effects of Nanoparticles on Cells After the Uptake
Engulfed nanoparticles can be toxic to the phagocytic cells. For instance, deposition
of positively charged nanoparticles in lysosomes can lead to the activation of proton
pump mechanism (Xia et al. 2008). In acidic environment positively charged
nanoparticles act as a proton sponge triggering the influx of protons and retention of
Cl ions and water. It causes swelling and eventual rupture of lysosomes which
release its content into cytoplasm. Among other mechanisms of nanoparticles
toxicity toward phagocytic cells is generation of reactive oxygen species
(ROS) (Jeong et al. 2011; Brown et al. 2004), which can induce organelle and DNA
damage (Di Bucchianico et al. 2013) and act as a second messengers inducing
signaling pathways leading to synthesis of proinflammatory cytokines (Brown et al.
2004; Singh and Ramarao 2012). Dissolution of nanoparticles and release of ions
could induce mitochondrial damage and could lead to apoptosis (Singh and
Ramarao 2012). It was shown that intracellular dissolution of silver nanoparticles is
50 times higher compared to this process in water due to high oxygen tension and
generation of free radicals inside the cells (Singh and Ramarao 2012). Perturbation
of cellular metabolic pathways is another mechanism of nanoparticles toxicity
(Triboulet et al. 2014). For example, after phagocytic uptake zinc oxide nanopar-
ticles directly bind and induce carbohydrate catabolic enzymes. The toxicity of
these particle is also thought to be due to their dissolution and ion release inside the
cell (Triboulet et al. 2014).
Several studies have demonstrated that protein binding to nanoparticle surface
affects interaction of the nanoparticle with cells and therefore influences particle
toxicity. For example, binding of brinogen and albumin to carbon nanotubes
prevented nanotube interaction with and activation of human platelets in vitro (De
Paoli et al. 2014). Likewise, binding of fetuin and albumin decreased cytotoxicity of
calcium phosphate nanoparticles to vascular smooth muscle cells (Dautova et al.
2014).
1.3 Methods Used to Study Protein Corona
Analysis of protein corona involves two major steps: protein isolation and protein
identication (Fig. 2). The process of identication of protein corona composition
starts from the separation of nanoparticle bound proteins from bulk plasma, serum,
or other protein containing biological fluid. Prevention of proteins dissociation from
nanoparticles is the main challenge during this step and has been reviewed else-
where (Aggarwal et al. 2009). Centrifugation, dialysis, size-exclusion chromatog-
raphy, and magnetic isolation are common techniques employed to separate
nanoparticle bound proteins from biological fluids. All of these methods have their
limitations. For instance, centrifugation is the most widely used technique. It is
easy, does not require large quantities of material and several samples can be
processed concurrently (Aggarwal et al. 2009; Monopoli et al. 2013). However,
washing steps and solution volumes may affect the outcome (Treuel and Nienhaus
2013). Some proteins may be lost from protein corona during washing steps
(Cedervall et al. 2007). Furthermore, repeated centrifugation may cause proteins
aggregation and sedimentation resulting in false identication of these proteins in
protein corona (Aggarwal et al. 2009). Dialysis-based methods require higher
sample volumes compared with other methods (Treuel and Nienhaus 2013).
Size-exclusion chromatography has limited throughput (Monopoli et al. 2013;
Treuel and Nienhaus 2013). Magnetic separation is applicable only to paramagnetic
nanoparticles. It is interesting that despite the differences in these methods (cen-
trifugation, magnetic separation, and size-exclusion chromatography) they gave
similar results of protein corona composition (Monopoli et al. 2013).
The second step involves identication of proteins bound to nanoparticle sur-
face. Several approaches can be applied during this step. They include polyacry-
lamide gel electrophoresis (PAGE), capillary gel electrophoresis, and liquid
chromatography. One of the most widely used approaches is one- or two dimen-
sional polyacrylamide gel electrophoresis, 1D or 2D PAGE, respectively (Gessner
et al. 2002; Monopoli et al. 2013). For successful identication of proteins a sample
gel is needed to be compared with reference map of human plasma proteins
(Aggarwal et al. 2009). The main advantages of this method are logistical simplicity
and low cost. However, this method has a couple of drawbacks: (1) results may be
Fig. 2 Methodological challenges with analysis of nanoparticle protein corona. Analysis of

protein corona includes two major steps: protein isolation and protein identication. Variety of
methods is available to perform each of these steps. Each methodology has its own strength and
limitations. This gure summarizes main challenges with individual methodologies used to
analyze nanoparticle protein corona
affected by inter-donor variability, blood collection process, and anticoagulant,

making it hard to match the gel of interest to the protein map (Aggarwal et al.
2009), (2) lack of the standardized gel-analysis software complicates the analysis
and introduces additional variability in the test results. In addition some proteins,
which have extreme isoelectric point, are not suitable for 2D-PAGE (Karmali and
Simberg 2011). For further identication, separated proteins can be analyzed by
Western blot, N-terminal sequencing or mass spectrometry (MS) (Dobrovolskaia
et al. 2009; Aggarwal et al. 2009; Chonn et al. 1992). None of these methods of
identication is ideal. For instance, detection with antibodies can be employed for
detection of very limited amount of proteins at the same time. MS is expensive and
requires specialized equipment and trained personnel. These and other challenges
have also been recently reviewed by Capriotti et al. (2014).
2 Conclusion
Understanding nanoparticle interaction with proteins is important for both biodis-

tribution and toxicity. Nanoparticle uptake by the cells, their interaction with cells
and mechanisms of toxicity are influenced by the protein repertoire bound to par-
ticle surface. In turn, nanoparticle surface may also modify the protein secondary
structure and activity, thus affecting its normal function. While total amount of
protein bound to nanoparticle surface can be used as a predictor of rapid
nanoparticle clearance from circulation by the MPS, single fact of the protein
presence on nanoparticle surface cannot be used to predict a change in the protein
function and nanoparticle toxicity. Specialized hematology, immunology, and cell
biology tests should be used to explore toxicities and their mechanisms.
Information about composition of the protein corona is important to understand
what protein may contribute to the observed toxicity and verify potential mecha-
nisms. Combined together understanding of how nanoparticle physicochemical
properties influence formation of protein corona, and knowledge about contribution
of individual proteins of the corona to nanoparticle toxicity will be helpful to design
nanoparticle drug carriers with improved safety prole.
Acknowledgments This project has been funded in whole or in part with Federal funds from the
Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract
HHSN261200800001E. The content of this publication does not necessarily reflect the views or
policies of the Department of Health and Human Services, nor does mention of trade names,
commercial products or organizations imply endorsement by the US Government.
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Chapter 16
Toxicological Aspects of Polymer
Nanoparticles
Juan M. Irache, Nekane Martn-Arbella, Patricia Ojer,

Amaya Azqueta and Adela Lopez de Cerain
Abstract This chapter describes the effects of some physico-chemical properties of

polymer nanoparticles influencing the development of toxicological effects. More
particularly, the effect of some parameters that may control the interaction of
polymer nanoparticles with the biological environment (such as their composition,
size, surface properties, and biodegradability) and, thus, be key factors of their
efcacy and toxicity, is discussed. In addition, the chapter also reviews the toxicity
results that have been found in the literature regarding the administration of
polymer nanoparticles as delivery systems by different ways of administration
including intravenous, oral, pulmonary, nasal, and ophthalmic routes.

Keywords Nanoparticles Toxicity Biodistribution Intravenous route Oral

delivery Pulmonary delivery Nasal delivery Ocular delivery
1 Introduction
In the past decades, polymer nanoparticles have demonstrated interesting capabil-

ities to develop applications that bring signicant advances in the diagnosis and
treatment of diseases. These applications include drug delivery, imaging,
nutraceuticals, and production of improved biocompatible materials (Duncan 2003;
De Jong and Borm 2008; Ferrari 2005). The reasons why these polymer
nanoparticles are attractive for medical purposes is based on their capability to carry
biologically active compounds and control their release as well as on their large
(functional) surface that can be used to link different ligands in order to modify their
J.M. Irache (&) N. Martn-Arbella P. Ojer

Department of Pharmacy and Pharmaceutical Technology, University of Navarra, C/Irunlarra,
1, 31080 Pamplona, Spain
e-mail: jmirache@unav.es
P. Ojer A. Azqueta A. Lopez de Cerain
Department of Pharmacology and Toxicology, University of Navarra, 31080 Pamplona,
Spain

DOI 10.1007/978-3-319-41421-8_16
522 J.M. Irache et al.
biodistribution and targeting properties. However, the same features that determine
the efcacy of these nanoparticles in the host (distribution, targeting and controlled
release properties) may contribute to their eventual accumulation and toxicity.
As a result, nanotoxicology has arisen as a new multi-interdisciplinary eld (as
part of the broader discipline of toxicology) to properly assess the safety and
toxicological issues of nanocarriers (Oberdorster et al. 2005a). However, till now,
most of this research has involved a limited group of nanoparticles, mainly inor-
ganic nanomaterials, for their environmental and public health treats concerns. In
fact, the toxicity of polymer nanocarriers (and other biodegradable devices) has
been studied to a lesser extent; although they are the most promising devices for
pharmaceutical applications. The main reason explaining this lack of information
would be related with the fact that the materials used to produce the so-called
polymer nanoparticles (e.g. polymers, lipids, proteins, polysaccharides) are, in
general, considered as safe. In fact, many of them possess a Generally Recognized
as Safe (GRAS) label or they are used as pharmaceutical excipients and
employed in other healthcare and pharmaceutical products. Thus, in most cases,
these polymer nanoparticles are considered safe as a result of the safety of the
source material used to their production. However, the properties and behaviour of
a material at the nano scale may be completely different from those observed in a
macroscopic scale. Consequently, the toxicological approach has to be substantially
different from the classical way to address the adverse health effects.
On the other hand, specic importance should be devoted to the toxicity of the
empty non-drug-loaded nanoparticles. For nanodevices used as drug delivery sys-
tems, the focus in most papers is mainly on obtaining a reduction of toxicity of the
incorporated drug, whereas the possible toxicity of the nanocarrier per se is not
considered or even ignored. However, particular attention must be paid in the case
of slowly degradable or non-degradable nanoparticles used for drug delivery. After
the delivery of the cargo, the residual components of these nanocarriers may
accumulate in the body causing possible adverse effects. Furthermore, in some
cases, the degradation products generated during the residence of nanoparticles in
the body should be considered as another toxicity risk factor.
In this context, to properly assess nanoparticle toxicity the following concepts
should be highlighted:
(I) After administration, the fate of polymer nanoparticles and their arrival to a
particular organs/cells in the body are dependent on their physico-chemical
properties.
(II) This distribution of polymer nanoparticles may lead to undesirable outcomes
such as crossing the blood barriers and activating the coagulation pathways.
(III) Apart from the physico-chemical characteristics of polymer nanoparticles,
their biodegradability within physiological conditions may determine their
elimination and, thus, their accumulation in the body.
(IV) This accumulation or prolonged residence time in direct contact with the
body tissues may result in a toxic response.
16 Toxicological Aspects of Polymer Nanoparticles 523
This chapter describes rst the main physico-chemical properties of nanoparti-

cles that may have an influence on a toxic response. Then, the chapter summarizes
the main toxicity concerns associate to the use of polymer nanoparticles adminis-
tered by different routes.
2 Factors of Polymer Nanoparticles Affecting Their

Toxicological Prole
The toxicity of polymer nanocarriers would be determined by their ability to reach

specic areas as their capability to accumulate within the body. In fact, the same
physico-chemical parameters determining their in vivo biodistribution, targeting
and efcacy would be involved in the toxicological effects. So, physico-chemical
parameters of polymer nanoparticles such as their composition, size, surface
properties, presence of ligands or shape, may result in different kinetic properties
and, thus, determine their fate when administered in vivo. Indeed, these
physico-chemical characteristics may facilitate the passage of nanoparticles through
membranes more easily and, thus, induce a toxic response when crossing these
barriers (Elsaesser and Howard 2012). This section reviews such physico-chemical
characteristics of polymer nanoparticles that may yield to the development of
toxicological effects.
2.1 Materials
A number of different polymers, macromolecules and lipids, both synthetic and

natural, have been utilized in formulating biodegradable nanocarriers. Many of
them are used as excipients for other pharmaceutical applications or they have the
consideration of GRAS; although, as indicated before, their transformation into
nanoparticulate devices may open the door to toxicological concerns.
The rst exhaustive toxicity studies involving polymer nanocarriers corre-
sponded to poly(alkylcyanoacrylates) (PACA) nanoparticles. Using different types
of cell cultures, the cytotoxicity of PACA nanoparticles was found to be dependent
on the length of the alkyl side chains. Overall, these nanoparticles induced a loss of
adhesion followed by dilation of the rough endoplasmic reticulum of the cells and,
nally, perforation of the cell membrane (Vauthier et al. 2003). In Swiss 3T3 cell
cultures, the cytotoxic effect of PACA nanoparticles decreased with increasing the
molecular weight, regardless of the kind of polymer used (Tseng et al. 1990a, b). In
line with these ndings, Lherm et al. (1992) show in L929 broblasts that only
nanoparticle with slow degradation kinetics (i.e. long alkyl side chain) was
non-toxic. This observation would be directed related to the release of degradation
products during the incubation of nanoparticles with cells (Muller et al. 1990).
Apart from the length of the alkyl chain of PACA and its influence on the degra-
dation rate of nanoparticles, cytotoxicity would be also mediated by the interaction
between PACA nanoparticles and cell membrane, which would facilitate the release
of the biodegradation products locally (Lherm et al. 1992).
Some of the most extensively investigated polymer nanoparticles are those based
on the use of poly(lactide) (PLA) and their derivatives with glycolic acid (PLGA)
(Lu et al. 2009). As polyesters in nature, all of them undergo hydrolysis of their
ester linkages in an aqueous environment, forming biologically compatible and
metabolizable moieties (lactic and glycolic acids) that are eventually removed from
the body by the citric acid cycle (Pandita et al. 2015; Mundargi et al. 2008). In
addition, their biodegradation products are formed at a very slow rate and, hence,
they would not affect the normal cell function (Pandita et al. 2015).
Other materials that have been extensively proposed for drug delivery purposes
include proteins (albumins), lipids and polysaccharides (chitosan). Overall, these
materials and their derivatives induce low toxicity effects (Merodio et al. 2002; Ojer
et al. 2013; Severino et al. 2014). However, information is scarce and usually limited
to some current cytotoxicity studies. Table 1 summarizes some of these studies related
with the evaluation of the cytotoxicity of polymer nanoparticles. In case of chitosan
nanoparticles, it has been demonstrated that they can open the tight junctions between
epithelial cells (Qi et al. 2005; Yeh et al. 2011). Therefore, the possibility that chitosan
nanoparticles enter into the circulation after their mucosal administration exists.
2.2 Size and Shape
The size of particulates has an important role in how the body responds to, distributes
and eliminates materials (Powers et al. 2006, 2007). Moreover, the particle size can
also affect the mode of endocytosis, cellular uptake, and the efciency of particle
processing in the endocytic pathway (Lanone and Boczkowski 2006; Rejman et al.
2004). All of this is related to the inverse relationship between particle size and surface
area exposed to the biological environment. As the size decreases, the surface area of
nanoparticles increases and, thus, the possibilities to interact with the biological media
also increases exponentially (Oberdorster et al. 2005a; Nel et al. 2006). In general, it is
considered that particles larger than 100 nm can only enter cells (and being potentially
toxic) by phagocytosis. Thus, these nanocarriers would mainly be taken up by mac-
rophages and other phagocytic immune cells that are mainly located in the liver and
spleen. On the contrary, polymer nanoparticles below 100 nm can be internalized by
(in principle) any cell by endocytosis and, therefore, they would have a considerable
higher toxicity risk (Verma and Stellacci 2010; Sahoo et al. 2002; Zauner et al. 2001).
In this way, it has been demonstrated that nanoparticles of about 100 nm show
2.5-fold greater uptake compared to 1 m and sixfold higher uptake compared to
10 m microparticles in Caco-2 cells (Desai et al. 1997). Furthermore, increased
uptake into certain tissues may lead to accumulation, where they may interfere with
critical biological functions (Lanone and Boczkowski 2006; Kreyling et al. 2006).
Table 1 Some examples of cytotoxicity studies with polymer nanoparticles

NP Characteristics Cell lines Observations References
PLGA NP 200350 nm Caco-2 Cell viability Semete et al.
of >75 % that was (2010a)
signicantly higher
than for ZnO NPs
PLGA NP Didodecyl Caco-2 NPs were not Bhardwaj
dimethylammonium cytotoxic et al. (2009)
bromide as stabilizer
PLA-TPGS 300330 nm HT-29; Caco-2 PLGA-TPGS NPs Zhang and
NP displayed 2-times Feng (2006)
higher cell uptake
efciencies than
classical PLGA NPs
CS-PLGA 150 nm Caco-2 The coating of NPs Zhang et al.
NPs Positively charged with CS did not (2012)
increase their
cytotoxicity
CS 80170 nm HT-29 Non-toxic effect on Hosseinzadeh
NP/CS-F127 Positively charged cells up to 20 M for et al. (2012)
NP 24 h incubation
CM-CS NP 200 nm NIH-3T3, L-929, In all cases, more Maya et al.
HEK-293 and than 80 % cells were (2012)
THP1 viable after 24 h
incubation with
150 g/mL. No
alteration of cell
morphology was
observed
PEG-CS NC 160250 nm Caco-2 Pegylation of CS Prego et al.
Positively charged reduced the (2006)
cytotoxicity. The NC
did not affect the
transepithelial
resistance of the
monolayer
Dextran CS 550 nm Caco2/HT-29/Raji Non-cytotoxic in cell Antunes et al.
NP B lines (24 h, (2013)
100 mg/mL)
PMM NP 130250 nm Caco2, HT29 NP were not Akhlaghi et al.
coated by Postively charged cytotoxic for HT29 (2010)
T-CS and Caco2
TMC-Cys 100200 nm Caco2 Lack of toxicity of Yin et al.
NP Positively charged these NP (2009)
LSC NP 315 nm Caco-2 Rekha and
Negatively charged Sharma
(2009)
(continued)
Table 1 (continued)
NP Characteristics Cell lines Observations References
HA-SLN 416 nm SK-OV-3 Both plain SLN and Mohammadi
Negatively charged HA-SLN were not Ghalaei et al.
cytotoxic (2014)
Zein NP 120140 nm Caco-2 NP showed no Luo et al.
cytotoxicity for 72 h (2013)
PLGA poly(lactide-co-glycolide), NP nanoparticles, ZnO zinc oxide, PLA-TPGS poly(lactide)-tocopheryl
poly(ethylene glycol succinate), PLA poly(lactide), CS chitosan, F127 Pluronic F-127, CM-CS
O-carboxymethyl chitosan, PEG poly(ethylene glycol), NC nanocapsules, PMM poly(methyl
methacrylate), T-CS chitosan-glutathione conjugate, TMT-Cys trimethyl chitosan-cysteine conjugate,
LSC lauryl succinyl, HA-SLN hyaluronic acid targeted solid lipid nanoparticles
The shape of polymer nanoparticles would also have an important effect on the
biological activity of nanocarriers. In an interesting study, spherical gold
nanocarriers displayed a higher capability to be taken up by HeLa cells than
nano-rod ones (Chithrani et al. 2006). More recently, Agarval and collaborators
(Agarwal et al. 2013) compared discoidal, rod-shaped and spherical poly(ethylene
glycol diacrylate)-based nanoparticles of equivalent volume and dimensions in
various cell lines (HeLa, HEK293, HUVEC and bone marrow dendritic cells). In all
cases, a discoidal shape facilitated the internalization of these hydrophilic
nanoparticles in all of these cells (Agarwal et al. 2013). However, in another study
with hydrophobic poly(styrene) nanoparticles, nano-rods and nano-discs displayed
a similar uptake in epithelial breast cancer cells (Barua et al. 2013). Curiously,
unlike for spherical nanoparticles, larged-sized nano-discs and nano-rods appear to
be internalized more efciently than their smallest counterparts (Agarwal et al.
2013). In any case, these differences could highlight the effect of other factors such
as the surface properties on the interaction between nanoparticles and the biological
substrate.
2.3 Surface Properties
Chemical composition at the surface of nanoparticles is another key parameter that

denes their interactions in vivo. Apart from the hydrophilic/hydrophobic proper-
ties of the surface of nanoparticles, other factors such as the presence of ionisable
groups or functional ligands may determine the capability of nanoparticles to
cross membranes and/or interact with biological components.
The use of pharmaceutical excipients is a popular strategy to improve the
physico-chemical stability of polymer nanoparticles; although, at the same time,
they can also modify the biodistribution and capability of these nanocarriers to
interact with the cells/tissues. As an example, the use of d--tocopheryl poly
(ethylene glycol) 1000 succinate (TPGS) as stabilizer of PLGA nanoparticles induced

a higher uptake of these nanocarriers by Caco-2 cells than when poly(vinyl alcohol)
(PVA) was used for the same purpose (Wing and Feng 2005). This nding is directly
related with the capability of PVA to render more hydrophilic the surface of the
resulting nanoparticles and, thus, to decrease their capture by cells (Sahoo et al. 2002).
In a similar way, Weiss and co-workers clearly displayed that poly (n-
butyl-2-cyanoacrylate) nanoparticles exhibited cytotoxicity as a consequence of their
capability to be internalized by cells. Thus, with Jurkat cells, it was demonstrated that
methoxypoly(ethylene glycol)-functionalized nanoparticles were taken up to a lesser
extent than either naked nanoparticles or phenylalanine-decorated nanoparticles
(Weiss et al. 2007). In a more recent work, Lira and collaborators, working with
fucoidan-coated poly(isobutylcyanoacrylate) nanoparticles demonstrated that the
coating degree dramatically affected the toxicity of the resulting nanoparticles. Thus,
in phagocytic cells, nanoparticles prepared with 100 % fucoidan appeared to be more
than four times more cytotoxic than nanoparticles prepared with 25 % fucoidan. These
results would be related to differences in the interaction prole of nanoparticles as well
as to their fate within the cells (Lira et al. 2011).
Another important factor that affects the interaction of nanoparticles with cells is
their charge. Overall, the uptake of nanoparticles by phagocytic cells increases by
increasing the net surface charge values (either positive or negative). However, when
the absolute values of zeta potential are similar, positively charged nanoparticles
appears to show a higher phagocytic uptake compared to negatively charged ones,
irrespective of their composition (He et al. 2010). This nding would be due to the
development of electrostatic interactions between nanoparticles and phagocytic cells
that would facilitate their internalization (Roser et al. 1998; das Neves et al. 2012).
With non-phagocytic cells, neutral and high positively charged nanoparticles seem, in
general, to be more efciently internalized (Gao et al. 2005; Lemarchand et al. 2006;
Tan et al. 2010). Nevertheless, this effect of the surface charge may also be modulated
by other factors such as the composition of material used to produce nanoparticles. In
this way, the surface properties of PLGA nanoparticles, coated with either chitosan
(positively charged) or poloxamer (negative), had just a mild effect on their cyto-
toxicity (Mura et al. 2011). In any case, positively charged nanoparticles are con-
sidered to be more toxic than negatively charged ones due to their high capability to
both interact and be taken up by cells (He et al. 2010; Klesing et al. 2010; Liu et al.
2011a). Moreover, the positively charged nanoparticles would induce DNA damage
and the activation of checkpoints in the cell cycle whereas the negatively ones do not
have obvious effects (Liu et al. 2011a).
The distribution of polymer nanoparticles can also be modulated by the covalent
binding of different excipients/ligands that are frequently used to generate blood
compatible carriers and/or for targeting therapy. Pegylation is one of the most popular
ways to modify the distribution of polymer nanocarriers. In this way, it has been
described that pegylation of poly(hexadecylcyanoacrylate) nanoparticles decreased
their cytotoxicity in J774 cells (Peracchia et al. 1999). This fact was explained by the
steric repulsion effect of poly(ethylene glycol) (PEG) chains, which would decrease the
interaction of nanoparticles with macrophages. More recently, Yu et al. (2012) have
also demonstrated that PEG coatings reduce nanoparticle cytotoxicity. Similar results
have been observed by decorating nanoparticles with other excipients, such as dextrans
and chitosan derivatives (Yu et al. 2012; Qi et al. 2010).
For targeting purposes, different ligands have been proposed for engineering the
surface of nanoparticles including monoclonal antibodies (Aydn 2013), lectins
(Moulari et al. 2014), hyaluronic acid (Yadav et al. 2008) or transferrin (Ren et al.
2010). However, the influence of such ligands on the toxicity properties of the
resulting nanoparticles has not been fully investigated.
Last but not least, in many cases, nanoparticles may interact with single mole-
cules and/or macromolecules (e.g. proteins) when they enter in contact with the
body fluids. Then, these compounds may form a layer (corona) surrounding the
nanoparticles that would dene their fate in vivo (Lynch et al. 2007; Cedervall et al.
2007). This fact is a supplementary factor that increases the complexity of the task
and makes it necessary to understand not only the nanomaterial, but also the
nanoparticle environment when testing for nanotoxicity (Lynch et al. 2009).
2.4 Biodegradability and Biocompatibility
Degradability of the material is an important component of acute and long-term

toxicity. In principle, non-degradable nanoparticles can accumulate in organs and
also intracellularly where they can cause detrimental effects, similar to that of
lysosomal storage diseases (Garnett and Kallinteri 2006). In contrast, biodegradable
nanomaterials would have a lower toxicity risk (Williams 2003; Nair and Laurencin
2007; Aggarwal et al. 2009); although they can also lead to unpredicted toxicity due
to unexpected toxic degradation products (Fischer and Chan 2007). Thus, the
degradation products generated during the erosion of polymer nanoparticles should
be harmless, as well as the modication of their physico-chemical properties. In
fact, during the biodegradation process the original physico-chemical properties of
nanoparticles (e.g. size, shape and surface properties) are modied and, thus, the
toxicological issues may also be affected during this process.
Recently, a guide for risk assessment of nanomaterials has been proposed based on
their biodegradability and size. As for the biopharmaceutical classication system
(BCS), this tool differentiates between four general classes of nanomaterials (IIV) from
low/no risk to high risk. In accordance with this nanotoxicological classication system,
Class I would comprise particulates with the lowest risk (biodegradable nanoparticles
with a size higher than 100 nm in diameter). On the contrary, Class IV would involve
those particulates with the maximum toxicity risks (non-biodegradable nanoparticles
smaller than 100 nm) (Keck and Muller 2013). This classication is superimposed by
biocompatibility (B) and non-biocompatibility (NB) of the nanoparticle surface,

resulting in a total of eight classes from I-B (best tolerated) to IV-NB (highest potential
risk) (Keck and Muller 2013).
3 Toxicity of Nanoparticles Administered

by the Intravenous Route
When polymer nanoparticles enter into the bloodstream, they encounter a complex
environment in which these foreign particulates may be covered with opsonin
proteins. In principle, immunoglobulins and components of the complement system
(e.g. C3, C4 and C5) are known to be common opsonins as well as other blood
serum proteins such as laminin, bronectin, C-reactive protein and type I collagen
(Frank and Fries 1991; Johnson 2004). This process, which is dependent on the
physico-chemical properties and composition of nanoparticles, can take anywhere
from few seconds to many days to complete. After opsonization, nanoparticles are
detected and engulfed by phagocytes. For polymer nanoparticles, phagocytosis
mainly occurs in tissues by resident phagocytes of the mononuclear phagocytic
system (MPS) including Kupffer cells in liver, dendritic cells (DCs) in lymph
nodes, macrophages and B cells in spleen. However, circulating monocytes, pla-
telets, and other immune cells present in the blood stream can also interact and
capture nanoparticles. In any case, this process of sequestration of nanoparticles
on the MPS organs is, in general, very rapid and typically a matter of minutes (Illum
et al. 1987; Gref et al. 1995; Panagi et al. 2001). One possible strategy to slow
down this process of recognition and to prolong the circulation of nanocarriers in
the blood stream is the generation of a hydrophilic corona in the outer external
surface of these particles (e.g. pegylation). Just as example, 24 h after adminis-
tration, 40 % of PEG coated PACA nanoparticles were found in the liver, while for
naked nanoparticles around 90 % of the given dose were found in the liver after
only 3 min (Peracchia et al. 1999).
Following endocytosis of nanoparticles by cells of the MPS, the phagocytes will
begin to secret enzymes and other oxidative-reactive chemical factors, such as
superoxides, nitric oxide and hydrogen peroxide, to break down the phagocytosed
material (Mitchell 2004). If nanoparticles cannot be degraded signicantly by these
cellular processes, they will remain stored in the MPS organs for a long period of
time. Another concern would involve the products generated from the degradation
of polymer nanoparticles. These products may only be eliminated by the renal
system if their molecular size is lower than the slit pores localized in the glomerulus
of the nephrons (Gagliardini et al. 2010). Last but not least, it cannot be forgotten
that polymer nanoparticles intravenously administered should be hemocompatible
without induction of haemolysis, trombogenicity and complement activation.
Figure 1 summarizes the possible effects of nanoparticles when administered
intravenously.
Fig. 1 Biological fate and potential toxicity of polymer nanoparticles intravenously administered
3.1 Haemolysis
The term haemolysis is commonly used to describe the release of haemoglobin to

the bloodstream due to any damage induced to red blood cells and potentially
life-threatening conditions such as anaemia. Using silica nanoparticles, it has been
reported that large external surface area and small curvature (i.e. 1/r2 for spheres) of
nanoparticles render haemolysis process thermodynamically favourable (Zhao et al.
2011). However, other factors such as the dose of nanoparticles and the density of
reactive groups also influence the haemolytic activity (Yu et al. 2011).
Regarding polymer nanoparticles, in general, low or no signicant degrees of
haemolysis have been detected for most of these nanocarriers including those based
on poly(3-hydroxybutyrate)poly(ethylene glycol)poly(3-hydroxybutyrate) (Chen
et al. 2008), poly(-caprolactone) (Espuelas et al. 2003), PLGA (Cenni et al. 2008),
human serum albumin (Fischera et al. 2003), or N-acyl chitosan (Lee et al. 2004). In
a similar way, pegylation of nanoparticles would be a good option to decrease the
haemolytic effects due to the intravenous administration of nanoparticles (Gajbhiye
et al. 2007; Casettari et al. 2012). On the other hand, cationic polymers such as poly
(ethylene imine), poly(L-lysine) or poly(diallyl-dimethyl-ammonium chloride)
induce important degrees of haemolysis when incubated with red blood cells
(Fischera et al. 2003; Luo et al. 2012).
3.2 Thrombosis
Thrombogenicity is the propensity of a material to cause or induce blood clotting

and partial or complete occlusion of a blood vessel by a thrombus (a mixture of red
blood cells, aggregated platelets, brin and other cellular elements) (Ilinskaya and
Dobrovolskaia 2013). Different studies have shown that some kinds of nanoparti-
cles (e.g. carbon nanoparticles, latex particles and diesel exhaust) with size ranges
smaller than 400 nm can directly impact the clotting system, including activation of
platelets and thrombi formation (Nemmar et al. 2004; Shah et al. 2012; Radomski
et al. 2005). This phenomenon would be related to the activation of the glycoprotein
integrin receptor GPIIb/IIIa in platelets by these nanoparticles (Radomski et al.
2005; Nemmar et al. 2009). However, the specic mechanisms through which
nanoparticles induce platelet aggregation are largely unknown.
On the contrary, solid lipid nanospheres (Koziara et al. 2005), PLGA nanocar-
riers or chitosan nanoparticles, at concentrations lower than 10 g/mL, do not affect
the aggregation of platelets (Li et al. 2009). In a similar way, decreasing particle
surface charge with a PEG coating decreases platelet aggregation and activation
(Gref et al. 1995). Nevertheless, some authors have suggested that nanoparticles
designed to offer prolonged periods of residence in the general circulation and be in
contact with blood components for a long period of time, may potentially amplify
the activation of the coagulation cascade and blood clotting (Ilinskaya and
Dobrovolskaia 2013).
3.3 Activation of the Complement
The complement system involves a group of proteins linked to each other in a

biochemical cascade. The opsonisation by complement proteins plays a critical role
in the uptake of microorganisms and foreign particles, and so, it is involved in the
initiation of both the innate and adaptive immunity (Huong et al. 2001; Zolnik et al.
2010). However, complement is also a key player in the activation of DCs and for
optimal T cell function (Kemper et al. 2010; Knopf et al. 2008). Obviously local
activation of the complement system (e.g. subcutaneously or intradermally) may be
desirable for enhancing antigen presentation and, thus, increase the efcacy of
vaccines (Reddy et al. 2007). On the contrary, on intravenous administration the
activation of the complement may be an important concern because of its
involvement in the genesis of hypersensitivity reactions and anaphylaxis. In
accordance with the last data, liposomes, nanoparticles, micelles and other
nanocarriers may induce hypersensibility reactions mediated by complement acti-
vation after their intravenous administration (Chanan-Khan et al. 2003; Moghimi
et al. 2004; Hamad et al. 2008, 2010). These reactions typically occur within
minutes after beginning the administration of nanoparticles and the most frequent
symptoms are flushing, rash, dyspnea, chest pain, back pain and subjective distress
(Szebeni et al. 2011). Fortunately, these reactions are mostly mild, transient and
preventable by appropriate precautions. However, in occasional patients, they can be
severe or even lethal (Szebeni et al. 2011). The recognition of nanocarriers by the
complement system would be related to their similar size and shape than pathogenic
microorganisms, as well as subcellular organelles and membrane vesicles detached
from cells, against which the nonspecic immune system develops efcient eliminatory
mechanisms (Szebeni 2005). Furthermore, different studies using lipid nanocapsules
(Vonarbourg et al. 2006), polysaccharide-decorated poly(isobutylcyanoacrylate)
nanoparticles (Chauvierre et al. 2003; Labarre et al. 2005) or polystyrene latex
(Nagayama et al. 2007) have shown that charged nanoparticles are more efcient
activators of the complement system than their neutral counterparts. On the contrary,
PEG and poloxamine 908 coatings have been proposed as a strategy to reduce com-
plement activation by nanoparticles (Vonarbourg et al. 2006; Al-Hanbali et al. 2006).
However, recently, it has been described that PEGylated nanoparticles may activate the
complement via a C1q-independent, mannose binding lectin-associated serine protease
dependent pathway (Hamad et al. 2010; Moghimi et al. 2010). On the other hand, the
coating of PACA nanoparticles with dextran (as well as the coating layer thickness) has
been associated with an increase in the capability of nanoparticles to activate the
complement (Bertholon et al. 2006). Moreover, the specic conformation of the
macromolecule localized on the surface of nanoparticles (e.g. dextran or chitosan) has
been also identied as another factor that may influence the complement activation
(Bertholon et al. 2006; Zandanel and Vauthier 2012). Thus, complement activation is
very sensitive to several aspects of nanoparticle surface coatings in that the type of
polymer coating, its size, density, conguration on the surface, and accessibility to
reactive groups may all play a role. However, most of these parameters are controlling
the access of complement proteins to the surface of nanoparticles and, thus, influencing
the capability of these devices to trigger the activation of the complement cascade
(Vauthier et al. 2009). For instance, important complement activations have been
observed with either naked PLA nanoparticles or stabilized with Pluronic F-68;
although, when these PLA nanoparticles possess a high poly (ethylene glycol) surface
density the activation of the complement was signicantly reduced (Vittaz et al. 1996).
In a similar way, Vauthier and co-workers demonstrated that protein C3 of the com-
plement system is repelled by diffuse shells that have a dense mesh of hydrophilic
chains (i.e. dextrans chains) (Vauthier et al. 2011).
3.4 Toxicity of Polymer Nanoparticles on MPS Organs
Probably the rst studies aiming to evaluate the in vivo toxicity of nanoparticles
were conducted by Fernandez-Urrusono et al. (1995, 1997). In these works, animals
received intravenous doses of PACA nanoparticles for 14 days (10 individual doses
of 20 mg/kg). After treatment, rat hepatocytes were isolated and both secretion of
inflammation proteins (alpha 1-acid glycoprotein) and the oxidative response were
observed. These ndings were related to the release of mediators from Kupffer
cells, in which these PACA nanoparticles concentrated after intravenous adminis-
tration (Fernandez-Urrusuno et al. 1995). Interestingly, these effects were reversible
15 days after the administration of the last dose of nanoparticles.
In an acute toxicity study in mice, LD(50) values of N-octyl-O-sulphate chitosan
micelles administrated by intravenous and intraperitoneal routes were calculated as
103 and 131 mg/kg, respectively (Zhang et al. 2008). After single intravenous
administration (13.4 mg/kg), these micelles distributed to liver, lungs and kidneys,
in which they appeared to concentrate and the polymer was excreted in urine over a
7 days period. Interestingly, at this dose (13.4 mg/kg) the micelles did not induce
haemolysis or hypersensibility reaction (Zhang et al. 2008).
In a more recent study, poly(anhydride) nanoparticles based on Gantrez AN
were intravenously administered to rats at a dose of either 50 or 150 mg/kg. At the
lowest dose, the animals showed no sign of apparent toxicity or abnormal beha-
viour. On the contrary, when nanoparticles were evaluated at the highest dose,
evident signs of toxicity characterized by absence of mobility and respiratory dis-
tress were observed. These acute episodes, probably due to the activation of the
complement system, were resolved within 3 h. Interestingly, pegylation of these
nanoparticles reduced these symptoms (unpublished results). Histopathological
evaluation of samples from different organs revealed the presence of foamy mac-
rophages in the spleen, lungs and liver of animals exposed to the highest dose of
nanoparticles. Similar effects have been observed with the administration of
cationic amphiphilic drugs (i.e. aminoglycosides) and some lipid-based delivery
vehicles (Reasor et al. 2006) that may induce phospholipodisis, which is considered
a lysosomal pathology (Schmitz and Grandl 2009).
In another interesting work, Liao and co-workers evaluated the subchronic
toxicity and immunotoxicity of monomethoxyPEG-PLGA-monomethoxyPEG
nanoparticles in rats (Liao et al. 2014). After 28 days of daily intravenous
administrations, nanoparticles with a mean size of 50 nm were found to be more
toxic than those with a mean diameter of 200 nm. Thus, 50 nm nanoparticles
induced some histopathological changes in the spleen, increased serum IgM and
IgG plasma levels, produced alterations in blood lymphocyte subpopulations and
enhanced expression of interferon- by splenocytes (Liao et al. 2014).
It is quite clear that polymer nanoparticles, intravenously administered, offer
lower toxicological concerns than other kinds of organic or mineral nanoparticles.
However, in all cases in which the intravenous route of administration is involved,
the potential toxicity of nanoparticles has to be evaluated in deep due to the
importance of the problems that can be induced. These studies should include not
only the evaluation of the haemocompatibility but also of their capability to form
thrombi and to activate the complement system. Moreover, the acute and sub-
chronic toxicity studies should be complemented with biodistribution studies in
order to identify the target organs and, thus, evaluate adequately their behaviour and
hypothetical accumulation in these areas. In the same way, biodegradation studies

under simulated in vivo conditions may be of interest to identify properly the
degradation products as well as to gain insight about the real rate of degradability
and eventual elimination from the body. In most cases, there is a real absence of
such studies in which the biodegradation of the material is evaluated in the
nanoparticle form.
4 Toxicity of Polymer Nanoparticles Administered Orally
By the oral route, polymer nanoparticles offer some advantages that can be of
interest to improve the oral bioavailability of the loaded drug or to promote immune
responses for vaccination and immunotherapy purposes. Again, most investigations
have been concentrated in the efcacy of nanoparticles; whereas toxicity aspects, if
studied, have usually restricted to a screening of their cytotoxicity. For this purpose,
Caco-2 cells (derived from a human colon carcinoma) have been used as a model of
intestinal epithelium to evaluate the toxicity of many nanoparticles. To better mimic
the gut, mucin/mucus can be added on the surface of Caco-2 cells cultures in
transwells; or Caco-2 cells can be co-cultured with HT29 (a cell line also derived
from human colon carcinoma that produce mucus when grows until post-confluent).
The use of primary cultured cells is also an option though it is not much used due to
demanding cell culture protocols and to the complexity of performing toxicity
assays with them (Ojer et al. 2015).
Regarding nanoparticles based on chitosan and its derivatives, different studies
have demonstrated the absence of signicant toxic effects when evaluated for short
periods of time (Hosseinzadeh et al. 2012; Huang et al. 2004; Zaki and Hafez
2012). However, when the incubation between chitosan nanoparticles and cells is
prolonged for at least 12 h, the viability of cells is compromised. Thus, Zaki and
Hafez, using Caco-2 cell monolayers and J774.2 macrophages, demonstrated that
after 24 h of incubation the viability of these cells decreased in a
concentration-dependent manner (Zaki and Hafez 2012). Similarly, the in vitro
viability of Caco-2 and HT-29 cell monolayers was analysed by exposing them to
chitosan nanoparticles at different concentrations for different incubation times
(Shrestha et al. 2014). For Caco-2 cells, nanoparticle concentrations up to
250 mg/mL yielded to cell viabilities higher than 80 %. However, after 12 h of
incubation, results show a concentration-dependent reduction in the cell viability.
For HT-29 cells, after incubation for 3 h, all the nanoparticles at all concentrations,
(except the highest concentration) showed cell viability values higher than 80 %
(Shrestha et al. 2014).
On the other hand, nanoparticles from chitosan derivatives appear to offer an
increased safety, compared to those based on the conventional polysaccharide.
Therefore, Yin and colleagues demonstrated the absence of toxicity of thiolated
trimethyl chitosan-cysteine nanoparticles in Caco-2 (Yin et al. 2009). A similar
absence of cytotoxicity has been described for lauryl succinyl chitosan nanoparti-
cles using Caco-2 (Rekha and Sharma 2009), O-carboxymethyl chitosan
nanoparticles in NIH-3T3, L-929, HEK-293 and THP-1 cell lines (Maya et al.
2012), dextran sulphate chitosan nanoparticles in Caco-2 and HT29 (Antunes et al.
2013) or thiolated chitosan nanoparticles coated with poly (methyl methacrylate) in
Caco-2 and MCF-7 cells (Akhlaghi et al. 2010).
As for chitosan, PLGA nanoparticles have been evaluated in several studies
(Table 1). Overall, the majority of these studies have demonstrated that these
nanoparticles are not cytotoxic. Additionally, genotoxicity of PLGA-poly(ethylene
oxide) (PLGA-PEO) nanoparticles has been recently evaluated (Kazimirova et al.
2012). In this study, these nanoparticles did induce neither DNA strand-breaks nor
oxidized DNA bases induction. However, using two experimental protocols for the
micronucleus assay, PLGAPEO nanoparticles displayed a weak but signicant
increase in the level of micronucleated binucleated cells (Kazimirova et al. 2012).
Nanoparticles based on the copolymer of methyl vinyl ether and maleic anhy-
dride (Gantrez AN) were also evaluated using Caco-2 cells. Thus, nanoparticles
functionalized on surface with either albumin or Sambucus nigra lectin displayed a
similar low cytotoxicity as control (non-functionalized) nanoparticles (Arbos et al.
2002). Interestingly, both types of nanocarriers displayed a different behaviour
when incubated with cells: albumin-coated nanoparticles displayed cytoadhesive
properties (remained adhered to the cell surface membrane) whereas
lectin-functionalized nanocarriers showed moderate capabilities to cross the cell
membrane and reach the cytoplasm (cytoinvasive properties) (Arbos et al. 2002). In
a more recent work, these poly(anhydride) nanoparticles combined with either
hydroxypropyl--cyclodextrin or PEG6000 did not affect the viability of Caco-2
cells after 24 h of incubation. Both types of nanoparticles displayed cytoadhesion to
the cell surface but not internalization (Ojer et al. 2013). This is an interesting
nding because, in principle, cytoinvasive properties in vitro may be associated
with more serious toxicity concerns in vivo.
Regarding in vivo toxicity studies of polymer nanoparticles for oral drug
delivery, it is important to highlight that most of these experiments have been
conducted with drug-loaded nanoparticles, and not always a group of animals
treated with empty nanoparticles (to check the effect associated to the carrier) has
been included. In this context, the oral daily administration of 1 mg/kg of
estradiol-loaded PLGA nanoparticles for 11 days in Sprague Dawley rats did not
affect organs such as the liver, spleen, and the intestinal segments (duodenum,
jejunum, and ileum) (Mittal et al. 2007). In a similar way, when tamoxifen was
encapsulated into PLGA nanoparticles, the presence of liver enzymes in plasma
(aspartate aminotransferase and alanine aminotransferase) remained within the
basal levels (Jain et al. 2011). Moreover, liver sections of rats treated with oral
tamoxifen-loaded PLGA nanoparticles showed normal histopathological appear-
ance (Jain et al. 2011), suggesting that these nanoparticles were not absorbed
from the gut. Conversely, Semete and co-workers have suggested that oral PLGA
nanoparticles enter into the circulation. Thus, the oral administration of PLGA
nanoparticles (size 200350 nm) daily to Balb/c mice for 10 days (Semete et al.
2010b) did not induce specic anatomical pathological changes or tissue damage in
animals. However, these PLGA nanoparticles, following oral administration,
entered into the circulation and, after 7 days, the particles remained detectable
mainly in the brain but also (in a less extent) in heart, kidney, liver and lungs
(Semete et al. 2010b). The absence of toxicity would be due to both the slow rate of
PLGA biodegradation and the biologically compatible properties of the degradative
products (lactic and glycolic acids), which can be removed by the citric acid cycle
(Campbell and Geis 1995).
In another work, PLGA nanoparticles coated with either chitosan or PEG were
orally administered to Balb/C mice in order to evaluate their immunological
response within 24 h. Twenty-four hours post-administration, the plasma levels of
different cytokines were measured. In all cases, the expression of pro-inflammatory
cytokines (IL-2, IL-6, IL-12p70 and TNF-) were low, whereas the amount of
anti-inflammatory cytokines (IL-10, INF-, IL-4, IL-5) remained at normal levels.
The only remarkable effect was the increased amount of monocyte chemoattractant
protein-1 (MCP-1) that was produced in the rst hour after administration (Semete
et al. 2010b).
The toxic effects of chitosan, poly (methyl methacrylate) and ethyl cellulose
nanoparticles on the functions of various tissues and organs in rats were also
evaluated after oral administration. After 30 days of nanoparticle administration,
the blood haematology and biochemistry parameters remained within the normal
levels. Similarly, histopathological analysis of different organs revealed that the
animals did not show any signicant changes or modications, demonstrating the
safety of the three kinds of polymer nanoparticles when administered by this route
(Lekshmi et al. 2011).
In another recent work, the toxicity of three types of nanoparticles based on the
copolymer of methyl vinyl ether and maleic anhydride were evaluated after their
oral administration to Wistar rats (Ojer et al. 2012). Naked poly (anhydride)
nanoparticles showed no sign of toxicity or abnormal behaviour in laboratory
animals in a single dose (2 g/kg) and at a dose-repeated study of 28 days (30 or
300 mg/kg daily) (Ojer et al. 2012). Similar results were obtained with pegylated
poly(anhydride) nanoparticles. In the sub-acute toxicity study, all the animals
survived the duration of the study, with no signicant changes in clinical signs,
food consumption or body weight. In these experiments, the haematological and
biochemical parameters of all the animals were found to be within the normal
ranges with no differences between the control and experimental groups. These
ndings were corroborated by the absence of lesions or abnormal structures in all
the evaluated organs (liver, spleen, thymus, kidney and intestinal segments) (Ojer
et al. 2012). Interestingly, these results were corroborated with biodistribution
studies after the radiolabelling of nanoparticles with technetium-99. The images
revealed that all these poly(anhydride) nanoparticles were located within the gut
with no evidences of distribution in other organs or nanoparticle translocation.
On the other hand, no toxic effects were also found in IRC mice treated with
100 mg/kg of chitosan-poly(glutamic acid) nanoparticles (Sonaje et al. 2009). In a
similar way, both zein and chitosan nanoparticles have been found to be safe when
fed to freshwater amphipod Hyalella azteca. Both kinds of nanoparticles showed no

signicant effect on the survival, growth or feeding behaviour of H. azteca (Gott
et al. 2014).
5 Toxicity of Polymer Nanoparticles for Pulmonary

Delivery
In the recent past, the concern for the presence and generation of airborne
nanoparticles has led to the evaluation of ultrane particles (dust, carbon black and
other pollutants) and their effects to the airways and lungs (Donaldson et al. 2002;
Schmid et al. 2009).
In principle, the size of the inhaled nanoparticles appears to be the main factor
determining their deposition within the respiratory tract (Asgharian and Price 2007;
Oberdorster et al. 2007). In humans, ultrane particles (<100 nm) may deposit in all
regions. Nevertheless, tracheobronchial deposition would be highest for particles
lower than 10 nm in size, whereas alveolar deposition would need nanoparticles
ranging from 10 to 20 nm in size. In a similar way, nanoparticles lower than 20 nm
may also efciently deposit in the nasopharyngeal-laryngeal region (Asgharian and
Price 2007; Oberdorster et al. 2007).
The main question is whether particles, in the lung, can cross the
air-blood-barrier and, thus, gain access to the rest of the body (Bennett 2002). In
spite of the defence mechanisms (mucus and mucociliary escalator, Geiser 2010),
nanoparticles seem to be capable to enter into alveolar epithelial cells via endo-
cytosis (Yacobi et al. 2010) and, in some extent, reach the liver, spleen, heart and
possibly other organs (Choi et al. 2010).
While the absorbed dose of such dust particles is considered, in general, low
(mostly less than 1 %) (Oberdorster et al. 2005b), nanoparticles administered for
therapeutic and/or diagnostic purposes must yield high deposition rates. For lung
delivery there are two main aspects which have to be considered; one is the acute
toxicity of the nanocarrier on the epithelia; and secondly the interaction of
nanoparticles with the alveolar environment.
Currently, there is a lack of available and standardized cell culture models to
mimic the epithelium permeability in the alveolar region, except for pneumocyte
monolayers in primary cultures (Kim et al. 2001). In spite of this, A549 (alveolar)
and BEAS-2B (airways) cell lines have been used to study the nanotoxicological
aspects of inhaled environmental pollutants (Forbes and Ehrhardt 2005;
Chairuangkitti et al. 2013). In this context, using A549 human lung epithelial cells,
Grabowski and collaborators (Grabowski et al. 2013) demonstrated that the cyto-
toxicity of PLGA-based nanoparticles was lower to that observed for titanium
dioxide nanoparticles. In addition, for all the nanoparticles, the levels of inflam-
matory cytokines released was low; although, the incubation of these cells with
negative charged PLGA nanoparticles (coated with Pluronic F68) led to a higher
inflammatory response, which appeared to be due to a higher uptake by A549 cells

(Grabowski et al. 2013). On the other hand, 16HBE14o- and Calu-3 cells have
shown to be suitable models for the bronchial epithelium. Using these cells,
Brzoska and collaborators demonstrated that polymer nanoparticles (from either
gelatin, human serum albumin or PACA) have little or no cytotoxicity and cause no
inflammation (Brzoska et al. 2004). Another important concern using nanoparticles
for lung delivery purposes is the interaction of these devices with the alveolar
environment. The alveolar space is covered with a thin surfactant lm (Gill et al.
2007), which is involved in the gas exchange and in lowering the surface tension in
the alveolar space. Any harmful effect at this level may compromise these functions
and, thus, might cause life-threatening consequences. In an interesting work, the
interactions between gelatin nanoparticles and dipalmitoylphosphatidylcholine
(DPPC), the main lipid component of the lung surfactant lm, were evaluated by
measuring modications in the surface pressure of the DPPC monolayer (Stuart
et al. 2006). Interestingly, the interactions between the nanoparticles and the lung
surfactant lm did not destabilize the monolayer.
Regarding in vivo studies, Dailey and collaborators (Dailey et al. 2006) studied
the potential of PLGA nanoparticles to provoke inflammatory reactions in mice
lungs after intratracheal instillation using, as control, polystyrene nanospheres (75
and 220 nm). Measuring different inflammatory parameters (lactate dehydrogenase
release, protein concentration, macrophage inflammatory protein-2 mRNA induc-
tion and polymorphonucleocyte recruitment in the bronchial alveolar lavage fluid),
they deduced that biodegradable nanoparticles may not induce the same inflam-
matory response as non-biodegradable polystyrene particles of comparable size
(Dailey et al. 2006).
In summary, the results of the different studies show that although there are some
concerns regarding the safety of ultrane inhalable nanoparticles, the inhalation of
biocompatible polymer nanoparticles used for drug or gene delivery might expose
little or no toxicity if the particle size is not smaller than 100 nm (Azami et al.
2008). In any case, to the best of our knowledge, polymer nanoparticles do not
induce severe symptoms such as acute inflammation responses or emphysema (as
observed for some kinds of metallic nanoparticles (Chen et al. 2006; Brown et al.
2014)) when administered through the respiratory tract.
6 Toxicity of Polymer Nanoparticles for Nasal Delivery
Conventionally, the nasal route has been exploited to deliver drugs that treat local
disorders and for systemic delivery of certain drugs (e.g. small molecular weight
drugs, peptides and proteins) as an alternative to their administration by parenteral
injections. As for other types of nasal preparations, formulations based on
nanoparticles have to demonstrate that the constituents do not negatively impact on
the cilia and their function as clearance system in the upper airways. Cilia are
mobile nger-like appendages extending from the surface of the nasal epithelial
cells and move in a well-organized and coordinated way to propel the overlying
mucus layer toward the throat, which contributes to the bodys primary nonspecic
defence mechanism by propelling potentially hazardous substances (Yan et al.
2013). In a very interesting work, Gao and co-workers demonstrated that
poly(ethylene glycol)poly(lactide) (PEGPLA) nanoparticles did not affect the
morphology and integrity of the cilia on the nasal mucosa as observed by electronic
microscopy when administered once a day during 6 days (Gao et al. 2006). In
another study, Amidi and co-workers evaluated the cytotoxic effect of N-trimethyl
chitosan (TMC) nanoparticles in Calu-3 cells and after the measurement of the
ciliary beat frequency of chicken embryo trachea. Cytotoxicity tests with Calu-3
cells showed no toxic effects of the nanoparticles, whereas a partially reversible
cilio-inhibiting effect on the ciliary beat frequency of chicken trachea was observed
(Amidi et al. 2006). More recently, PEG-PLA nanoparticles functionalized with
concanavalin A were also in vivo evaluated in rats (Shao et al. 2013). These
nanoparticles did not show signicant toxicity as the nasal mucosa displayed intact
and dense cilia in comparison with the positive control group of animals that
presented the cilia in a state of chaos with abscission and frequent disruptions (Shao
et al. 2013).
During the last few decades, the nasal route has also been applied in attempts to
deliver drugs to the brain for the treatment of specic brain diseases. In fact,
nanoparticles would be capable of reaching the brain via migration along the
olfactory or trigeminal nerve endings after deposition on the olfactory mucosa in
the nasal region (Oberdrster et al. 2004). This route was evidenced with model
particles such as carbon, gold and manganese oxide in experimental inhalation
models in rats (Oberdorster et al. 2005a; Oberdrster 2004; Elder et al. 2006) and
with wheat germ agglutinin (WGA) functionalized PEG-PLA nanoparticles (Gao
et al. 2006). Obviously, this new and interesting route to deliver drugs to the central
nervous system (CNS) (by-passing the blood brain barrier) may also be an
important concern from a toxicological point of view. The inhalation of ambient air
nanoparticles and metallic nanoparticles has been shown to induce the production
of reactive oxygen species and oxidative stress (Nel et al. 2006; Elder et al. 2006) as
well as pro-inflammatory cytokines (Campbell et al. 2005) in the brain. All of these
effects have been implicated in the pathogenesis of neurodegenerative diseases such
as Parkinsons and Alzheimers diseases (Calderon-Garciduenas et al. 2004).
However, till now, very few reports have evaluated the toxicity of polymer
nanoparticles in the brain. In this context, the toxicity of PEG-PLGA nanoparticles
coated with odorranalectin was evaluated on Calu-3 cell lines and rat nasal mucosa
(Wen et al. 2011). The brain targeting indexes of these nanoparticles when
administered nasally were signicantly higher than when administered intra-
venously. Moreover, the toxicity assessment suggested good safety of these tar-
geted nanoparticles both in vitro and in vivo (Wen et al. 2011). In a similar
approach, using WGA as targeting ligand of PEG-PLGA nanoparticles, slight signs
of brain toxicity was observed when intranasally administered to rats for 7
continuous days (Liu et al. 2011b). This toxicity was evidenced by increased
glutamate levels in brain and enhanced lactate dehydrogenase (LDH) activity in the
olfactory bulb (Liu et al. 2011b).
7 Toxicity of Polymer Nanoparticles for Ocular Delivery
Chitosan nanoparticles have been proposed as carriers for ophthalmic drug delivery.
These nanoparticles were found to be safe when evaluated in human conjunctival
epithelial cells (IOBA-NHC) in spite of their ability to be taken up in a large extent
by these cells (Enrquez de Salamanca et al. 2006). This capability of chitosan
nanoparticles to penetrate into corneal and conjunctival epithelia has also been
observed in vivo (de Campos et al. 2004). In rabbits, after exposure to the
nanoparticles, the animals did not show signs of discomfort and the ocular surface
was normal without any macroscopic sign of inflammation or surface alteration.
These results were conrmed by pathological studies conrming the presence of
normal ocular surface structures in both control and treated eyes (Enrquez de
Salamanca et al. 2006). In a more recent work, hyaluronic acid-chitosan nanopar-
ticles also displayed a very low toxicity in both human corneal epithelial (HCE) and
IOBA-NHC cell lines (De la Fuente et al. 2008). After ophthalmic administration in
rabbits, these nanoparticles did not induce signs of ocular irritation and no signif-
icant effects on the tear production system were observed (Contreras-Ruiz et al.
2010). In a similar way, solid lipid nanoparticles (Seyfoddin et al. 2010) and
nanoparticles based on polysaccharides from plants (e.g. fenugreek, isphagula and
mango bark) were observed to be non-irritant and non-toxic in vitro and in vivo up
to a concentration of 2 mg/mL (Pathak et al. 2014).
More problematic from a toxicological point of view is the parenteral delivery of
nanoparticles in the eye, including periocular and intravitreal routes. The potential
risks associated with these routes of administration include inflammation,
immune-stimulation and immune-suppression (Zolnik et al. 2010; Dobrovolskaia
and McNeil 2007), membrane disruption (Panessa-Warren et al. 2009), and
oxidative stress generation (Medina et al. 2007). These responses could further lead
to vitreous haze, epithelial damage, membrane opacity, haemorrhage, brosis, and
retinal damages such as traction, thickening, and degeneration in the back of the eye
(Kompella et al. 2013). In an interesting work, the in vivo toxicity of chitosan
nanospheres, poly[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene)
ammonium iodide] ethyl phosphate (PCEP) nanocarriers and magnetic nanoparti-
cles was evaluated (Prow et al. 2008). When administered in the vitreous, chitosan
nanoparticles induced inflammatory reactions (90 % of the treated eyes), opacity
(60 %) and haemorrhages (30 % eyes); whereas low toxicity effects were observed
for PCEP nanoparticles and magnetic devices (only induced haemorrhages in 30 %
of eyes) (Prow et al. 2008).
Using albumin nanoparticles, Mo and collaborators demonstrated the absence of

cytotoxicity in ARPE-19 cells at concentrations up to 5 mg/mL for 96 h (Mo et al.
2007). After intravitreal administration, albumin nanoparticles remained in the
vitreous cavity for at least 2 weeks, forming a thin layer overlying the retina and in
the area close to the blood aqueous barrier (Merodio et al. 2002). In spite of their
prolonged residence time in the eye, albumin nanoparticles did not induce
inflammatory reactions or alterations in the tissue architecture (i.e. cellular inl-
trations or vascular inflammation) (Merodio et al. 2002).
More recently, nanoparticles from methoxy poly(ethylene glycol)-poly(-
caprolactone) (*50 nm) were proposed as candidates for ocular drug delivery. In
vitro cytotoxicity assays showed that these carriers had no apparent cytotoxicity
against HCE, human lens epithelial cells, and retinal pigment epithelial cells. In
rabbit eyes, after a single intracameral injection, these nanoparticles did not affect
the intraocular pressure. In addition, histopathological studies demonstrated that
there was absence of any obvious changes in microstructure of the corneal tissue
and retina after a single intracameral or intravitreal injection of these carriers (Xu
et al. 2013). Similarly, in vivo, poly(anhydride) nanoparticles obtained from
Gantrez AN appears to be well tolerated after both sub-tenon ocular and intrav-
itreal injections (Prieto et al. 2012). However, all of these results are preliminary
and needs to be completed.
8 Conclusions and Future Challenges
Nanoparticle toxicology is a relatively young eld, and the bulk of reports have
focused on inorganic nanoparticles as well as airborne and pollutants particulates.
On the contrary, a relatively low amount of reports are devoted to the evaluation of
the toxicity/safety of polymer nanoparticles. Some reasons may explain this lack of
information. First, polymer nanoparticles are usually produced with GRAS com-
pounds (proteins, polysaccharides, lipids) and/or pharmaceutical excipients and
there is the temptation of assuming that the resulting device would be safe due to its
composition. Second, in some cases, the use of polymer nanoparticles for drug
delivery purposes aims to minimize the arrival of the drug to particular
organs/regions in which they can induce toxicological effects. Thus, the points of
interest are the biodistribution properties of the carriers and the toxicity induced by
the drug. Last but not least, in many cases, the toxicity concerns (usually due to
Regulatory requirements) only appear during the last steps of the preclinical
development.
On the other hand, a large majority of studies are concentrated in the evaluation
of the cytotoxicity and acute toxicity. Long-term toxicity of polymer nanoparticles
and examination of chronic exposure are critical to understanding the behaviour of
these devices in vivo. This is perhaps the main challenge regarding the elucidation
of the real conditions in which nanomaterials can safely be used as therapeutics and
as diagnostic tools.
Acknowledgments The authors wish to thank for support by the European Communitys Seventh
Framework Programme via the large project Alexander (FP7-2011-NMP-280761).
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Chapter 17
Theranostics: In Vivo
Viktorija Herceg, Norbert Lange and Eric Allmann
Abstract Theranostics is a portmanteau of thera(py) and (diag)nostics. It is an

interdisciplinary eld of research that unites pharmaceutical technology, chemistry,
imaging, and medicine with the purpose of creating a single drug delivery system
able to diagnose, treat, and monitor disease. In this chapter, the reader will be
presented with an overview of the state-of-the-art developments in theranostics in
which clinically relevant imaging modalities are combined with polymers,
lipid-based systems, inorganic assemblies, antibody conjugates, and gene delivery
vehicles. Here, special emphasis will be placed on the systems tested in vivo.

Keywords Theranostics In vivo Polymer systems Lipid-based systems

Inorganic assemblies Gene therapy
1 Introduction
In order to overcome the pitfalls of conventional xenobiotics, such as high toxicity,

adverse effects, multiple-drug resistance (MDR), low drug diffusion into the target
sites, and rapid clearance from the body, a rational approach to designing
nanoconstructs with multiple functions was developed. In this context, nanomedi-
cine can be dened as the application of nanotechnology for in vitro and in vivo
medical purposes. The advent of nanomedicine began in the mid-1990s when
liposomal doxorubicin formulations (Doxil and Daunoxome) gained market
approval as anticancer therapeutics. This was directly followed by paclitaxel-loaded
V. Herceg N. Lange E. Allmann (&)

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Rue
Michel-Servet 1, 1211 Geneva 4, Switzerland
e-mail: Eric.Allemann@unige.ch
V. Herceg
e-mail: Viktorija.Herceg@unige.ch
N. Lange
e-mail: Norbert.Lange@unige.ch

DOI 10.1007/978-3-319-41421-8_17
552 V. Herceg et al.
Table 1 Classication of theranostics

Therapeutic approach Treatment Imaging modality References
mechanism
Conventional Active OI/MRI/PET/SPECT Agulla et al. (2014a), Alexiou
treatment pharmacological et al. (2000), de Smet et al.
ingredient (2011), Grange et al. (2010),
Kaida et al. (2010), Lammers
et al. (2009), Lee et al. (2013),
Nurunnabi et al. (2010), Ponce
et al. (2007), Quan et al. (2011),
Ranjan et al. (2012), Topete
et al. (2014), Wu et al. (2014),
Yu et al. (2008)
Photothermal therapy Heat OI/MRI/US Bardhan et al. (2010), Charan
et al. (2012), Cheng et al. (2012,
Gao et al. (2014), ONeal et al.
(2004), Peng et al. (2011)
PDT PS/light/O2 OI Topete et al. (2014), Gabriel
et al. (2009), Jang et al. (2011),
Zeisser-Labouebe et al. (2009)
Gene therapy Oligonucleotides OI/MRI/PET/SPECT Wang et al. (2012), Wang et al.
(2013)
Radioimmunotherapy X-rays OI/PET/SPECT Bander et al. (2005), Stillebroer
et al. (2013), Tagawa et al.
(2013)
nanoparticles (Abraxane) (Modery-Pawlowski and Gupta 2014). Most impor-

tantly, due to their intrinsic multimodal nature, nanoparticulate drug delivery sys-
tems have opened new doors toward the development of theranostic agents. In such
systems, a therapeutic and an imaging agent are contained in the same delivery
vehicle. Theranostics provides the real-time monitoring of a given treatment in a
noninvasive manner. It grants patients a better therapeutic outcome by maximizing
the safety proles. Depending on the design and characteristics of theranostic
systems, they can be classied as treatments with an active pharmaceutical ingre-
dient, photothermal therapy, photodynamic therapy (PDT), gene therapy,
radioimmunotherapy, or combinations of the abovementioned strategies (Table 1).
In this sense, supramolecular nanoconstructs as drug/imaging agent carriers provide
potential advantages over small molecules, such as improved bioavailability,
decreased systemic toxicity, and reduced severity of adverse effects (Hoste et al.
2004). Moreover, the use of nanocarriers provides a way to efciently deliver
poorly water-soluble drugs and enables the incorporation of multiple imaging tags
in a single platform. Most importantly, the use of polymer nanosystems allows the
encapsulation of multiple agents in a conned space even when the therapeutic and
imaging agents are distinct chemical entities. Taken together, these advantages
make polymer systems good candidates for theranostic purposes. A polymer-based
nanotheranostic system is composed of a polymer, therapeutic agent, and an
imaging agent. Targeting ligands may be added to these structures to enhance the
treatment specicity (Fig. 1).
17 Theranostics: In Vivo 553
Fig. 1 Schematic
representation of
polymer-based
nanotheranostic systems
PEG for stealth properties

Active pharmacological ingredient
Imaging agent
Targeting moiety
NP
There are two ways of delivering nanoparticles (NPs) to the desired site of
action: passive and active targeting. The former depends on the enhanced perme-
ability and retention (EPR) effect, rst reported by Matsumura and Maeda (1986).
EPR is typically described in tumor biology and is characterized by the combination
of the leaky architecture of angiogenic blood vessels with fenestrations between
adjacent endothelial cells and a poor lymphatic drainage inside the tissue
(Hagendoorn et al. 2006). As a result, NPs are able to better extravasate and
accumulate inside solid tumors. Numerous factors, including anatomical and
microscopic defects in the tumor vasculature, the presence of molecular factors in
the extracellular matrix, such as vascular endothelial growth factor (VEGF), bra-
dykinin, prostaglandins, nitric oxide, peroxynitrite, and proteolytic activity, can
have an impact on the EPR effect (Iyer et al. 2006). Improvement of the theranostic
agents may be achieved by adding active targeting moieties.
Active targeting strategies can be classied into the following categories (Fig. 2):
1. attaching ligands onto the surface of NPs, such as antibodies, antibody frag-
ments, afbodies, peptides, nucleic acids, and others (vitamins and carbohy-
drates), that bind to the receptors of the targeted cells (Peer et al. 2007; Accardo
et al. 2013);
2. using of tissue-specic physiological triggers, such as enzymatically cleavable
(Gabriel et al. 2009) or pH-responsive linkers; and
3. physical targeting by externally applied magnetic elds, ultrasound, light
(McCarthy et al. 2010), or heat (Ganta et al. 2008).
Recently published literature on theranostics have reported on systems that were
not necessarily yet tested in vivo (Chen et al. 2011; Mura and Couvreur 2012; Xie
et al. 2010; Janib et al. 2010; Svenson 2013; Luk and Zhang 2014). The purpose of
the present chapter is to address examples of theranostic systems with an emphasis
Fig. 2 Active targeting strategies. 1 Attachment of specic ligands to the nanoparticles. 2 Use of
tissue-specic triggers such enzymatic digestion or pH-responsive linkers. 3 Physical targeting by
external impulses
on their in vivo assessments for both therapeutic and diagnostic functions. We will
focus our attention on the imaging modalities used in clinical practice in combi-
nation with a wide range of NPs including polymer conjugates and complexes,
liposomes, micelles, inorganic assemblies, theranostic NPs used for gene delivery,
and antibody conjugates for radioimmunotherapy. The review will be useful to
understand which functionalities may be incorporated into polymer-based NPs to
design tools for theranostic application.
2 Imaging Modalities
Various imaging modalities are available in clinical practice for disease diagnosis
and treatment monitoring. In this chapter we will cover examples of ultrasound
(US), optical imaging, X-ray imaging computed tomography (CT), magnetic
Table 2 Commonly used imaging modalities

Imaging Agent used Spatial Advantages Disadvantages
modality resolution
US Microbubbles 50 m High sensitivity, Low depth penetration
no radiation,
quantitative
Optical Fluorochromes 15 mm High sensitivity, Low depth penetration
imaging no radiation
CT Iodine or 50 m High spatial Requires high atomic
barium-based resolution, speed mass contrast agents that
agents have to be administered
at molar concentration
MRI Gd chelates, 10 High resolution, Long imaging time
Mn, iron oxide 100 m no radiation,
nanoparticles unlimited tissue
penetration,
quantitative
18
PET F, 15O, 13
N, 12 mm High sensitivity, Radiation, limited spatial
11
C, unlimited tissue resolution
penetration,
quantitative
99
SPECT mTc, 123I, 12 mm High sensitivity, Radiation, limited spatial
131
I, 111In unlimited tissue resolution
penetration,
quantitative
resonance imaging (MRI), and radionuclide-based imaging such as positron

emission tomography (PET) and single-photon emission computed tomography
(SPECT). Table 2 summarizes the main characteristics of the commonly used
clinical imaging modalities. The successful implementation of clinical imaging for
therapeutic monitoring relies on high signal-to-background ratios (SBRs) to reliably
discriminate the diseased and surrounding healthy tissues (Frangioni 2008). With
the current state of nanotechnology, all agents of the aforementioned imaging
techniques can now be incorporated into nanoconstructs for theranostic purposes.
2.1 Ultrasound
Ultrasound imaging is broadly available, noninvasive, relatively easy to implement,

and is one of the most employed diagnostic techniques for the examination of soft
tissues in clinical practice. An US transducer placed on the skin of a patient emits
sound waves that penetrate the body. US images are subsequently created from
backscattered waves coming from different tissue structures. For further contrast
enhancement and differentiation between healthy and diseased tissues, microbubbles
with unique acoustic properties can be used. Microbubbles are typically 1 to 4 m

spheres of perfluorocarbons surrounded by phospholipids, proteins, or polymers
(Dijkmans et al. 2004; Kiessling et al. 2012) and are designed to circulate within blood
vessels but not extravasate. US imaging has a high enough sensitivity allowing for the
detection of a single microbubble (Deckers and Moonen 2010). Although
microbubbles have been reported as a promising platform for drug delivery (Eisenbrey
et al. 2009, 2010a, b; Cochran et al. 2011), their gas core and the single phospholipid
outer layer of bubbles is unsuitable for the efcient encapsulation of therapeutic
molecules. Another limitation for their application in theranostics is that US-mediated
drug release causes signal loss upon microbubble disruption (Deckers and Moonen
2010). Therefore, subsequent administrations of microbubbles would be required for
treatment monitoring. Although interesting, the concept of using two separate for-
mulations for active drug delivery and for therapeutic monitoring is beyond the scope
of this chapter and will not be further discussed.
2.2 Optical Imaging
Optical imaging uses photons emitted from different sources to visualize living
tissues. Optical imaging can be further categorized as fluorescence imaging, pho-
toacoustic imaging, and absorption-based imaging. Due to its high sensitivity,
fluorescence imaging is the primary optical image technique used in clinical
practice. However, photoacoustic imaging and diffusion-based techniques have
recently gained the attention of clinicians (Choe et al. 2009; OSullivan et al. 2013;
Leproux et al. 2013). Unlike MRI, SPECT, PET, or CT, optical imaging permits the
monitoring of the treatment during surgical interventions (Keereweer et al. 2011;
van Driel et al. 2014; van Dam et al. 2011; Heath et al. 2012). Optical imaging is
safe for patients, relatively simple to use, and can be repeated frequently. The major
limitation for optical imaging is the low penetration of light into deeply seated sites
due to light scattering in the tissue. Furthermore, if improperly selected, the
fluorescent signal of the contrast agent may interfere with tissue autofluorescence.
Today, most of the optical imaging studies are directed toward near-infrared
(NIR) markers, which offer better results for in vivo diagnoses and treatment
monitoring. The advantages of NIR imaging include the increased penetration of
light into tissues and an increased SBR due to a low tissue autofluorescence at these
wavelengths (Keereweer et al. 2011; Adams et al. 2007). Currently, there are
numerous studies describing NIR-guided open surgery, laparoscopy, thoracoscopy,
and robot-assisted surgery (Vahrmeijer et al. 2013; Keereweer et al. 2013). Agents
that are routinely employed in intraoperative fluorescence imaging are methylene
blue, indocyanine green (ICG), and 5-aminolevulinic acid (5-ALA) (Vahrmeijer
et al. 2013). Intravital microscopy and whole-body photonic imaging are techniques
in which optical fluorescence imaging is employed in vivo (Mulder et al. 2007).
Photoacoustic imaging (PAI) is a newly developed technique that provides
high-resolution images in tissues beyond the limit of optical diffusion. PAI uses
chromophores, which induce a thermoelastic expansion in the tissue when excited.

This generates an acoustic pressure waves that can be measured by an ultrasound
transducer (Ng et al. 2014; Huynh et al. 2014).
NIR diffuse optical tomography and spectroscopy are two rapidly developing elds
for disease detection and diagnosis. These absorption-based imaging techniques
employ low-power NIR light to measure wavelength-dependent optical absorption
coefcients in tissues. The results obtained by this measurement provide direct
information about physiological properties, such as blood dynamics, total hemoglobin
concentration, oxygen saturation, and water and lipid concentrations (Choe et al. 2009;
OSullivan et al. 2013; Leproux et al. 2013; Konecky et al. 2009).
2.3 X-Ray Computed Tomography
X-ray CT enables 3D reconstructions based on the attenuation of an X-ray beam

that passes through the body. The generated CT images provide anatomical
information due to differences in the X-ray absorption of different tissues (Janib
et al. 2010). The benets of using CT are its high spatial resolution, availability, and
speed. To improve contrast, iodinated contrast agents are routinely used for imaging
vasculature (i.e., angiography) (Hasebroock and Serkova 2009) and barium-based
agents for gastrointestinal investigations. However, one major drawback for the
creation of usable nanotheranostics for CT imaging is that a suitable contrast agent
must have a high atomic mass and be delivered at very high doses (Frangioni 2008).
2.4 Magnetic Resonance Imaging
Magnetic resonance imaging is a highly versatile, noninvasive diagnostic technique

with high temporal and spatial resolutions. However, the major drawbacks of this
imaging modality include its low sensitivity and high cost (Terreno et al. 2010).
MRI signals are detected from water protons in tissues when the body is placed in a
magnetic eld generated by an MR scanner. Hence, the contrast of an MR image is
determined by the proton density in tissues and their relaxation times following a
radio frequency pulse. Different paramagnetic, superparamagnetic, and ferromag-
netic contrast agents are used to enhance MRI signals by shortening the relaxation
time of water in the tissues. T1 agents, also called positive or bright contrast agents,
such as gadolinium (Gd) and manganese (Mn) chelates, increase the signal inten-
sity. T2 contrast agents decrease the signal intensity and are accordingly referred to
as negative or dark contrast agents (Weishaupt et al. 2006; Davies et al. 2013). T2
contrast agents are mainly iron oxide-based particles. Overall, the most frequently
used contrast agent is Gd (Davies et al. 2013; Caravan 2006). However, free Gd is
highly neurotoxic and thus, is required to be tightly chelated in molecules such as
tetraazacyclododecane tetraacetic acid (DOTA) (Davies et al. 2013). To overcome
the low sensitivity of MRI, high doses of contrast agents need to be delivered. For
this purpose, various NPs have been developed that encapsulate high payloads of
Gd to amplify the signal contrast and to optimize the relaxivity of the imaging agent
itself (Terreno et al. 2010, 2012). However, the use of Gd-based contrast agents
may lead to severe adverse effects, such as nephrogenic systemic brosis
(Hasebroock and Serkova 2009).
2.5 Radionuclide-Based Imaging, PET, and SPECT
The basis for both PET imaging and SPECT imaging is the emission of gamma
-rays from exogenously administered radioactive tracers. In PET imaging, the
radionuclide undergoes positron emission decay. Then, the emitted positron loses
its kinetic energy, and by encountering an electron, an annihilation process occurs.
The produced pairs of -photons move in opposite directions and are detected by
PET cameras to create an image (Fig. 3). 18F, 15O, 13N, and 11C are radionuclides
commonly used for PET imaging (Massoud and Gambhir 2003), and 99mTc, 123I,
131
I, and 111In are commonly used for SPECT imaging (Peng et al. 2011) (Table 3).
Because of an increased glucose metabolism, the glucose mimetic, 2-deoxy-2-[18F]
fluoro-D-glucose (18FDG), is readily taken up by the brain or by cancerous cells and
is used in various diagnostic procedures. Compared to PET agents, which have to
be administered shortly after their synthesis because of their short half-lives,
radionuclides used in SPECT are easier to prepare and have longer half-lives, which
makes them suitable for treatment monitoring for hours or days after the
Fig. 3 Principle of PET imaging process

Table 3 Commonly used Type of imaging Radionuclide Half-life

radionuclides for PET and 18
SPECT imaging PET F 110 min
15
O 2 min
13
N 10 min
11
C 20 min
99m
SPECT Tc 6h
123
I 13 h
131
I 8 days
111
In 2.8 days
administration of the imaging agent (Rahmim and Zaidi 2008; Beer and Schwaiger
2008). Additionally, in SPECT imaging, it is possible to simultaneously use several
radionuclides (Janib et al. 2010). Both modalities possess unlimited depth pene-
tration, but have limited spatial resolution. An advantage of PET over SPECT
imaging is its two to three orders of magnitude higher sensitivity (Rahmim and
Zaidi 2008). Radionuclide-based imaging for theranostic purposes remains quite
limited not only because of the short half-lives of most isotopes, but also because of
the strict radioactive safety requirements.
3 Theranostic Nanosystems
3.1 Polymer Systems
In the mid-1970s, H. Ringsdorf proposed an ideal model of polymer prodrugs. An

early version of minimal theranostic system is described in his model, which
consisted of a biodegradable polymer backbone as a carrier onto which spacers for
drug conjugation, imaging agents, and targeting moieties were attached (Fig. 4)
(Ringsdorf 1975). Several polymers have already been successfully tested in the
development of polymer therapeutics, including vinyl polymers such as poly(N-
(2-hydroxypropyl) methacrylamide) (PHPMAm) (Kopecek and Kopeckova 2010;
Duncan 2003); poly(styrene-co-maleic acid/anhydride) (PSMA) (Maeda 2001);
synthetic poly(-amino acids), such as poly(L-lysine) and poly(L-glutamic acid);
polysaccharides, such as dextran and chitosan (Liu et al. 2008); and proteins, such
as human serum albumin (HSA) (Hoste et al. 2004; Miele et al. 2009).
Polymer conjugates include rationally designed macromolecular drugs, poly-
merdrug, and polymerprotein conjugates (Duncan 2003; Khandare and Minko
2006). The chemical assembly depends on the properties of the polymer carrier, the
targeting moiety, and the drug and imaging agent to be attached (Veronese et al.
2005; Furgeson et al. 2006). Due to the huge variation of physicalchemical
properties of the individual building blocks, the preparation of the nal product can
be challenging. The choice between homopolymers, graft, or block polymers for the
conjugation of the drug and imaging agents and the chemical yield is dependent on
Fig. 4 Model of a polymer prodrug. A polymer backbone serves as a carrier for drugs, imaging
agents, and targeting moieties. A hydrophilic unit, such as PEG, enhances the solubility and
provides stealth properties
the chemical nature, molecular weight, steric hindrance, and reactivity of the polymer
and the drug and imaging agents (Khandare and Minko 2006). One of the main
requirements in polymer systems is a good stability between the carrier and its payload
under physiological conditions. However, the carrier and payload should be rapidly
cleaved once the target site is reached (Veronese et al. 2005). By using enzymatically
cleavable or pH-responsive linkers, this can be exploited as an active targeting strategy
for the controlled release of therapeutic agents in the region of interest. For instance, it
is known that upregulated proteolysis is found in cancer, cardiovascular, neurode-
generative, and inflammatory diseases among others (Gabriel et al. 2011). In cancer,
the breakdown of the extracellular matrix and subsequent extravasation is largely
facilitated by tumor-associated cells that are abundantly expressing several proteases.
These proteases can be identied as a hallmark of neoplastic growth. In this case, the
most prominent proteases are matrix metalloproteinases, cysteine proteinases, plas-
minogen activators, and cathepsin D (Gabriel et al. 2011). This enzymatic machinery
can be used for the activation of prodrugs and imaging agents (Gabriel et al. 2011;
Harris et al. 2008). Another means to trigger drug release is by exploiting changes in
the pH of the tumor environment. This has been shown to be successful in solid
tumors, which produce large amounts of lactic acid due to anaerobic glycolysis and
acidify interstitial fluid. Thus, suitable target pH values for the release of the attached
agent can be tuned to the physiological values at the targeted location, i.e., the
extracellular domain of the cancerous cells or their intracellular compartments, such as
endosomes or lysosomes.
Polymer chemistry research for biomedical purposes has focused on the syn-
thesis of nontoxic and biodegradable polymers. Examples of such polymers in
polymer prodrugs include, but are not limited to, poly(lactide) (PLA) (Kulkarni
et al. 1971) and poly(lactide-co-glycolide) (PLGA) (Danhier et al. 2012). Because
of its stability in systemic circulation and its nontoxicity and low immunogenicity,
HPMAm polymer is also frequently used for both complexations (Vasey et al. 1999)
and conjugations of drugs and imaging agents (Hoste et al. 2004; Janib et al. 2010;
Kopecek and Kopeckova 2010). Lammers et al. (2008) reported a series of experi-
ments with a supramolecular assembly in which cytostatic drugs doxorubicin (DOX),
gemcitabine (GEM), or both (Lammers et al. 2009) were conjugated via a lysosomally
cleavable peptide linker to the polymer backbone of an HPMAm copolymer. First,
PHPMAm-bound DOX or GEM were tested in combination with radiotherapy
(Lammers et al. 2008). To monitor the in vivo biodistribution, the HPMAm copolymer
was labeled with Gd and imaged by MRI. As expected, the MRI images showed an
enhanced accumulation of the construct in tumors over time. However, because there
was no therapeutic agent attached, these constructs cannot be classied as theranostics.
In the subsequent study, an HPMAm polymerdrug conjugate was designed, carrying
6.4 wt% GEM and 5.7 wt% DOX. Tyrosinamide units were incorporated into the
copolymer to allow radiolabeling with 131I. The in vivo study carried out in the
Dunning AT1 tumor model conrmed the simultaneous delivery of both drugs to the
tumors (Lammers et al. 2009). It would have been interesting to see the potential of the
aforementioned PHPMAm construct labeled with Gd for theranostic applications.
Both ndings shed new light on the simultaneous multimodal approaches of cancer
treatment and its future theranostics implementation.
PDT and photodiagnosis (PD) are other exciting elds that have been introduced in
clinical practice with high efcacies for both diagnostic and therapeutic purposes.
Three factors govern the PDT mechanism: a photosensitizer (PhotoS), light, and
molecular oxygen in the target tissues (Lange 2003). Irradiation with light excites the
administered photosensitizer, which converts molecular oxygen into toxic singlet
oxygen molecules and reactive oxygen species (ROS) (Gabriel et al. 2011). Moreover,
upon light activation, the emitted fluorescence from the PhotoS can serve as a diag-
nostic tool. Most conventional PhotoSs are generally hydrophobic and prone to
aggregation in aqueous environments. Therefore, their administration in pharmaceu-
tically acceptable formulations is impeded. Furthermore, their bioavailability is often
very low without an appropriate formulation. To overcome these obstacles, PSs have
been conjugated to peptides, proteins, carbohydrates, and polymers or loaded into
micelles, dendrimers, liposomes, or other types of polymer NPs (Sibani et al. 2008).
One such example is the encapsulation of a potent natural PhotoS hypericin into
polymer PLA NPs, as reported by Zeisser-Labouebe et al. (2006). Hypericin-loaded
NPs obtained by nanoprecipitation were tested in vivo on an ovarian cancer xenograft
in Fischer rats F-344. Fluorescence endoscopy and tissue analyses showed the
selective accumulation of hypericin in ovarian micrometastases when the NPs were
used in comparison with the free drug (Zeisser-Labouebe et al. 2009). In addition to
cancer, rheumatoid arthritis (RA) is another disease in which PDT has been suc-
cessfully tested in vivo. RA is a chronic autoimmune disease characterized by the
inflammation of the synovial tissues of joints, tendon sheets, and bursae that lead to
the progressive destruction of the articular cartilage. Thrombin is a serine protease of
the coagulation cascade converting brinogen into brin. In RA patients, the prote-
olytic activity of thrombin has been found to be upregulated. Gabriel et al. (2009)
Fig. 5 a Schematic representation of protease-mediated activation of thrombin-sensitive polymer

photosensitizer prodrug (T-PhotoS). Coupled to the poly-lysine backbone, PhotoSs remained
quenched, but upon thrombin cleavage of the peptide linker, they were freed and could be light
activated, which resulted in fluorescence emission and ROS production; b schematic represen-
tation of T-PhotoS. Adapted from Gabriel et al. (2009)
developed a thrombin-sensitive polymer photosensitizer prodrug (T-PhotoS) for the

photodynamic eradication of the hyperplastic synovium in RA. In their prodrug,
multiple PhotoS units were tethered to a polymer (poly-lysine) backbone via
thrombin-cleavable peptide linkers. Moreover, the polymer backbone was modied
with single high molecular weight PEG (20 kDa) (Fig. 5). The close proximity of the
PhotoSs attached to the copolymer resulted in the inactivation of the fluorescence
signal and the photodynamic activity due to self-quenching. The in situ activation of
T-PhotoS was demonstrated in vitro and in vivo using a collagen-induced arthritis
mice model. T-PhotoS was administered intravenously and the fluorescence intensity
in inflamed joints was observed by whole animal fluorescence imaging.
A fluorescence increase over time was observed to occur selectively in arthritic joints,
whereas the fluorescence emissions in non-arthritic paws remained close to baseline.
The fluorescence rapidly increased during the rst 8-h post administration and then
reached a plateau. Furthermore, the fluorescence intensity after i.v. injection of
T-PhotoS at a dose of 1 mg/kg pheophorbide equivalence correlated with established
clinical scores (Fig. 6). A similar approach was followed for the targeting of
urokinase-like plasminogen activator (uPA), which is involved in the progression of
prostate cancer, and was tested in vitro and in vivo (Zuluaga et al. 2012, 2013). The
selectivity of the corresponding PhotoS in PC-3 tumors was conrmed by the colo-
calization of bioluminescence and fluorescence imaging. Furthermore, uPA-mediated
PDT resulted in the cure or partial remission of tumors in the PDT group only.
Fig. 6 a Quantitative in vivo fluorescence images acquired before, 4, 8, and 12 h post i.v. injection of
T-PhotoS at a dose of 1 mg/kg pheophorbide equivalents. Selective fluorescence increase can be seen
in the arthritic paws (A), but not in the non-arthritic paws (NA). b Image of the normal versus inflamed
joints in mice. c Fluorescence time proles show a rapid increase of fluorescence in arthritic paws
during the rst 8 h after T-PhotoS administration and subsequent plateaus. Here, fluorescence
enhancement was related to the clinical score of inflammation. Adapted from Gabriel et al. (2009)
Photothermal therapy is another promising approach to treat diseases that cir-

cumvent the adverse side effects caused by conventional drugs. It is based on the
optical properties of certain molecules or NPs that dissipate energy as heat upon the
absorption of light. A double targeting strategy explored in a theranostic
supramolecular photothermal agent between an NIR dye squaraine (SQ) and bovine
serum albumin (BSA) indicated promising results for both tumor ablation and
noninvasive imaging. An SQBSA construct was compared to another carrying
folate as a targeting moiety (SQBSAFA). Folate is a critical nutrient for DNA

synthesis, methylation, and repair and its receptor is often overexpressed in rapidly
dividing cells, such as in the case of cancers (Gao et al. 2014; van Dam et al. 2011).
SQBSAFA was found to be preferentially retained in tumors due to passive
targeting and active retention. Extravasation of administered formulation is by its
nature always passive, but the following retention process is active due to the
interaction between the receptor and targeting ligand. Photothermal treatment with
both products resulted in the inhibition of tumor growth of up to 32 % for SQBSA
and up to 78 % for SQBSAFA. The benets of active targeting were conrmed
both by NIR imaging during the treatment and later by histology (Gao et al. 2014).
Photothermal cancer treatment was also proven possible by using PEGylated
nanoconstructs based on a poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate)
(PEDOT:PSS) polymer mixture covered with poly(allylamine hydrochloride)
(PAH) and poly(acrylic acid) (PAA). The PEG termini of PEDOT:PSS-PEG were
labeled with a fluorescent dye Cy5 to follow the in vivo behavior in 4T1 murine
breast-cancer-bearing mice. Fluorescent imaging was performed at 1, 4, 8, 24, and
48 h after NP administration. The increasing fluorescence intensity with time in
tumors conrmed the uptake of NP by the EPR effect. To investigate the therapeutic
potential of these agents, mice were divided into four groups. The rst group received
PEDOT:PSS-PEG at a dose of 1 mg/ml and was exposed to an 808-nm laser at a
power density of 0.5 W/cm2 for 5 min 48 h post-injection. The other three groups are
untreated mice, laser only, and PEDOT:PSS-PEG without laser irradiation served as
controls. The mice injected with PEDOT:PSS-PEG and exposed to the laser showed a
complete eradication of tumors after only 1 day of treatment. No noticeable toxicity
was reported (Cheng et al. 2012). These ndings conrmed the potential of a pho-
tothermal tumor ablation approach for theranostic purposes.
4 Lipid-Based Nanosystems
4.1 Liposomes
Liposomes are aqueous vesicles surrounded by a phospholipid bilayer. As drug

delivery systems they have been exploited mainly with sizes ranging between 50 and
200 nm (Patil and Jadhav 2014). Depending on the preparation and purication
techniques, liposomes may vary in structure from unilammellar vesicles to
multi-layered formulations (Patil and Jadhav 2014). Because they are composed of a
hydrophilic core and a hydrophobic lipid bilayer, liposomes are well-suited for the
delivery of both hydrophilic and hydrophobic compounds. This makes them perfect
candidates for theranostic delivery systems. Specic ligands can be easily attached on
the liposomal surface to achieve active targeting. Various labels for diagnostic imaging
can also be conjugated to the surfaces (Torchilin 2005). Although liposomes are one
of the most widely used delivery systems, their main drawback is their rapid
opsonization and uptake by the cells of the reticuloendothelial system (RES) in the
liver and the spleen. One of the most employed strategies to avoid uptake and
clearance by the RES is the incorporation of hydrophilic PEG chains onto the surface
of liposomes (Harris and Chess 2003; Torchilin et al. 1994). Due to the association of
water molecules, PEG acts as a shield against enzymatic degradation and opsonization
by plasma proteins. Overall, the attachment of PEG improves the in vivo pharma-
cokinetics (Chen et al. 2004), prolongs the circulation time of particles, and gives
liposomes stealth properties (Harris and Chess 2003; Torchilin et al. 1994).
Liposomes have been designed to target integrins, a family of heterodimeric cell
surface receptors involved in cell adhesion, motility, growth, and survival. One of the
mostly targeted proteins of this family is the 3 overexpressed on the vascular
endothelium and signicantly involved in angiogenesis. Peptides containing the
arginineglycineaspartic acid (RGD) motif are often used for the specic targeting of
the 3 integrin on angiogenic endothelial cells (Beer and Schwaiger 2008; Chen
et al. 2004; Mittra et al. 2011; Teesalu et al. 2013). Another target for anti-angiogenic
treatment is neural cell adhesion molecules (NCAMs). NCAM is a protein that
belongs to the superfamily of immunoglobulins (Ig). Its role in cellcell interactions is
found to be important in tumor-associated angiogenesis (Bussolati et al. 2006) and is a
valuable target for high-afnity NCAM-binding peptides (Geninatti Crich et al. 2006).
Grange et al. (2010) prepared NCAM-binding peptide (C3d)-coated PEG liposomes
encapsulated with DOX and a lipophilic (Gd)-DOTAmonamide (DOTAMA)
derivative. In vivo studies with these liposomes were carried out in a model of
Kaposis sarcoma in SCID mice. The results indicated a more efcient tumor
regression and a lower toxicity when using NCAM-targeted liposomes compared with
untargeted counterparts. Interestingly, the MRI signals were higher for the untargeted
liposomes. This was attributed to the efcient internalization of targeted paramagnetic
vesicles into the cells that limited the relaxation enhancement due to the reduced
exchange of water molecules across the intracellular compartments.
Theranostic liposomes were developed by Agulla et al. (2014a) for the treatment
of stroke. Their formulation encapsulated citicoline and contained rhodamine for
fluorescence and Gd ions for MR imaging. Active targeting was achieved by
conjugating the protein HSP72, a biomarker of the peri-infarct region. Both the
diagnostic and the therapeutic potential of the liposomal formulation were assessed
on an in vivo rat model of ischemic stroke. The MRI T1 mapping of the brain of
ischemic rats was performed 1 day before and 1, 3, and 7 days post-intervention.
Promising results were obtained with respect to the targeting potential of the vec-
torized liposomes in the delineation and the follow-up of the peri-infarct tissue. To
further evaluate the therapeutic potential of anti-HSP72 liposomes, a permanent
focal cerebral ischemia was performed in six groups of Sprague-Dawley rats. The
results showed the highest therapeutic effect in rats intravenously injected with
HSP72-targeting liposomes encapsulated with citicoline at t = 45 min and 6, 12,
24, and 30 h post-surgery. Lesion volumes were signicantly smaller in comparison
with the controls at day 1, day 2, and day 7 post-surgery, and compared with
citicoline encapsulated in nontargeted liposomes (Agulla et al. 2014a).
An active targeting with liposomes is also possible by taking advantage of acidic
pH changes in subcellular compartments. A pH-sensitive liposome can be achieved
by modifying their lipid composition, incorporating pH-sensitive polymers, or a

combination thereof. This type of liposome prevents the premature release of an
active principle or an oligonucleotide during gene therapy (Torchilin 2005). The
release of the cargo from pH-sensitive liposomes is triggered by the acidic endo-
somal environment as was demonstrated by a kinetic model of the cellular uptake
and release of MRI agents (Delli Castelli et al. 2010).
One of the most promising theranostic agents and certainly the most advanced in
terms of clinical development is the thermosensitive DOX-loaded paramagnetic
liposome designed for MRI (Langereis et al. 2013). These liposomes not only cir-
cumvent the undesirable side effects of DOX, but also enable the assessment of
therapeutic efcacy. In thermosensitive liposomes (TSLs), the cytotoxic drug, and the
imaging agent are entrapped within the aqueous lumen at normal body temperatures
and are released in cancerous tissue upon the application of local hyperthermia (de
Smet et al. 2011). It was demonstrated that hyperthermic conditions enabled the
efcient extravasation of small liposomes (Kong et al. 2000; Kong et al. 2001). Lipids
selected for the preparation of TSLs have relatively low melting phase transition
temperatures. When this temperature is reached at mild hyperthermic conditions (39
42 C), a liquidcrystalline phase transition occurs and increases the permeability and
release of its contents (de Smet et al. 2011; Langereis et al. 2013). Pioneering reports
suggesting the feasibility of such drug delivery systems for tumor treatment were
published in the late 1970s by Yatvin et al. (1978), Weinstein et al. (1979). With
modern-designed lipids and stealth liposomes, hyperthermia-induced drug release is
now a realistic goal. A large number of studies have been published in the past decade
investigating safer techniques and practices for use in patients. Hyperthermia can be
induced by high intensity focused ultrasound (HIFU). MRI-guided HIFU is a non-
invasive procedure already used in clinical practice (Tempany et al. 2003). It increases
local temperatures deep inside targeted tissues by focusing US waves over a small,
well-dened area (Deckers and Moonen 2010; Moonen 2007). The MRI-directed
guidance enables in situ target denition and a highly localized treatment that spares
healthy tissue (Tempany et al. 2003; Moonen 2007). Over the course of 15 years,
Dewhirst and colleagues developed paramagnetic liposomes for the delivery of
chemotherapeutic agents into tumors under mild hyperthermia (Ponce et al. 2007;
Needham et al. 2000; Needham and Dewhirst 2001; Viglianti et al. 2004, 2006;
Negussie et al. 2011). In an extensive longitudinal study, they elaborated the concept
of drug dose painting. This concept uses different protocols for hyperthermia with a
real-time control of intratumoral drug distribution. Three hyperthermia protocols that
used lysolipid-based temperature-sensitive liposomes (LTSLs) containing DOX and
Mn as an MRI tracer (DOX/Mn-LTSLs) were evaluated in tumor-bearing rats (Fig. 7).
In the rst protocol, hyperthermia was initiated 15 min before the intravenous
administration of DOX/Mn-LTSLs. For the second protocol, DOX/Mn-LTSLs were
administered 15 min before hyperthermia. In the third protocol, half of the
DOX/Mn-LTSL dose was administered before hyperthermia initiation and the other
half was administered 15 min after a thermal steady state was reached. They found
that rats administered with DOX/Mn-LTSLs during hyperthermia showed the best
tumor response and demonstrated an MRI-monitored real-time drug dose painting
Fig. 7 a Schematic representation of a DOX/Mn-LTSL, theranostic temperature-sensitive

liposome containing therapeutic DOX, and an MRI contrast agent, MnSO4. b In vivo MRI
images showing a tumor drug distribution after i.v. injection of DOX/Mn-LTSL to rats-bearing
brosarcomas. a DOX/Mn-LTSL administered during steady-state hyperthermia. Radial lines
depict the orientation of DOX concentration prole. b DOX/Mn-LTSL administered 15 min
before the onset of hyperthermia resulted in central enhancement of DOX concentration.
c Injection of DOX/Mn-LTSL both before and during hyperthermia show uniform drug
distribution. Adapted from Ponce et al. (2007)
(Ponce et al. 2007). Unfortunately, the use of toxic Mn for MRI-mediated monitoring
of the treatment response has thus far prevented the clinical development of
DOX/Mn-LTSLs. In a proof-of-concept study in 9L glioblastoma-bearing rats, de
Smet et al. developed temperature-sensitive liposomes (TSLs) encapsulated with DOX
and [Gd(HPD03A)(H2O)] complexes (de Smet et al. 2010). A HIFU-triggered release
of imaging agents was monitored with interleaved T1 mapping of the tumor and
correlated with DOX release. The results showed a good correlation between the
averaged R1, changes across the tumor tissue, and the uptake of DOX. Because of
high variations of inter-tumoral DOX concentrations, this study suggested that the
uptake of therapeutic agents depended on the type of tumor and its pathophysiology
(vascularization, permeability, and existence of a necrotic core) (de Smet et al. 2011).
The feasibility of MR-HIFU using DOX-loaded LTSLs was recently shown to be
efcient on a Vx2 rabbit tumor model (Ranjan et al. 2012).
During the development of MRI theranostics, the payload of contrast agents
plays a critical role with respect to their toxicity. Agulla et al. compared the in vivo
contrast for liposomal formulations containing different Gd concentrations. In their
study, the intensity of the T1 effect was not linearly proportional with the liposomal
Gd content. The liposomal formulation with the lowest concentration of Gd had the
highest longitudinal relaxivity (Agulla et al. 2014b). This was in agreement with the
claim that the longitudinal relaxivity of paramagnetic liposomes was modulated in

living cells by compartmentalization effects (Langereis et al. 2013).
4.2 Micelles
Micelles are self-assembling systems made of amphiphilic polymers with

hydrophobic chains in the core and outward-facing hydrophilic moieties forming a
corona. As is the case for liposomes, depending on the amphiphilicity of its con-
stituents, micelles can have stealth properties and can be functionalized for active
targeting (Torchilin 2004). Micelles used for drug delivery purposes have a
diameter typically less than 100 nm (Straathof et al. 2011; Blanco et al. 2009;
Torchilin 2007). Polymer micelles are stable, biocompatible systems that can sol-
ubilize a large palette of poorly water-soluble compounds (Blanco et al. 2009;
Torchilin 2007; Matsumura et al. 2004). Different copolymers can be used for the
preparation of micelles. Poly(propylene oxide) (PPO), PLA, poly(glycolide),
PLGA, poly(-caprolactone) (PCL) (Blanco et al. 2009), poly(amino acid), and poly
(trimethylene carbonate) (PTMC) are among the most widely used polymers in the
hydrophobic core. The corona is mainly composed of hydrophilic polymers, such as
PEG (Blanco et al. 2009; Torchilin 2007), poly(N-vinyl-2-pyrrolidone)
(PVP) (Torchilin 2007), and PHPMAm (Talelli et al. 2010). Because micelles lack
the aqueous core found in liposomes, both drug and an imaging agent can either be
bound to the polymer, conjugated via an anchor molecule, or entrapped in its
hydrophobic core (Janib et al. 2010; Torchilin 2007).
Kaida et al. prepared theranostic micelles incorporating gadoliniumdiethylenetri-
aminepentaacetic acid (GdDTPA) and (1,2-diaminocyclohexane) platinum
(II) (DACHPt; i.e., the parent complex of an anticancer agent oxaliplatin) into the
hydrophobic moiety of poly(ethylene glycol)-block-poly(glutamic acid) [PEG-b-P
(Glu)]. Compared with the free drug, these micelles showed a prolonged circulation
time and strong anticancer effect in an orthotopic human pancreatic cancer model.
Furthermore, an enhanced micelle-mediated MRI contrast of the tumor tissue was
compared with that of the GdDTPA alone (Kaida et al. 2010).
Recently, polymer micelles incorporating radionuclides for SPECT and PET
imaging have gained interest. Peng et al. reported on a multimodal system designed
for the photothermal therapy of cancer that incorporated a NIR dye, IR-780, for
fluorescence imaging, and 188Re for SPECT imaging (Peng et al. 2011). The in vivo
results of photothermal therapy induced by irradiation with a light dose of
540 J/cm2 of NIR light showed an 82.6 % inhibition of tumor growth after 27 days.
Treatment was successfully monitored with both NIR fluorescence imaging and
micro SPECT/CT.
An entirely different approach for using micelles as theranostic vehicles was
explored in the work of Wu et al. who described a rst NIR dicyanomethylene-4H-
pyran (DCM)-based prodrug, PEGPLA/DCM-S-camptothecin (CPT), for the
monitoring of the anticancer treatment in living animal models (Fig. 8). The
Fig. 8 a Scheme of proposed CPT release mechanism from the activated prodrug; b fluorescence
intensity images of DCM-C-CPT and c DCM-S-CPT in internal organs after administration.
Antitumor activities of PBS, CPT, and PEGPLA micelles loaded with the C or S form of the
prodrug administered at a dose of 5 mg/kg (CPT equivalents) to BCap-37 mice tumor model. As it
can be seen from the histogram, PEGPLA/DCM-S-CPT micelles inhibited tumor growth
(IRT) when compared with the PEGPLA/DCM-C-CPT and free CPT (*p < 0.05; **p < 0.01).
Adapted from Wu et al. (2014)
prodrug itself was composed of the NIR fluorophore DCM and the anticancer drug
CPT joined by a disulde linker. The prodrug was loaded into PEGPLA micelles
to improve its water solubility and to achieve tumor targeting by the EPR effect
(Wu et al. 2014). An advantage of the system was that both the fluorescence and
cytotoxicity were initially quenched. When the micelles reached the tumor cells, the
disulde bonds were cleaved because of elevated intracellular glutathione levels.
This enabled the drug release, which was monitored by the unquenched NIR
fluorescence. The antitumor activity of these micelles was assessed in a BCap-37
tumor xenograft mouse model. Mice were intravenously administered with PBS,
CPT, PEGPLA/DCM-S-CPT, and PEGPLA/DCM-C-CPT (with an alkane bond
instead of a disulde bond), at a CPT equivalent dose of 5 mg/kg. PEG
PLA/DCM-S-CPT showed a tumor inhibition rate of 96.4 % compared with 16.3
and 88.9 % for PEGPLA/DCM-C-CPT and CPT alone, respectively. No severe
systemic side effects were observed upon PEGPLA/DCM-S-CPT treatment (Wu
et al. 2014).
5 Inorganic Assemblies
In addition to organic polymers and lipids, various inorganic materials may be used
in the preparation of NPs for drug delivery and imaging. These range from metals,
such as iron, silver, or gold, and quantum dots (QDs), to carbon- and silica-based
matrices. In the next section of this chapter, we will cover the area of iron oxide and
gold NPs used in theranostics.
5.1 Iron Oxide NPs (IONPs)
Iron oxide NPs (IONPs) are made from hematite (Fe2O3) or magnetite (Fe3O4).
These particles are paramagnetic, can serve as good T2 MRI contrast agents, and
possess attractive physical properties for hyperthermic treatment.
Superparamagnetic iron oxide NPs (SPIONs) are small IONPs (120 nm), mostly
made from magnetite. The large specic surface area of IONP and SPIONs makes
them suitable for the attachment of drugs and other ligands (Choi et al. 2012).
Furthermore, their biodegradability, biocompatibility, and unique targeting possi-
bilities controllable by an external magnetic eld make them candidates for ther-
anostic purposes (Santhosh and Ulrih 2013). Standard methods for the preparation
of IONPs include co-precipitation (Xie et al. 2010; Santhosh and Ulrih 2013),
microemulsication, thermal decomposition of iron precursors (Xie et al. 2010;
Santhosh and Ulrih 2013), and hydrothermal methods (Santhosh and Ulrih 2013).
New approaches for the production of monodispersed NPs use microwaves and
sonochemical routes (Santhosh and Ulrih 2013). Silica (Algar et al. 2011),
phospholipids, or polymers, such as PEG, dextran, chitosan, PVP, and polyaniline,

are typically added during their preparation (Xie et al. 2010; Santhosh and Ulrih
2013) to minimize oxidation, avoid NP aggregation, and tailor their surfaces for
further attachment of functional groups and drugs (Xie et al. 2010).
Considering IONPs, we rst examine the results of passive targeting nanosystems.
Then, different active targeting strategies will be discussed. Quan et al. (2011)
developed human serum albumin (HSA)-coated IONPs loaded with DOX
(DOX-HSA-IONPs). NPs were prepared by adding DOX and dopamine-coated
IONPs into an HSA-containing aqueous solution. Because HSA has a good
ligand-binding capacity, a high loading rate was achieved for both DOX and IONPs
[the DOX/Fe/HSA ratio was 1:2:20 (w/w/w)]. The efcacies of the
DOX-HSA-IONPs, commercial Doxil liposomes, and free DOX were evaluated on a
4T1 murine breast cancer model. Particle uptake upon administration was monitored
by MRI. MR images taken 1 and 4 h after the injection of DOX-HSA-IONPs showed
a signal decrease as a result of NP accumulation in the tumor area. The efcacy of
DOX-HSA-IONPs in tumor suppression was comparable with that of Doxil and
outperformed free DOX treatment (Quan et al. 2011).
Another passive targeting nanotheranostic system was developed by Yu et al.
who incorporated DOX into the polymer shell of thermally cross-linked
(TCL) SPIONs (TCL-SPIONs) through electrostatic interactions with polymer
coating layers. For the validation of the therapeutic efcacy of DOX-TCL-SPIONs,
mice-bearing Lewis lung carcinoma was used. It was demonstrated that
DOX-TCL-SPIONs primarily localized in tumors. Because of their SPION content,
these nanosystems were precisely localized by MRI. In this case, the
DOX-TCL-SPIONs gave a superior therapeutic outcome when compared with free
DOX. No systemic toxicity was reported (Yu et al. 2008).
As mentioned above, the surface of iron oxide particles can be functionalized with
specic moieties targeting overexpressed proteins. IONPs targeting the receptor of uPA
(uPAR) were conjugated with the amino-terminal fragment (ATF) peptide of the
receptor-binding domain of uPA and the chemotherapeutic drug gemcitabine
(GEM) via the cathepsin B cleavable peptide linker (GFLG). In vivo experiments were
carried out in the orthotopic tumor xenografts with MIA PaCa-2 human pancreatic
cancer cells in nude mice. Mice were divided into groups administered with free GEM,
ATF-IONP-GEM, and IONP-GEM (without any targeting moieties). A 2 mg/kg
GEM-equivalent dose was given to mice twice a week for ve times. T2 MR images
were acquired before the treatment and 1 and 2 weeks following the treatment (Fig. 9).
Mice treated with ATF-IONP-GEM showed an approximate 50 % inhibition of tumor
growth in comparison with 30 and 23 % by free GEM and nontargeted IONP-GEM,
respectively. Moreover, the conjugation of GEM to the NPs improved the stability of
the drug in vivo by preventing its deactivation by cytidine deaminase (Lee et al. 2013).
This study is one of many that showed the effectiveness of active targeting.
Additionally, it showed the potential of using IONPs to monitor the accumulation of
nanosystems in targeted tissues and conrmed a sustained drug release.
Magnetic drug targeting is a term introduced by Alexiou et al. (2000). In their
experiments, mitoxantrone (MTX), an antineoplastic agent, was bound to
Fig. 9 Axial T2-weighted MRI images of tumor-bearing mice before, 1 (1 W) and 2 weeks (2 W)
upon NPs administration. Post 1W and post 2W images were obtained 48 h after second and third
injections, respectively. Pink-dotted circles mark tumor location and size, whereas red arrows
show the MRI contrast change in the spleen. Adapted from Lee et al. (2013)
multi-domain NPs formed from iron oxides and hydroxides (ferrofluids), which
were then coated with starch polymers. The colloidal dispersions of the
ferrofluid-MTX were administered to female New Zealand White rabbits inoculated
with VX-2 squamous cell carcinoma. An inhomogeneous magnetic eld produced
by an electromagnet was used to direct the ferrofluid-MTX to the tumor. High
concentrations of the construct in the tumor tissue were conrmed by MRI and
histology. This magnetic drug targeting approach led to the complete remission of
the tumors with reduced doses of 20 and 50 % by the ferrofluid-MTX relative to the
conventional MTX dose. The therapy was well-tolerated and showed no signs of
toxicity (Alexiou et al. 2000). Although promising in animal studies, this approach
seems difcult to be translated to humans.
5.2 Gold NPs (AuNPs)
Gold NPs (AuNPs) range in size between 1 and 100 nm. Depending on their size
and shape, their optical properties are characterized by strong absorption and light
scattering in the visNIR region (Algar et al. 2011; Alkilany et al. 2012; Boisselier
and Astruc 2009; Chen et al. 2010; Khlebtsov et al. 2013; Zhang et al. 2013).
Another important feature of AuNPs is the phenomenon of localized surface
plasmon resonance (LSPR) (Khlebtsov 2008). Upon light irradiation at the proper
frequency, the electrons in the conduction band of gold atoms are excited and begin
to oscillate, which leads to extensive light extinction. For gold nanospheres, a broad
LSPR band is observed at approximately 520 nm. In gold nanorods (AuNRs), the
LSPR band splits into a transverse band and a longitudinal band. Their positions are
dependent on the length and width of AuNRs. Additionally, AuNRs can be used for
the photothermal therapy. AuNPs have been tested for the photodynamic treatment
of pathogenic bacteria and photothermal cancer therapy (Khlebtsov et al. 2013).
Furthermore, AuNPs have been used to quench the fluorescence of different
fluorophores (Algar et al. 2011) and are capable of increasing the fluorescence
quantum yield of NIR dyes up to 80 % (Bardhan et al. 2009). Gold is considered an
inert metal, but there are a number of synthetic methods to tune the size and shape
of AuNP and to chemically bind various molecules to their surface, such as MRI,
fluorescence reporters, and therapeutic moieties, for multimodal theranostic appli-
cations (Alkilany et al. 2012). These characteristics make AuNPs suitable contrast
agents for in vivo fluorescence imaging and valuable theranostic entities.
One of the most advanced nanosystems in use in photothermal tumor ablation by
NIR-absorbing AuNPs is AuroLaseTM. This system is a PEGylated gold nanoshell
formulation prepared by the absorption of colloidal gold to amine groups on silica
NPs (ONeal et al. 2004). Results observed after i.v. administration to both rodents
(ONeal et al. 2004) and dogs (Schwartz et al. 2009) showed the accumulation of
nanoshells in tumor tissue via the EPR effect. Irradiation with light at 80 nm led to
heat dissipation by the AuNPs and consequently, tumor eradication by thermal
ablation, as indicated by MRI based on the temperature-dependent proton resonance
frequency shift (Schwartz et al. 2009).
The search for multimodal imaging techniques to further improve diagnosis and
monitoring is intensifying. One way to overcome the limitations of single imaging
modalities is to design platforms to carry several complementary imaging probes
(Louie 2010). One example is a platform using gold nanoshells covered with a layer of
silica entrapping Fe3O4 NPs and the NIR fluorophore indocyanine green (ICG). These
particles were intended to have dual diagnostic features and photothermal ablative
properties. Because silica enables the attachment of various moieties, additional
anti-HER2 antibodies were conjugated to the particles (Bardhan et al. 2009).
Furthermore, PEG was added to decrease their immunogenic potential and to increase
their blood circulation time (Bardhan et al. 2010). Tests were performed in mice
inoculated with either BT474AZ cells highly overexpressing HER2 or MDAMB231
cells with low HER2 expression. NIR images were obtained at 0.3, 2, 4, 24, 48, and
Fig. 10 Imaging of nanocomplex delivery. a NIR fluorescence images of mice with HER2
overexpressing BT474AZ xenografts and MDAMB231 (control) xenografts. Images were taken at
0.3, 2, 4, 24, 48, and 72 h post-injection. b T2-weighted images of BT474AZ and MDAMB231
mice before and 0, 4, 24, 48, and 72 h after administration. Tumors are marked with red circles.
Adapted from Bardhan et al. (2010)
72 h post-injection with highest fluorescence intensity observed at 4 h. The results

showed signicant variations between the two xenograft models indicating the efcacy
of the antibody-mediated active targeting strategy. This result was conrmed by
T2 MRI (Fig. 10). However, a certain difference in NP accumulation across time
points can be seen when the fluorescence images were compared with the MRI. The
authors explained these discrepancies by the intrinsic differences between the NIR and
MR imaging. In short, this study provided an example of a theranostic agent for
thermoablation with simultaneous NIR and MR imaging features for the active tar-
geting of HER2-positive breast carcinoma (Bardhan et al. 2010).
As mentioned above, active targeting is often achieved by attaching antibodies to
NPs. A probe made from AuNR covalently linked to a low-molecular weight
chitosan (Mw 5000), capped with 11-mercaptoundecanoic acid (MUA) and con-
jugated to the epithelial growth factor receptor (EGFR) monoclonal antibody, was
described by Charan et al. (2012). In vitro and in vivo studies showed the selectivity
of this theranostic probe for the specic targeting of cancer cells overexpressing
EGFR. Furthermore, no signicant toxicity was observed in mice injected with the
AuNR nanocomplexes.
Gold particles can also be used for PDT. PEGylated AuNRs loaded with a
phthalocyanine capable of dual photothermal and PDT were tested in xenograft
mice (Jang et al. 2011). Treatment was monitored by NIR imaging. For mice
injected with AuNR and irradiated with NIR light, terminal deoxynucleotidyl
transferase dUTP nick end labeling technique (TUNEL) showed a high degree of
tissue damage and apoptosis across the tumor area compared with mice treated with
PBS (control) or PDT only.
Another complex theranostic nanoconstruct was recently created by Topete et al.
(2014). It was composed of a PLGA matrix loaded with DOX and covered with a
porous shell of gold. To provide the nanoparticle with stealth properties, the surface
was functionalized with HSA. ICG was attached for both imaging and PDT pur-
poses. Active targeting was provided by the covalent conjugation of folic acid.
Preliminary in vivo results were obtained on BALB/c nude mice inoculated with
folic acid receptor-overexpressing MDA-MB-231 breast cancer cells. NIR
fluorescence was monitored intravitally during treatment. An accumulation of these
theranostic nanoconstructs was observed in tumor tissue and in the brain, which
was surprising and would deserve conrmation (Topete et al. 2014).
6 Quantum Dots (QDs)
Quantum dots (QDs) are nanocrystals (210 nm) made from semiconducting mate-
rials. For biomedical applications, the QD cores are usually made from CdSe or CdTe
and coated with ZnS for improvement of luminescent properties (Algar et al. 2011).
Compared with standard organic fluorophores, QDs have up to 100 times improved
brightness, photostability, and resistance to photobleaching. Additionally, QDs have
wide excitation bands and narrow emission windows (Janib et al. 2010). QDs are now
extensively investigated for their incorporation in imaging nanoconstructs. However,
synthetic routes for QDs have resulted in insoluble and hydrophobic materials.
Therefore, bifunctional ligand coating and bifunctional covalent attachment have been
employed to overcome these limitations (Algar et al. 2011). Furthermore, efforts have
been made to increase the stability, biocompatibility, and blood circulation of QDs.
NIR QD-loaded PEG-10,12-pentacosydonic acid micelles equipped with trastuzumab
conjugates for active targeting showed 77.3 % tumor growth inhibition when injected
into athymic BALB/c-nu/nu nude mice-bearing HER2 positive tumors. NIR images
showed a rapid distribution of micelles and their accumulation at the tumor sites
(Nurunnabi et al. 2010). Although, these theranostic micelles did not show signs of
toxicity, more experiments are required to assure the safety and complete clearance of
QDs from the body.
7 Theranostic Application of Antibodies
Trastuzumab (Herceptin), rituximab (Rituxan), bevacizumab (Avastin), cetux-

imab (Erbitux), and panitumumab (Vectibix) are among many examples of Food
and Drug Administration in the United States of America (FDA) approved
therapeutic antibodies for cancer treatment (Accardo et al. 2013; Fleuren et al.
2014). Antibodies can be modied with radioactive tracers and fluorescent dyes for
use in disease diagnosis and monitoring. This approach facilitates the selection of
patients who are likely to respond to targeted antibody treatments. A comprehensive
review on the theranostic applications of antibodies in oncology was recently
published by Fleuren et al. (2014). Here, we will address only a few examples of
these theranostic agents. 177Lu-J591 is a humanized radiolabeled monoclonal
antibody against prostate-specic membrane antigen (Bander et al. 2005).
177
Lu-J591 emits both and -rays, which make this antibody conjugate well-suited
for theranostic purposes. -rays will enable the disease treatment, while -rays will
serve as imaging tracers. In a phase II clinical trial, the response rate in patients was
assessed. 177Lu-J591 administration was well-tolerated and exhibited accurate
tumor targeting capabilities. Additionally, prostate-specic antigen responses were
in accordance with dose response (Tagawa et al. 2013). Another example of a
potentially successful theranostic agent for treatment prediction is a chimeric
antibody 177Lu-girentuximab (177Lu-cG250), which was developed for the treat-
ment of metastatic clear cell renal cell carcinoma. This antibody binds to a
heat-sensitive transmembrane glycoprotein carbonic anhydrase IX (CAIX). A phase
I clinical study showed that 177Lu-cG250 was well-tolerated in patients, could be
repetitively administered, and could prevent the progression of the disease
(Stillebroer et al. 2013). Apart from their application as theranostic agents, the use
of antibodies, especially those having fluorescent tags, is also potentially applicable
for imaging-assisted surgery (Heath et al. 2012; Muselaers et al. 2014). However,
this is beyond the scope of this chapter.
8 Nanotheranostic Systems for Gene Therapy
Gene therapy was initially intended to be used to treat hereditary diseases; however,
it also has high potential in cancer treatment (Devi 2006). Vectors for gene delivery
are viral or non-viral. Although good results of gene transfer were obtained with
viral vectors, their use still raises important issues about their safety because a few
vectors may induce mutagenesis and can cause unwanted immune responses in
patients. Functionalized gold NPs, carbon nanotubes, polymer NPs, and cationic
polymers are examples of non-viral gene vectors (Wang et al. 2012; Mok and Park
2012). Among them, polymer particles have shown potential in delivering nucleic
acids to cells and are now being investigated for theranostic purposes. One example
of these complex structures is a coreshell NP produced by the SPION loading of
mPEGPLA micelles for the delivery of plasmid DNA coated with cationic
polymers (chitosan and PEI). These NPs are designed for T2-weighed MRI and
gene delivery and have shown efcient cell transfection in vitro and in vivo (Wang
et al. 2012). Multifunctional chitosan magnetic-graphene NPs are also promising
theranostic gene/drug delivery vehicles (developed by the same group). These
SPION-loaded NPs simultaneously deliver plasmid DNA and DOX to implanted
tumors in mice and were suitable for MRI (Wang et al. 2013). The gene Survivin,
an inhibitor of apoptosis, is upregulated in many types of cancer. Because this gene
is not expressed in normal cells, it presents a valuable target for gene silencing by
siRNA. Theranostic liposomes were prepared from the combination of following
compounds: Gd-DOTA-DSA, CDAN, DOPC, DSPE-PEG2000, and DOPE rho-
damine through the rehydration and sonication of lipid lms. Anti-Survivin siRNA
was then added by intensive vortex mixing. Liposomes were intravenously
administered into mice carrying OVCAR-3, human ovarian cancer cells. T1 MR
images showed increased signal intensity upon treatment. Tumor growth was
reduced in comparison with the non-silencing negative controls. Fluorescence
microscopy imaging conrmed the colocalization of liposomes and siRNA in the
tumor (Kenny et al. 2011). These multifunctional nanocarriers are promising sys-
tems for the future of gene delivery approaches for the treatment of cancers.
9 Discussion
Early diagnosis is of utmost importance in providing efcient treatment to patients

suffering from diseases such as cancer. With respect to cancer detection, the most
encountered problems with most imaging modalities are low detection limits. In
general, tumor masses must contain at least 109 cells (having a volume of approxi-
mately 1 cm3) (Frangioni 2008) to be detectable by conventional imaging techniques.
The difculty in developing a successful theranostic agent is in optimizing the con-
centration of the imaging probe to provide a sufcient SBR and a sufcient concen-
tration of active pharmaceutical ingredient in a single vehicle. The recent development
of nanotechnology provides the opportunity to conne multiple imaging modalities
within the same carrier. Today, we are able to visualize pathophysiological processes,
such as changes in enzyme regulation or DNA and RNA expressions at cellular and
molecular levels. This so-called molecular imaging will be important for the early
diagnoses of diseases where phenotypic changes in cells are not yet evident
(Weissleder 1999, 2002; Mulder et al. 2007). Theranostic agents have the ability to
monitor and control the location, dose, and time of drug delivery and provide a
real-time feedback of a given treatment. This enables the adjustment of subsequent
treatments for an individualized therapeutic approach. Multimodal theranostic NPs
enable molecular imaging because of the complementary functions of two or more
imaging agents colocalizing at the target site (Louie 2010; Thorsen et al. 2013).
Examples of these multi-imaging constructs are particles capable of simultaneous MRI
and fluorescence imaging. MRI provides excellent anatomical visualizations at high
resolutions, but is limited by nonspecic interactions and requires large quantities of
imaging agent. Optical fluorescence is very sensitive, has a high SBR, and can detect
molecular changes at the cellular level (Mulder et al. 2007). Multimodal delivery
systems provide advantages in single administration and have the same pharma-
cokinetic prole for each modality found in NPs. However, the development of this
type of nanosystems requires careful planning of synthesis procedures in order to
attach two entities with different chemical properties or imaging probes whose sen-
sitivities may vary among each other up to three orders of magnitude (Louie 2010).
The encapsulation of small molecules into nanocarriers or their conjugation with
polymers changes their properties. It increases the solubility of hydrophobic
molecules by orders of magnitude; alters the pharmacokinetic prole, biodistribu-
tion, and clearance properties; and reduces systemic toxicity. Nevertheless, there are
a number of requirements that need to be fullled for a clinically applicable nan-
otheranostic system. In this sense, polymer NPs present a valuable option for the
future development of theranostic agents as long as the polymers used in their
preparation fulll the following criteria: the manufacturing process should be easily
scaled up, linkages on the NP surface should be stable, and the specic activities of
the attached moieties should not be affected during the NP preparation. Moreover,
coupling chemistry protocols should be compatible with future medical applications
(Algar et al. 2011). Additionally, nanosystems need to be sterile. Up to date, it is
still difcult to have sterilization protocols that do not alter the physico-chemical
properties of the nanotheranostic formulations. Another challenge in the develop-
ment and the approval of polymer nanosystems is the natural heterogenicity of
polymers. This, along with the need to incorporate a large number of entities to NP
surface for the theranostic application, ultimately leads to complex characterization
methods (Duncan 2003; Yuan et al. 1995).
As mentioned earlier, the chances of better therapeutic outcome lie in the active
targeting of diseased tissues. Successful active targeting is very difcult to achieve due
to the limited number of highly specic cell surface epitopes expressed at the
appropriate locations. One of the key factors for the development of an efcient
therapy is regulating the particle sizes of the constructs to control their ability to cross
barriers, including basement membranes, endothelial cell linings, and in some cases
the bloodbrain barrier. It has been shown that the accumulation of drug delivery
systems in solid tumors depends on the tumor type and on the fenestration of tumor
blood vessels (Yuan et al. 1995). Recent progress in the eld of molecular biology and
organic synthesis has made it possible to identify and couple small ligands with
specic targeting capabilities onto the surface of NPs. The attachment of small
molecules generally favors penetration into tissues, induces fewer immunogenic
reactions, and provides better pharmacological and pharmacokinetic properties
(Weissleder 2001). In addition to size, other physical characteristics of administered
NPs, such as the polymer used in their synthesis, PEGylation, and attached ligands, all
play important role in the ability of formulations to reach and accumulate at the tumor
site (Iyer et al. 2006). Nevertheless, the EPR effect has mainly been proven in small
animals and rarely shown in humans. Murine tumors are induced by a subcutaneous or
orthotopical injection of previously cultured cancer cells which are not submitted to
immune pressure. These fast-growing tumors lack genetic diversity and they form
blood vessels unlike those in the human body. In this case much care is required when
extrapolating results from preclinical in vivo pharmacokinetics and biodistribution
studies to humans (Nichols and Bae 2014).
There are many questions concerning the safety of nanomedicines. This par-
ticularly applies to metals and polymers used in the composition of nanoconstructs.
During initial phases of the NP development, cellular assays such as the 3-

[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) or lactate
dehydrogenase (LDH) assays are usually employed to determine the toxic potential
of their components (Haglund et al. 2009). However, these types of assays do not
reflect the real situation. Hence, acute and chronic toxicity tests should be con-
ducted in all cases.
10 Conclusion and Future Perspectives
The eld of nanomedicine is growing rapidly giving rise to fascinating new plat-
forms and constructs that might provide new solutions for the expansion of ther-
anostics. Recent avenues in nanomaterials are in the eld of photopolymerizable
lipids (Puri and Blumenthal 2011). Liposomes made from this type of lipid can be
used for light-triggered drug release as shown by Yavlovich et al. (2011).
Moreover, a new imaging modality is emerging for the early detection of cancer
based on Raman spectroscopy (Nijssen et al. 2009). Nanoparticle-based combina-
tion chemotherapy with particles loaded with two drugpolymer conjugates have
also been recently reported (Aryal et al. 2011). This controlled dual drug loading
may serve in creating improved polymer platforms for theranostics.
It has been shown that the enhanced nanoparticle retention at tumor sites is
achieved when an active targeting approach is used. Hence, the future of theranostic
nanomedicine lies in a hetero-multivalent ligand design of NPs. This would enable
co-operative binding interactions for further improving targeting capabilities
(Modery-Pawlowski and Gupta 2014). Another solution for the efcient active
targeting of multimodal NPs could be found in a protease-removable polymer
coating that veils attached ligands. Once the hydrophilic polymer is cleaved, the
attached moieties become exposed and can be activated (Harris et al. 2008).
Additionally, Frster resonance energy transfer (FRET)-activated
self-immolative linker designs (Redy and Shabat 2012) show promise for prodrug
activation monitoring in vivo and can provide information about the specic
location and concentration of activated drugs in tissues.
Dendrimers, a class of well-dened multifunctional polymer nanoconstructs,
may also be a good solution for the development of theranostics. Dendrimers are
hyperbranched polymer macromolecules suitable for the controlled orthogonal
attachment of different moieties for drug transport, imaging, and targeting (Ornelas
et al. 2011; Cai et al. 2013). Many patients have already been led and then papers
published on dendrimer synthesis and their use in theranostics for photothermal
therapy (Li et al. 2014), PDT (Sibani et al. 2008; Taratula et al. 2013; Klajnert et al.
2012), and conventional drug targeting (Huang et al. 2014). In spite of their high
potential, dendrimer synthesis is a relatively expensive multistep process that may
present problems for scale-up productions.
As mentioned, microbubbles are another possible platform for drug delivery
(Eisenbrey et al. 2009, 2010a, b; Cochran et al. 2011). As one of the major causes
of death in the world, stroke has limited timeframe for treatment application.
Currently, the recombinant tissue plasminogen activator (rtPa) is the only throm-
bolytic drug approved by the FDA and European Medicines Agency (EMA) for the
treatment of ischemic stroke (Petit et al. 2012a). Petit et al. (2012b) observed a
synergistic effect of US, microbubbles, and rtPa on clot lysis. Although it is not
exactly the type of theranostics described in this chapter, microbubbles loaded with
rtPa and equipped with a targeting ligand could potentially provide better treatment
outcomes than the aforementioned theranostic procedures. Additionally,
microbubbles are interesting for multimodal imaging purposes. Recently, Huynh
et al. (2014) reported a microbubble formulation prepared by the substitution of
50 % molar regular phospholipid (DSPC) with a porphyrinlipid. These mi-
crobubbles were injected into mice-bearing KB tumor xenografts and imaged by
US, PAI, and fluorescence. Although the microbubbles burst upon US application,
the porphyrinlipid composition enabled the real-time tracking using PAI and
fluorescence imaging. Microbubbles with the abovementioned properties are
promising theranostic agents.
Although theranostics shows great potential for the development of medicine,
there have only been a few theranostic agents that have reached clinical trials. The
most advanced ones seem to be thermosensitive liposomes for HIFU treatment,
AuNPs for tumor thermoablation, and Ab conjugates used in radioimmunotherapy.
Acknowledgments NLs work is supported by the Grants Nos. 205320_138309,

CR32I3_129987, CR32I3_147018, 31003A_149962, and CR32I3_150271 of the Swiss Science
Foundation.
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Part IV
From Lab to Prescription Desk
Chapter 18
NanomedicinesA Scientic Toy
or an Emerging Market?
Matthias G. Wacker
Abstract In recent years, signicant effort has been made in the development and
synthesis of polymer nanoparticles for the targeted delivery of drugs. Although
many of these nanocarriers have attracted the attention of the pharmaceutical
industry, only a few of them have been approved so far. The growing knowledge of
their interactions with biological surfaces enables an adoption of the unique
properties of the nanoparticle to the physiological environment. The following
section describes the criteria that need to be considered for the development and
optimization of these versatile drug delivery systems and the requirements for their
translation into novel nanomedicines.

Keywords Nanocrystals Nanomedicines Industry Market Regulations

Developability Drugability
1 Introduction
Over the past decades many efforts have been made to employ nanotechnology in
formulation design and manufacture of new medicinal products. Since liposomal
drug delivery systems, diagnostic nanoparticles, and nanocrystals entered the
market (Wacker 2013), the regulatory framework has been adjusted and some of the
most important requirements for a successful product development have been
identied (European Medicines Agency (EMA) 2013a, b, c; Wacker 2014).
Nanocrystals are only one example for application of nanotechnology in phar-
maceutical product development. They take advantage of the increased dissolution
rate of small particles focusing active pharmaceutical ingredients (API) from the
M.G. Wacker (&)

Project Group for Translational Medicine and Pharmacology (TMP), Department of
Pharmaceutical Technology and Nanosciences, Fraunhofer-Institute for Molecular Biology
and Applied Ecology (IME), Max-Von-Laue-Strae 9, 60438 Frankfurt/Main, Germany
e-mail: matthias.wacker@ime.fraunhofer.de

DOI 10.1007/978-3-319-41421-8_18
592 M.G. Wacker
classes II and IV of the biopharmaceutical classication system (BCS). These API

are characterized by poor aqueous solubility and bioavailability.
Elan Nanosystems has a family of patents led in this area, including tech-
nologies for peroral drug formulations and injectables. By applying wet bead
milling to the nanonization of API, the company was successful in licensing their
technology to global pharma companies including Merck & Co, Johnson &
Johnson, Roche, or Wyeth (see Sect. 2.2.2, Table 1).
In 2011, the primary patents covering Elans NanoCrystal technology expired in
the United States of America (USA). In the following years more and more com-
panies started to implement nanocrystal technology into their formulation pipeline.
Especially during the early toxicity studies in rodents, formulations with high drug
load are needed. Wet bead milling is a rapid and simple method that allows the
synthesis of formulations for parenteral and peroral administration containing high
doses of the API in a liquid dosage form (see Sect. 4).
For many nanoformulations several stabilizing agents are required. Depending
on the route of administration specic issues arise during formulation development
such as limitations in the use of novel excipients. Traditionally, excipients were
inert substances that were mainly used as manufacturing aids, coatings, or diluents.
This denition has been challenged by modern high technology products (Pinco
and Sullivan 2006).
Similarly to drug discovery, drugability assessment has become an essential
part of formulation development in the pharmaceutical industry (Butler and
Dressman 2010). This developability approach allows the prioritization of for-
mulation characteristics, the descriptors, with respect to the intended therapeutic
application. By identifying descriptors with high impact on the therapeutic out-
come, a rational formulation design can be applied and the requirements for suc-
cessful product development can be identied. In the following some of the most
important criteria for a successful formulation development of polymer nanopar-
ticles will be discussed. Risks and benets of this outstanding approach in com-
petition with other technologies are considered.
Table 1 Nanocrystal products in the market and technologies applied for their manufacture at
large scale
Drug formulation Technology Compound

Rapamune NanoCrystal (Elan Drug Technologies) Sirolimus
Emend NanoCrystal (Elan Drug Technologies) Aprepitant
TriCor NanoCrystal (Elan Drug Technologies) Fenobrate
MegaceES NanoCrystal (Elan Drug Technologies) Megestrolacetate
TriglideTM DissoCubeTM (SkyePharma) Fenobrate
Invega, SustennaTM NanoCrystal (Elan Drug Technologies) Paliperidone palmitate
18 NanomedicinesA Scientic Toy or an Emerging Market? 593
2 Descriptors
For more than 30 years extensive research activities focused the investigation of
interactions between nanomaterials and biological interfaces, e.g., in the blood
(Labarre et al. 2005), the lungs (Lehmann et al. 2011; Schleh et al. 2014), the brain
(Wagner et al. 2012), the gastrointestinal (GI) tract (Beck et al. 2007), or at the skin
(Raber et al. 2014).
After having entered systemic circulation, colloids are distributed by ltration or
diffusion-based mechanisms in the respective organs. In specic, particles of a
certain size accumulate in the ne capillaries of lungs, liver, and kidney (Geiser and
Kreyling 2010). Another fraction is rapidly taken up by the macrophages after
opsonization with proteins of the immune system (Owens and Peppas 2006) or
eliminated after passing the glomerular lter (e.g., smaller drug conjugates).
Facing this biological environment, surface structure of the particles is modied by
procedures of adsorption or desorption of proteins (Labarre et al. 2005; Calatayud
et al. 2014; Sempf et al. 2013), aggregation, erosion, degradation, and metabolism.
In principle, there are two scenarios that have attracted the attention of the
pharmaceutical industry. Nanocrystal formulations have been utilized to optimize
bioavailability of compounds by increasing the dissolution pressure
(Merisko-Liversidge and Liversidge 2011; Merisko-Liversidge et al. 1996) or to
enable the parenteral administration of high doses of API, e.g., in toxicity studies.
Under such conditions solubility and permeability are of major importance for drug
release and bioavailability. But they do not affect the biodistribution of the
compound.
Contrasting such immediate release formulations, nanocarriers are specically
designed to target compounds to their site of action. A sustained release of the API
from the nanocarrier is essential to fulll the drug delivery paradigm (Wacker
2013): interaction of the matrix material with its physiological environment con-
trolling biodistribution of the API (see Fig. 1).
Fig. 1 Dissolution and release from polymer nanoparticles used as nanocarriers

594 M.G. Wacker
Nanoparticle
formulation
Nanocarriers Nanocrystals
Active drug Passive drug

Particle size Release rate
targeting targeting
Surface Surface
Drug load / Particle size/ Drug load/ Particle size/
charge/ charge/
Release rate shape Release rate shape
Hydrophilicity Hydrophilicity
Specific
binding
affinity
Fig. 2 Decision tree for optimization of nanoformulations based on therapeutic purpose and
major descriptors
While a number of mechanisms take influence on biodistribution and the

pharmacokinetic prole, an optimal nanoparticle design is achieved by applying a
rational formulation design supported by all necessary analytical tools. A selection
of formulation descriptors is based on the intended therapeutic application and
formulation approach (see Fig. 2).
At present, there are only few products on the market that claim a selective
targeting such as the liposomal formulation Doxil or the albumin-based product
Abraxane. In both cases, the release of the API is controlled by poor aqueous
solubility of the compound and not by the matrix material (Csuhai et al. 2015).
These formulations have been pioneering an emerging landscape of innovative
pharmaceuticals. A growing knowledge about the interplay between drug release,
formulation parameters, and the in vivo performance of nanosized dosage forms
will be a strong basis for a rational formulation design and, with this, improved
drugability of polymer nanoparticles as nanocarriers.
2.1 Drug Load and Encapsulation Efciency
2.1.1 General Considerations
The drug load is a critical parameter determining availability of the API at the site
of action. Although many nanocarrier devices have been specically designed to
locally deposit the drug substance (Wacker 2013; Barenholz 2012; Kratz 2008),
rst calculations of the effective dose should be based on the same criteria which
have been applied to the free drug.
Nanocarriers for parenteral application have been postulated to accumulate in

solid tumors due to the enhanced permeability and retention (EPR) effect (Maeda
et al. 2001). For other routes of administration a higher tissue penetration was
expected, e.g., at the skin (Raber et al. 2014; Mller et al. 2002) or into the gut wall
(Beck et al. 2007; Lautenschlager et al. 2013).
However, thus far, the primary justication for approving liposomal parenterals
has been their ability to reduce drug-related toxicity, rather than to enhance ther-
apeutic efcacy by enhanced tumor accumulation. Myocet, a liposomal formu-
lation of Doxorubicin demonstrated response rates comparable to the free drug,
while the incidence of cardiac events was signicantly reduced (Wacker 2013;
Barenholz 2012; Kratz 2008; Lammers et al. 2011).
For the nanoparticulate formulation Abraxane an increase in the administered
dose from 175 to 260 mg/m2 compared to Taxol was responsible for its clinical
success.
While toxicity and solubility of the compound are limiting the maximum tol-
erated dose, it is more difcult to predict the impact of nanoformulations on the
clinical outcome of drug therapy.
Generally speaking, improving drug therapy by using nanocarriers is more likely
for those compounds, that are flexible in dose, but limited by availability or side
effects that occur due to drug penetration into specic tissues. For Doxorubicin
improved therapeutic outcome was accompanied by a decreased volume of distri-
bution varying between the different formulations, e.g., Doxorubicin solution
(*1500 L), Myocet (*65 L), Doxil (*5 L).
Irrespective of the route of administration, adjusting the drug load of
nanomedicines requires precise knowledge of the therapeutic dose (D), maximum
acceptable excipient amount (cEx), and volume available at the site of administra-
tion (V). From these parameters the required drug load (dmin) may be calculated (see
Eq. 1).

mg Dmg
dmin mg 1
mg cEx mL V mL
Regarding the drug load, nanocrystals are consisting of crystalline API and
contain only limited amounts of polymer excipients.
Their drug load of 7090 % allows the formulation of high drug amounts.
Polymer nanoparticles used as nanocarriers are composed of a polymer matrix
system that contains higher amounts of excipient. The drug load varies between 1
and 30 % ([mg] API per [mg] matrix material) and is a limiting parameter in the
formulation design (see Fig. 3).
Aside the drug load, encapsulation efciency has major impact on the in vivo
performance of nanoformulations. This parameter will be discussed in Sect. 2.2.
596 M.G. Wacker
Fig. 3 The impact of encapsulation on the release prole of nanoformulations with different drug
load
2.1.2 Parenteral Nanocarriers
Commonly, a volume between 15 and 1000 mL is used for parenteral infusion of

drugs. Most liposomal preparations and the nanoparticle formulation Abraxane
fall into that range. In case of Abraxane the nal preparation contains 5 mg/mL of
Paclitaxel and 40 mg/mL of partly undissolved human serum albumin (European
Medicines Agency (EMA) 2007). Differently to other polymer nanoparticle for-
mulations, the soluble protein acts as a stabilizing agent (Wacker 2013). The solid
phase primarily consists of Paclitaxel and is stabilized by high albumin concen-
trations in the liquid phase. Typically, a concentration between 5 and 10 mg/mL of
solid material (irrespective of the exact composition of the solid) and an infusion
volume below 150 mL are well accepted in the clinical treatment of cancer. They
have been addressed by the formulation design.
The importance of the drug load for parenteral dosage forms is revealed by the
calculation of the applicable dose for a drug load of 10 %, a well-tolerated solid
concentration of 5 mg/mL, and a maximum infusion volume of 150 mL.
For this combination only 75 mg of the API can be administered. For the
subcutaneous and intramuscular injection higher concentrations of solid micro- or
nanoparticles are acceptable. Unfortunately, the tissue forms a strong diffusion
barrier at the site of administration and is efciently holding back the formulation
from systemic circulation. Thus, nanocarrier-based drug delivery approaches
focusing these administration routes have not been efcient enough to enable tissue
targeting.
2.1.3 Peroral Nanocarriers
High solid content is well accepted by patients when the peroral route of admin-
istration is addressed. Nanocarriers have been employed for the targeted delivery in
the GI tract for two reasons: local accumulation of nanocarriers in the stomach,
intestine, or associated tissues (Lautenschlager et al. 2013; Dembri et al. 2001) and
the enhanced uptake of molecules by utilizing their particulate form to enter sys-
temic circulation (Ensign et al. 2012).
Both of these strategies require a nanosized carrier formulation that persists under
the physiological conditions found in the gastrointestinal environment. Commonly, a
poor drug-to-polymer ratio guarantees a sustained release rate which has major
influence on the effectiveness of such drug delivery devices (Beyer et al. 2015).
Due to increased acceptable solid concentration a relatively high excipient
concentration can be used to adjust the required dose in a liquid dosage form. The
long-term stability of liquid dosage forms is limited. Therefore, dry sirups, powders,
or tablets for the in situ formation of the suspensions after (or shortly before) the
administration are preferred by the pharmaceutical industry.
Spray or freeze drying can be applied to convert the nanoparticle suspensions
into solid powders (Beyer et al. 2015). While spray drying allows the production of
relevant amounts in a high throughput and is well accepted in the industrial-scale
production, freeze drying is relatively expensive due to limited capacity and high
energy cost.
However, since the addition of excipients for stabilization of nanosuspensions
reduces the volume available for excipients required in the tablet compression
process, it can only be applied to compounds in a certain dose range (see Fig. 4).
A dose reduction as it was seen in the drug formulation Tricor (from 300 to 145
mg of Fenobrate) is likely for nanocrystal technology only. For nanocarriers, these
effects are only of minor importance.
2.1.4 Other Administration Routes
Many different routes of administration have been addressed by using polymer

nanoparticles, e.g., nanoparticles for transdermal delivery (Raber et al. 2014),
inhalation (Sadhukha et al. 2013), vaginal (das Neves et al. 2014) or ophthalmic
application (Das and Suresh 2010).
Fig. 4 Composition of nanoparticle-containing tablets and impact on total compression volume

598 M.G. Wacker
For all of these systems the uptake, diffusion, dissolution, and release properties
at the site of administration are depending on the microenvironment of the carrier.
Therefore, new requirements have to be dened for every route of administration
based on information about physiological parameters with impact on the particulate
system.
2.2 Release Rate
2.2.1 General Considerations
Release testing is an important tool that allows to monitor the quality and to predict
the in vivo performance of new drug formulations. At present, the in vitro tests
compliant to the existing compendial equipment are the dispersion releaser tech-
nology (Pharmatest AG, Hainburg, Germany) and the dialysis adapter A4D
(Sotax AG, Aesch, Switzerland) that is used in combination with the flow-through
cell. A variety of formulation parameters can have influence on the release prole
including the particle size, hydrophilicity, drug load, or excipient composition.
On the one hand release models can be applied to detect differences in phar-
maceutical quality, discriminating between formulations or batches (Villa Nova
et al. 2015). In these cases, a high robustness of the test system, short release
intervals, and a good discrimination capability are of importance.
On the other hand, biorelevant release models are employed to enable a corre-
lation between in vitro and in vivo performance by simulating the physiological
conditions with impact on the pharmacokinetic prole (Juenemann et al. 2010).
2.2.2 Nanocrystals
Nanocrystals have been utilized to enhance peroral or parenteral bioavailability of

poorly soluble API in a number of formulations (Muller et al. 2011). With
decreasing particle size, an increase in dissolution rate occurs, that is described by
the NoyesWhitney equation (Noyes and Whitney 1897). Furthermore, an
increased surface to volume ratio of smaller drug particles leads to an increasing
saturation solubility of the API as rst described by Ostwald (1900).
Both effects result in a rapid release from the nanosized dosage form and support
the absorption of poorly soluble API (Merisko-Liversidge and Liversidge 2011;
Merisko-Liversidge et al. 1996). Nanocrystal technology is well accepted by the
pharmaceutical industry and has been utilized for solubility enhancement in several
marketed products (see Table 1). By assessing the drug release of nanocrystals,
crystal growth or the formation of new polymorphic forms with impact on drug
solubility can be detected. Even after freeze or spray drying of liquid nanoformu-
lations such changes may have remarkable impact on bioavailability of the API.
A number of polymer excipients have been used to reduce agglomeration or crystal

growth over the time of storage (Wang et al. 2013). In contrast to nanocarriers, these
polymers are highly soluble in aqueous media. However, in some cases a sustained
release occurs due to the poor aqueous solubility of the API. For the parenteral route of
administration such slow-dissolving nanocrystals have been used for drug delivery
purposes as in the albumin nanoparticle formulation Abraxane. Generally speaking,
solubility enhancement is the most important application of nanocrystals and there are
only few cases where targeted drug delivery has been achieved.
2.2.3 Nanocarriers
In contrast to nanocrystals, nanocarriers have been employed to transport API to a

specic site of action. Interactions with biological surfaces are controlled by the
carrier material.
Since the pharmacokinetic prole and body distribution of a drug delivery
system strongly depends on the amount of encapsulated and nonencapsulated drug
substance, the release rate is an important parameter determining their in vivo
performance.
During early formulation development, high encapsulation efciency indicates
strong interaction between compound and carrier material. By testing the drug
release of these nanocarriers with and without sink conditions, more detailed
information about the stability of the drug delivery system and the free fraction of
the API are available. Nanocarrier formulations utilize polymer excipients at a
concentration of more than 70 % in order to adjust the release prole and
physicochemical characteristics. By this the drug load is signicantly decreased.
Parenteral Route of Administration
Nanocarriers for intravenous injection rapidly accumulate in liver, lungs, spleen,

and kidney (Wacker 2013; Leu et al. 1984). After adsorptive or covalent coating
with poly(ethylene glycol) (PEG), about 20 % of the particles remain in the blood
stream for more than 30 min (Leu et al. 1984).
Protein nanoparticles consisting of bovine serum albumin that were modied
with PEG chains on their surface demonstrated a prolonged circulation time in
tumor-bearing mice (Kaul and Amiji 2004). After 2 h about 2040 % of core
particles and 80 % of covalently surface-modied particles were found in the
plasma. Elimination of these drug delivery systems occurred after 34 h (Kaul and
Amiji 2004). For the PEGylated liposome formulation Doxil an initial half-life of
13 h was observed in men. Doxil reduces cardiotoxicity of Doxorubicin and
enhances the uptake of the drug into solid tumors. Therefore, a slow release over a
long period of time is desirable. Two thirds of the administered dose are slowly
cleared from the plasma with a second half-life of 4246 h (Gabizon et al. 1994).
The initial distribution is followed by tumor accumulation.
600 M.G. Wacker
Differently, nanocarrier devices have been designed to take advantage of their

rapid uptake into the liver, e.g., Livatag (Bioalliance Pharma 2009). Without
surface modication these colloids are efciently accumulating at the site of action.
Under these conditions a fast release is acceptable.
Altogether, a sustained release during the rst 412 h after administration is
favorable to fulll the drug delivery paradigm for the parenteral route of admin-
istration. The acceptable release rate varies with every formulation and strongly
depends on the eld of application. For the targeting of solid tumors a prolonged
circulation time should be accompanied by sustained release properties in order to
assure efcacy of the formulation.
Since only few biorelevant media are available, the in vitro testing of release rate is
difcult. Most in vitro tests are conducted in buffer media at a pH of 7.2 or 7.4
(Seidlitz and Weitschies 2012). Earlier studies conrmed the impact of serum proteins
on the release rate (Gido et al. 1993, 1994). Therefore, recent investigations included
serum proteins as part of the release medium (Villa Nova et al. 2015).
Peroral Administration Route
For the peroral route of administration the release rate is dened by the gastroin-
testinal transit time varying between 24 and 35 h. It differs in the fed and in the
fasted state. For targeting approaches in the upper GI tract, e.g., the stomach or the
duodenum, a sustained release over the rst 12 h is sufcient to allow distribution
of the API in its particle-bound form. For formulations addressing targets in the
colon, a slower release prole is desirable.
Commonly, a sustained release over 4 h assures an efcient drug targeting
strategy. For a longer release time there is a certain risk that the nanosized dosage
form is cleared from the target site before the active form of the compound has been
released. There are a variety of biorelevant media and release models available for
simulating physiological conditions GI tract (Jantratid et al. 2008). The physio-
logical conditions in the GI tract support a rapid release from nanosized dosage
forms which makes formulation development more difcult. Especially, when small
particles are required to target the inflamed tissue, the interaction between API and
carrier material plays a major role for drug delivery applications (Beyer et al. 2015).
Other Administration Routes
As mentioned before, many routes of administration have been addressed with

nanocarriers. Specic biorelevant release media have been employed to simulate the
physiological environment, e.g., tear fluid, vaginal fluid or pulmonary fluid
(Marques et al. 2011). These media have not been listed in the pharmacopoeia and
in most cases there is no standard setup available for dissolution testing.
Release models have to be individually adapted to physiology at the site of
administration. Further, the optimal release proles vary for each of these drug
delivery systems.
2.3 Particle Size, Shape, and Size Distribution
The physicochemical characterization of nanomedicines includes the assessment of

particle size and size distribution and was introduced to the regulatory framework
by the European Medicines Agency (EMA) (2013a, b, c) and the Food and Drug
Administration in the United States of America (FDA) (2002). Depending on the
route of administration minor changes in these characteristics affect the therapeutic
prole.
Despite the fact that there are a number of analytical technologies in place to
quantitatively determine particle size distribution, only few principles are applied.
All of these techniques are limited with regard to the measured size range, material,
or medium composition (Wacker 2013).
Light scattering and diffraction methods are used for powder diffraction analysis,
dynamic light scattering, eld flow fractionation, nanoparticle tracking analysis,
and many other technologies. They are the most applied principles for determina-
tion of particle size in the pharmaceutical industry. Recently, signicant differences
in the particle size distribution have been reported for particles manufactured by
nanoprecipitation in presence and in absence of glycofurol as revealed by a com-
bination of analytical ultracentrifugation and transmission electron microscopy
(TEM) measurements (Beyer et al. 2014). These nanocarriers (see Fig. 5) appeared
to be similar in size measured by light scattering technique.
For non-spherical particles such misleading results occur more often due to the
theory of spherical particle shape that is applied to calculate the particle size from
the detected signal. This in combination with a small effective sample volume and
poor sensitivity for small particle fractions make process validation extremely
difcult with regards to particle size distribution as one important formulation
parameter.
In addition to this analytical challenge, the relative sample size often is a critical
issue during the scale-up. Indeed, with increasing scale of production variations in
some critical process parameters such as homogenization efciency, pumping rates,
Fig. 5 Transmission electron microscopy of Eudragit RS 100 nanoparticles obtained by

nanoprecipitation with (left) and without (right) glycofurol as part of the formulation composition
602 M.G. Wacker
or hydrodynamics within the reaction chamber may produce particles outside the
expected size range (Wacker et al. 2011). At a larger production volume it is more
difcult to detect these smaller fractions of nanoparticles which may differ in their
biodistribution from a product within the selected specication range.
Nanocarrier devices for intravenous injection are cleared from the plasma to the
respective tissues by various ltration mechanisms (Wacker 2013). Particles larger than
300 nm rapidly accumulate in the ne capillaries of lungs, liver, and kidney (Geiser and
Kreyling 2010). Another fraction is taken up by the macrophages after opsonization with
proteins of the immune system (Owens and Peppas 2006) or is eliminated after passing
the glomerular lter (e.g., smaller drug conjugates). By increasing the particle size from
molecules to colloids, the penetration depth into the capillary bed is effectively con-
trolled. After the uptake of nanoparticles into macrophages more specic distribution
mechanisms occur. The distribution of nanocarrier and compound is controlled by both,
intracellular degradation of the matrix (causing release of free API) and the distribution
of macrophages within the human body. However, since all of these mechanisms are
affected by the particle size, a constant product quality regarding this critical formulation
parameter is essential for the therapeutic outcome.
Also in peroral drug delivery, penetration depth into the gut wall is strongly
affected by the particle size (Lautenschlager et al. 2013). Even drug delivery devices
that provide a more specic binding afnity to their pharmacological target are pas-
sively distributed. Therefore, a narrow size distribution with a dened upper and lower
size limit is mandatory to assure the efcacy and safety of medicinal products.
As already pointed out, none of the technologies in the market allows to measure
particle size for each particle species without being affected by shape or material
composition. In 2011, the European Commission recommended a denition of
nanomaterials including a threshold for number size distribution which has been
implemented into the European regulatory framework (Wacker 2014). Since then, a
number of guidelines deal with the issue of particle size measurement in complex
preparations such as cosmetics and food products (EFSA Scientic Committee
2011). They provide assistance to manufacturers from these industries. The
European Food Safety Authority (EFSA) discussed the use of at least two different
technologies for assessing the particle size distribution based on two different
principles with at least one of them based on electron microscopy (see Fig. 6).
Furthermore, particle shape plays a pivotal role with regard particle toxicity and
efcacy of drug delivery systems. In some cases, by creating new nanostructures
such as nanorods or discs a more specic interaction between nanocarrier and target
cells has been achieved (Venkataraman et al. 2011). These ndings provide a broad
basis for the development of next generation nanomedicines.
At present, most of the industrially applicable bottom-up and top-down tech-
nologies produce colloids of spherical shape allowing a prediction of body distri-
bution based on their diameter and surface properties.
However, since other shapes are still difcult to produce or to analyze with the
existing standard technologies (e.g., for size measurement), these ideas have not
been a breakthrough in the area of drug delivery. Even the recently developed
PRINTTM technology does not provide the capacity or the precision for
Dynamic light
scattering
Atomic force
Powder diffration
microscopy
Transmission
Electron
Particle size electron
microscopy
microscopy
Analytical Scanning electron

ultracentrifugation microscopy
Field flow
fractionation
Fig. 6 Analytical technologies available for measuring the particle size of various materials
GMP-compliant manufacture of nanocarriers at a signicant batch size. Not sur-

prisingly, up to now, only few pharmaceutical manufacturers showed their interest
in the technology provided by Liquida Technologies.
2.4 Surface Charge and Hydrophilicity
The surface charge and hydrophilicity of particles have major impact on their
interaction with biological surfaces (Lartigue et al. 2012) and liquids (Labarre et al.
2005; Calatayud et al. 2014; Sempf et al. 2013). For positively charged polymer
drug delivery devices an enhanced uptake into human macrophages was observed
(Brandhonneur et al. 2009). Liposomal preparations were cleared more rapidly
when zeta potential shifted from 9.64 to 46.37 (Levchenko et al. 2002).
Interestingly, the pharmacokinetic prole was quite similar for liposomes with a
zeta potential of +8.48 and 9.64 (Levchenko et al. 2002).
After intravenous administration a variety of interactions (e.g., ionic, unspecic)
enable the binding to cellular surfaces and adsorption of proteins from human
plasma (Labarre et al. 2005; Sempf et al. 2013). The binding of opsonines is
followed by the uptake into the reticuloendothelial system (RES) (Owens and
Peppas 2006). A hydrophilic surface modication reduces these interactions and
increases circulation time and tumor accumulation (Maeda et al. 2001; Kaul and
Amiji 2004; Gabizon et al. 1994). The chain length of liposomes has been varied by
604 M.G. Wacker
using PEG chains with a molecular weight of 750 and 5000 kDa. There was no
signicant difference in circulation time observed (Levchenko et al. 2002). For
liposomal preparations carrying a strong negative charge, further modications by
introducing a PEG chain to the particle surface had only minor impact on circu-
lation time (Levchenko et al. 2002).
Recently, drug delivery systems for targeting inflamed tissues in the GI tract
have been developed. The increased hydrophilicity of PEGylated poly
(lactide-co-glycolide) (PLGA) nanoparticles supported their deposition in the
respective tissues (Lautenschlager et al. 2013). Furthermore, the hydrophobicity of
particles has been discussed as one of the most important parameters with regards to
particle toxicity. In the lungs, the hydrophobicity of inhaled particles plays a major
role in the inflammation process (Dailey et al. 2014).
Altogether, surface charge and hydrophilicity are key parameters in the particle
design controlling the accumulation and residual time at a specic site of action
from which depends the pharmacological activity and the toxicological issue.
2.5 Specic Binding Afnity
Nanoparticles have been modied by using a variety of drug targeting ligands such
as antibodies (Low et al. 2011), peptides (Gao et al. 2014) and, proteins (Zensi et al.
2010) in order to enable a selective accumulation of API in target tissues or cells.
By targeting transporters at their surface, biological barriers such as the blood
brain barrier (BBB) have been addressed (Zensi et al. 2010). Since these targeted
nanocarrier devices also undergo a passive targeting in the initial phase of their
biodistribution, penetration efciency across the BBB increases with increasing
circulation time. Under such conditions API with a long half-life in the plasma and
a moderate brain penetration may exhibit a better brain penetration, compared to the
colloidal drug delivery system which is cleared more rapidly from the plasma.
Therefore, such an approach is particularly interesting with API having poor pen-
etration across the BBB.
Furthermore, the loading capacity of nanocarriers is limiting local availability at
the target site, making the technology more attractive for highly potent compounds
(see Sect 2.1).
In specic, those technologies that may be applied with a variety of different
indications and compounds provide an improved developability. However, the
example of BBB targeting points out the individual limitations of each technology
which have to be considered.
Contrasting the drug conjugates the dose range administered by using nanosized
carriers is more flexible and potentially higher because of the increased drug
binding capacity of the colloidal matrix. However, the development of nanocarriers
for active drug targeting is time-consuming and includes complex manufacturing
and conjugation processes. Passive drug targeting has been achieved by using
nanocarriers composed of well-dened materials with a long tradition in
pharmaceutical technology. In comparison, the regulatory requirements for

ligand-modied nanomatrices would be similar to those of new drug molecules. For
such excipients information about quality and purity has to be provided at the same
level of detail expected for drug substances ((FDA) 2002).
3 Safety and Risk Assessment
Over the past decades, approximately 40 % of the failures in drug discovery were
attributed to poor pharmacokinetic properties of drug candidates. In 11 % of cases,
the toxicological prole was hindering their success (van de Waterbeemd and
Gifford 2003). Nanotechnology offers exciting opportunities by enabling the for-
mulation of high drug amounts as seen for nanocrystal formulations (Muller et al.
2011) or by reducing the toxicity of the API encapsulated into nanocarrier matrices
(Wacker 2013; Kreuter 2007). Both aspects have been addressed by the drug for-
mulation Abraxane taking advantage of the nab technologyTM. By replacing
Cremophor EL with a nontoxic excipient, side effects have been reduced
signicantly.
Unfortunately, there are only few examples where the scientic ideas could be
translated into new nanomedicines. At present, the experience of the pharmaceutical
industry and the regulatory authorities is limited to liposomal and nanocrystal
formulations. A strategy for risk assessment has to be set up in order to successfully
introduce new products to the market.
Since the drug substance, the excipient composition and the formulation have
impact on the toxicological prole of polymer nanoparticles all of these aspects
Toxicological profile
Drug substance Formulation Excipients
Pharmacodynamics Pharmacokinetics Pharmacodynamics
Pharmacokinetics Pharmacokinetics
Fig. 7 Factors with influence on the toxicological prole of polymer nanoparticle formulations
606 M.G. Wacker
have to be addressed in the risk assessment strategy to assure patient safety (see
Fig. 7).
3.1 Compound
Even for those manufacturers disposing the knowledge and the infrastructure to
assure pharmaceutical quality of nanomedicines, the routine production under good
manufacturing practice (GMP) conditions is challenging (Barenholz 2012).
The use of new excipients or a modied biodistribution (see Sect. 5) increase the
risk of specic toxicities in preclinical studies or the clinical trials which are the
most expensive part of product development.
Actually, only few technologies have been used in pharmaceuticals (e.g., liposome
technology, nab technologyTM, NanoCrystal technology). Remarkably, all liposomal
formulations which have entered the market between 1990 and 2000 were based on
API that already passed clinical evaluation in at least one other dosage form (Wacker
2013). Similar observations have been made for nanocrystal formulations (Wagner
et al. 2006). This follows a very simple risk management strategy, avoiding the
combination of high level technologies with high-risk substances.
Consequently, the use of new technologies in a new drug application
(NDA) should be avoided because of the limited experience on both sides: the
manufacturer and the regulatory authorities.
Recent developments have demonstrated outstanding potential of polymer drug
delivery systems in combination with RNA- and DNA-based biologics (Draz et al.
2014). These molecules undergo rapid degradation after administration and have
been stabilized by using various nanocarrier technologies (Draz et al. 2014; Gaca
et al. 2013).
Despite all advances in nanoparticle technology, rst clinical trials have been
undertaken with liposomes (Draz et al. 2014; Schultheis et al. 2014). Since this
formulation concept has a 25-year old tradition in the pharma market, it is likely
that the next generation of siRNA products will be based on liposomes rather than
on polymer nanocarriers.
The more information becomes available about the fate of polymer nanocarriers
in vivo, the more interesting the technology will be for combinations with new drug
substances. By balancing the use of new excipients, innovative formulation
approaches, and new API an appropriate safety prole can be assured for the
clinical trials.
3.2 Excipients
Generally speaking, there is no procedure in place within FDA or EMA to evaluate

the safety of pharmaceutical excipients irrespective if they are nanomaterials or not.
Novel polymers are introduced to the market by approval in either a NDA or an

Abbreviated New Drug Application (ANDA) for use in a dened formulation
concept and concentration (Pinco and Sullivan 2006). The limitations arising from
their regulatory status are one major obstacle to the commercialization of polymer
nanoparticles.
Nanoparticles are characterized by a high dissolution pressure and a high specic
surface area. The drug release is controlled by interactions between API and
polymer. Therefore, the physicochemical properties of the material have major
impact on the in vivo performance of the formulation. For excipients with high
impact on therapeutic prole, information about quality and purity has to be pro-
vided at the same level of detail expected for drug substances ((FDA) 2002).
The high requirements for excipients in combination with the risk of unexpected
toxicities associated with their use make it more difcult for manufacturers of drug
products to introduce new polymers to the formulation design. On the one hand the
evaluation of new materials is mandatory to appropriately adjust the properties of
these drug formulations. On the other hand the regulatory framework does not
support the idea of a toxicologically safe excipient composition by setting up a
procedure for approval.
3.3 Formulation
Since body distribution of polymer nanoparticles strongly differs from the free drug,
toxicities may occur after enabling the drug substance to enter specic compart-
ments. A risk assessment that is based on information collected from preclinical
data with excipient or free drug does not reflect effects that derive from deposition
mechanisms, e.g., in the brain or the kidney.
Therefore, the toxicological prole should cover formulation characteristics and
their impact on patient safety. Over the last decade, nanotoxicity assessment has
become an important aspect in this area. For Doxil, one of the oldest liposome
formulations in the market, specic adverse reactions have been found when lipid
components have been administered parenterally in a nanosized dosage form
(Szebeni et al. 2007).
The more information becomes available about different particle systems, their
body distribution and their toxicity, the more interesting they will be for product
development in the pharmaceutical industry.
608 M.G. Wacker
4 Theory and Practice: Manufacture, Scalability

and Pharmaco-Economical Aspects
Tremendous efforts have been made in the synthesis of nanocarriers for drug tar-
geting applications. By optimizing the surface design and by applying a variety of
physical and chemical methods, the structure of these versatile carriers has reached
a molecular complexity similar to those seen in drug discovery processes.
However, with increasing complexity of drug formulations, manufacturing under
GMP conditions has become more challenging (Wacker 2013). In principle, the
production of nanocarriers by chemical synthesis would allow their manufacture in
large scale.
Unfortunately, there is no procedure in place for approval of novel excipients
(see Sect. 3.2). While the development of medicinal products is based on drug
approval, which allows bringing a number of products with the same API into the
market, excipients are treated on a case-by-case basis by the authorities. Only when
applied in a number of drug formulations, there is certain evidence, that an
ingredient is regarded as safe by EMA or FDA. Further, the price range for such
excipients would be based on a therapeutic or technical benet and increased sales
associated with their use.
Therefore, the pharmaceutical industry tends to use well-known excipients in
combination with some robust production methods, assuring scalability and
reproducibility of the nanoformulation at low cost.
4.1 Wet Pearl Milling
Wet pearl milling (WPM) has turned out to be a robust technique for manufacture
of nanocrystals in formulation development and GMP production. A number of
products for peroral administration are based on Elans NanoMill process. High
shear forces break drug crystals down to nanoparticles. By adjusting drug amount,
number and size of the milling pearls, milling speed, grinding medium, milling
time, and temperature a constant product quality is achieved at different scales of
production (Merisko-Liversidge and Liversidge 2011; Merisko-Liversidge et al.
1996).
4.2 High Pressure Homogenization
High pressure homogenization (HPH) takes advantage of cavitation forces when

API suspended in milling medium passes a very small homogenizer gap. Since
pressure gradient and liquid flow inside the homogenizer impact the product
characteristics, the scale-up is a critical step in product development.
4.3 Nanoprecipitation
Nanoprecipitation has been used in a variety of lab scale approaches for the syn-
thesis of nanoparticles and has been translated into process technology by SOLIQS,
a drug delivery unit of Abbott (Ludwigshafen, Germany). This bottom-up technique
is marketed under the trade name NanoMorph and applies to the nanonization of
API. Bottom-up technologies produce nanoparticles of narrow size distribution and
can be applied to the synthesis of amorphous nanosuspensions with high stability.
Residues of crystalline API are found in most formulations generated by applying
top-down strategies.
Further, microfluidic devices have been used for the manufacture of nanocarriers
at medium- and large scale (Villa Nova et al. 2015). MJR Pharmjet GmbH uses the
microjet reactor technology enabling the synthesis of nanocarriers and nanoparticles
under controlled conditions (Wacker 2013). An optimized chamber design allows
the production of nanoparticles in a relatively simple processing unit, the microjet
reactor. Scale-up is achieved by increasing the number of mixing chambers.
5 Pharmacological Background and Developability
In recent years, signicant progress has been made in understanding the interactions
between nanomedicines and biological surfaces and fluids that enable successful
drug delivery. Aside the optimization of formulation characteristics under GMP
conditions, the adjustment of the therapeutic concept plays a major role for de-
velopability of polymer nanoparticles.
In 2007, with Abraxane, a rst polymer nanoparticle formulation for intra-
venous injection entered the market in the USA. Nanocarrier formulations have
been utilized earlier for the treatment of tumors, taking advantage of the reduced
toxicity and enhanced deposition at the site of action. During clinical development a
reduction of side effects, increased response rates, and survival time have been
observed. Although Paclitaxel was highly effective in the treatment of tumors, the
excipients used in the standard treatment Taxol had negative impacts on life
quality and patient compliance (Gelderblom et al. 2001). The therapeutic need was
essential for the success of Abraxane.
Exploring the criteria that have to be considered for the successful marketing of
polymer nanoparticles, the API used in these formulations provide at least some of
the following characteristics: poor aqueous solubility, a flexible dose range to buffer
occurring toxicities in compartments of accumulation (e.g., the liver), poor pene-
tration into the dened target site or organ (e.g., liver, macrophages, tumor), an
unfavorable body distribution that is controlled by diffusion, and an expensive or
inefcient standard treatment.
At present, the production of nanoparticles involves specic knowledge and
process technology, making the development rather expensive. Only formulations
610 M.G. Wacker
that succeed in competition with other therapies can justify these efforts. When
primary patents covering Elans NanoCrystal technology expired in the USA and
some other countries between 2011 and 2012, another aspect with impact on
nanomedicines has been revealed.
The market potential of innovative dosage forms is closely related to the intel-
lectual property (IP) rights covered by innovator and the competitors. Aside patent
protection for own products and ideas, an emerging market of patent claims for a
specic technologies is motivating technological progress in the pharmaceutical
industry. From an economical perspective, this in addition to product-related value
determines the economic success of research projects. Creating own IP rights is
multiplying both, the economic value of the product (e.g., of the nanoparticulate
formulation that is currently under development) and the technology platforms
which cover a wide range of methods.
Since 2011 a variety of competitors adapted process technology, creating an
emerging market for nanocrystals. Today, a number of innovative and generic
products are under development in the pipelines of pharmaceutical companies
taking advantage of nanomilling technology.
Similar to new drug candidates, the intellectual property rights for these man-
ufacturing processes were a limiting factor in the development of nanoparticle
formulations.
6 Conclusion
Polymer nanoparticles have a long history in medical research and became part of
formulation development in the pharmaceutical industry. By adjusting their prop-
erties with respect to the physiological environment, a high therapeutic efcacy can
be achieved. Finding a balance between innovative character and patient safety for a
specic application decreases the risk of failure during the clinical trials.
However, the pharmacological background plays a major role for the marketing
of these versatile drug delivery systems. In competition with other therapies, nan-
otechnology allows the production of highly specic therapeutics but they are
relatively expensive. Consequently, the number and the quality of the therapeutic
alternatives decide over their success.
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Chapter 19
Regulatory Perspective
on the Development of Polymer
Nanomaterials
Xiaoming Xu and Mansoor A. Khan
Abstract Combination of nanomaterials with pharmaceutical sciences to produce

lifesaving drug products is a rapidly growing eld. Intense interest and evergrowing
nancial support in this area have propelled the research and development to
become increasingly mature. In the current chapter, recent advances in the eld of
polymer nanomaterial are presented, including various denitions and considera-
tions. Regulatory challenges and some of the recent policy developments associated
with nanomaterial drug product are also discussed.
Keywords Nanomaterial Regulatory ANDA NDA
1 Introduction
Nanomaterial refers to a class of material that has been deliberately manipulated to

be within the nanometer size range to render new or altered physical, chemical,
and/or biological properties. Nanomaterials have been around for over a millen-
nium. As early as medieval time gold nanoparticles were used in stained glass,
while nanotubes were found in blades of swords made in Damascus (Reibold et al.
2006). However, it is until the invention of high-powered microscopes that truly
allow us to see things at the nanoscale and begin taking advantage of phenomenon
occurring naturally at this scale.
X. Xu
Division of Product Quality Research, Ofce of Testing and Research, Ofce of
Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug
Administration, 10903 New Hampshire Ave, Silver Spring, MD 20993, USA
M.A. Khan (&)
Formulations Design and Development Core Laboratory, Texas A&M Health Science Center,
Irma Lerma Rangel College of Pharmacy, Reynolds Medical Building, Suite 159, College
Station, TX 77843-1114, USA
e-mail: mkhan@pharmacy.tamhsc.edu

DOI 10.1007/978-3-319-41421-8_19
616 X. Xu and M.A. Khan
Use of nanomaterial enables the development of new drug products, helps

improving, and even saves peoples life. In recent decades, uses of nanomaterials in
pharmaceutical drug products have seen great success. The unique physicochemical
properties of nanomaterial, such as small size, large surface area to mass ratio, high
reactivity can be useful to overcome some of the limitations found in traditional
therapeutic and diagnostic agents. For example, reducing the size of a crystalline
drug into nanometer range can signicantly improve the dissolution rate of poorly
water-soluble drugs (Kipp 2004; Verma et al. 2009). Delivery of drug using
nanocarriers also helps in prolonging release of a drug or in a responsive manner
thus decreasing the frequency of administration. Sometimes this system is used to
target the drug to its site of action and thereby decreasing exposure of the drug to
other tissues and thus minimizing its side effects (Allen 1994; Papahadjopoulos
et al. 1991). Drug product using nanotechnology can also be used in combination
therapy, in which, two or more biomolecules are released simultaneously to gen-
erate a synergistic effect and suppress drug resistance (Zhang et al. 2007).
Undoubtedly, the potential benets of these nanomaterials are enormous and the
process of realizing those potentials is just getting started. Yet, the very changes in
biological, chemical, and other properties that can make nanomaterial so magical
and exciting may also bring in unintended adverse consequences, such as product
safety (Aillon et al. 2009; Dobrovolskaia and McNeil 2007), effectiveness, or other
attributes. To ensure the patients receive safe, effective, and high-quality drug
products, a robust regulatory framework based on sound science is a prerequisite.
It should be noted that from a regulatory perspective, polymer nanomaterials
present similar regulatory challenges as nanomaterials and invoke similar sets of
regulatory considerations. In this chapter, unless otherwise specied, discussion
will be centered on nanomaterial in general with special considerations to those of
polymer origin.
2 Denitions and Considerations
With the anticipated increase in the applications of nanotechnology, there is an

urgent need to identify what can be considered as a nanomaterial by clear
unequivocal descriptions. In various industry and research sectors, terms related to
nanotechnology are understood with different meanings, which may lead to con-
fusion and uncertainty. The following section describes the effort by various
international institutions to provide clear denitions and considerations.
The International Organization for Standardization (ISO) provides the following
denitions (ISO/TS 2008):
nanoscale (or nano range): size range from approximately 1 to 100 nm;
nano-object material with one, two or three external dimensions in the nanoscale
nanoparticle nano-object with all three external dimensions in the nanoscale;
particle A minute piece of matter with dened physical boundaries;
19 Regulatory Perspective on the Development of Polymer Nanomaterials 617
nanomaterial material with any external dimension in the nanoscale or having

internal structure or surface structure in the nanoscale
In October 2011, European Union Commission adopted the denition of a nano-
material as (European Commission Policy on N. http://ec.europa.eu/nanotechnology/
policies_en.html 2014): A natural, incidental or manufactured material containing par-
ticles, in an unbound state or as an aggregate or as an agglomerate and where, for 50 %
or more of the particles in the number size distribution, one or more external dimensions
is in the size range 1100 nm. It further states that In specic cases and where
warranted by concerns for the environment, health, safety or competitiveness the number
size distribution threshold of 50 % may be replaced by a threshold between 1 and 50 %.
This denition is primarily used to identify materials for which special provisions might
apply (e.g., for risk assessment or ingredient labeling).
In the United States, since 2000 the National Nanotechnology Initiative Program
(2014) denes nanotechnology as the understanding and control of matter at
dimensions between approximately 1 and 100 nm, where unique phenomena enable
novel applications.
In 2006, American Society for Testing and Materials (ASTM) provides the
following denitions (E2456-06 A):
nanoscale: having one or more dimensions from approximately 1100 nm.
nanoparticle a subclassication of ultrane particle with lengths in two or three
dimensions greater than 0.001 m (1 nm) and smaller than about 0.1 m
(100 nm) and which may or may not exhibit a size-related intensive property.
ultrane particle a particle ranging in size from approximately 0.1 m (100 nm)
to 0.001 micrometers (1 nm).
In June 2011, the Food and Drug Administration in the United States of America
(FDA) issued a Draft Guidance to articulate the principles that could be used to consider
whether a product contains nanomaterials, which was later updated as a Final Guidance
in June (2014). While the considerations listed in the Guidance do not constitute formal
denitions, they set forth the boundaries for products (with the use of nanomaterial or
nanotechnology) within which questions about product safety, efcacy, quality, or
public health impact may be raised. There are two primary considerations as follows:
1. Whether a material or end product is engineered to have at least one external
dimension, or an internal or surface structure, in the nanoscale range (approx-
imately 1100 nm);
2. Whether a material or end product is engineered to exhibit properties or phe-
nomena, including physical or chemical properties or biological effects, that are
attributable to its dimension(s), even if these dimensions fall outside the
nanoscale range, up to one micrometer (1000 nm)
These two considerations apply not only to new products, but also when changes
to manufacturing processes alter the dimensions, properties, or effects of an
FDA-regulated product or any of its constituent parts. A list of recent policy
documents related to nanotechnologies is provided in Table 1.
Table 1 Published guidance documents related to nanotechnology

Year Guidance document Link
2014 Final Guidance for Industry: http://www.fda.gov/RegulatoryInformation/
considering whether an Guidances/ucm257698.htm
FDA-regulated product involves the
application of nanotechnology
2014 Final guidance for industry: safety of http://www.fda.gov/Cosmetics/
nanomaterials in cosmetic products GuidanceRegulation/GuidanceDocuments/
ucm300886.htm
2014 Final guidance for industry: assessing http://www.fda.gov/Food/
the effects of signicant GuidanceRegulation/
manufacturing process changes, GuidanceDocumentsRegulatoryInformation/
including emerging technologies, on ucm300661.htm
the safety and regulatory status of
food ingredients and food contact
substances, including food
ingredients that are color additives
2014 Draft guidance for industry: use of http://www.fda.gov/downloads/
nanomaterials in food for animals AnimalVeterinary/
GuidanceComplianceEnforcement/
GuidanceforIndustry/UCM401508.pdf
Despite the fact that the Draft Guidance used the same nanoscale denition as
other standard/regulatory institutions (i.e., with upper size limit of 100 nm), it
recognizes that FDA-regulated products may conceivably exhibit unique/novel
properties or phenomena attributable to the use of nanotechnology even if their
smallest components are larger than 100 nm (more on this topic later). Accordingly,
in the absence of a suitable bright line test, setting of the size range boundary for
nanomaterial should be based on function/performance rather than a clear-cut limit.
3 Regulatory Overview in US
3.1 Drug Regulation Laws
The rst regulation of drugs in the United States was to curb the importation of
adulterated drugs under the provision of Drug Importation Act of 1848. It was only
after 1906 that Pure Food and Drugs Act gave the authority to FDA to regulate
misbranding. The act was later amended by Shirley Amendment to regulate ther-
apeutic claims by patent medicine rms. However, there were many shortcomings
in that act. To address those shortcomings, in 1938 Food, Drug, and Cosmetic Act
(FD&C Act) was passed to give FDA the authority to regulate drugs and other
medical products. The act required manufacturers to le a New Drug Application
(NDA) with the FDA and to obtain FDAs pre-approval before marketing in the U.
S. Initially product safety was the main focus of these applications. Efcacy was
later included into the FD&C Act with the Kefauver-Harris Amendment. These
statutes or legislations are laws passed by Congress, and FDA is mandated to
implement the statutes via regulations, which are published in Code of Federal
Regulations (CFR). For example, the CFR Title 21 pertains to drugs, with sec-
tion 312, 314, and 601 deals with Investigational New Drugs (IND), NDA, and
Biological License Application (BLA), respectively, while section 211 deals with
the current Good Manufacturing Practices (cGMPs). In addition to new drugs,
generic drug product applications are also reviewed by FDA, with appropriate
modications to consider various types of dosage forms. The next section will focus
on NDA review process at Center for Drug Evaluation and Research (CDER).
3.2 Drug Review Process: Principles Governing

the Regulation of the Drug Products
Under current regulations in the United States, use of a human drug product not
previously authorized for marketing in the United States requires the submission of
an IND to the Agency. FDAs regulations of 21 CFR 312.22 and 312.23, respec-
tively, contain the general principles underlying the IND submission and the
general requirements for content and format. Section 312.23(a)(7)(i) requires that
an IND for each phase of investigation include sufcient CMC information to
ensure the proper identity, strength or potency, quality, and purity of the drug
substance, and drug product. The type of information submitted will depend on the
phase of the investigation, the extent of the human study, the duration of the
investigation, the nature and source of the drug substance, and the drug product
dosage form. Pre-IND meeting with sponsor and FDA takes place before IND is
submitted for review. IND review is mainly for safety consideration, but also take
into consideration of efcacy to determine if the drug is efcacious to justify further
development. INDs are not approved but they are called open INDs once they
become effective. After this stage, pharmaceutical rm may begin clinical trials.
Clinical trials are normally divided into three separate phases to address different
regulatory concerns. Phase-1 study is centered on safety of drug, while Phase-2
study seeks to establish the efcacy of the drug. Phase-3 study is then conducted to
reconrm the safety and efcacy prole of the drug in a larger population.
As the clinical trials progress, design, development, scale-up, process controls,
labeling, packaging, etc, are carried out by the pharmaceutical rms. Sponsors le
NDA with the FDA seeking an approval to market the new drug product.
NDA is a registration document through which drug sponsors formally propose
that the FDA approve a new pharmaceutical for sale and marketing in the United
States. The NDA is submitted under section 505(b)(1) or 505(b)(2) of the Act and
applicable Code of Federal Regulations (21 CFR.314). In principle, focus of the
NDA review is to assess whether the proposed drug product is safe and effective
and whether the benets outweigh the risk.
Fig. 1 NDA review process
NDA submission must provide all relevant data and information that a sponsor
has collected during the products research and development to support the safety,
efcacy, quality, purity, and manufacturability claims. Although the quantity of
information and data submitted in NDAs can vary from submission to submission,
the components of NDAs are more uniform and are, in part, a function of the nature
of the subject drug and the information available to the applicant at the time of
submission (Fig. 1).
The review process involves reviewers conrming and assessing the sponsors
conclusion that a drug is safe and effective for its proposed use and has adequate
Chemistry Manufacturing Control (CMC) to demonstrate that it would continue to
meet the standards of identity, strength, quality, purity, and potency through the
proposed shelf life as established in the NDA.
In addition to CMC data, NDA should also contain pharmacology, toxicology,

metabolism, clinical safety, and efcacy data, as well as proposed labeling infor-
mation. The NDA review process is schematically depicted in Figure. Upon
acceptance of data at all review and administrative levels the NDA application is
approved and the sponsor can then legally market the NDA drug product in the
United States.
During FDAs review of newer dosage forms from emerging technologies, the
agency may call for additional data from the sponsor needed to support the
applications, if not supplied in the original applications. FDA requests information
when the agency considers such information relevant to determining whether a
particular drug product is safe and effective. If FDA determines such data that are
needed for a class of drugs, FDA may issue guidance to applicants recommending
that they may be submitted in the original application.
3.3 Special Considerations for Nanomaterials
For product containing nanomaterials, including polymer nanomaterials, FDA does

not make a categorical judgment that nanotechnology is intrinsically benign or
harmful. Rather, for nanotechnology-derived and conventionally manufactured
products alike, FDA considers the characteristics of the nished product and, as
applicable, its safety, effectiveness, or other product attributes.
Nanomaterial in general possesses unique physicochemical properties due to its
dimension, including small size, large surface area to mass ratio, high reactivity,
etc., the assessment of benet and risk of nanomaterial can be distinctively different
from those of bulk material. In an effort to better understand the ramications or
risks associated with the use of nanomaterial in its regulated products, FDA has
established Nanotechnology Task Force across seven centers (Center for Drug
Evaluation and Research, Center for Biologics Evaluation and Research, Center for
Devices and Radiological Health, Center for Tobacco Products, Center for Food
Safety and Applied Nutrition, Center for Veterinary Medicine, and National Center
for Toxicological Research) in order to better understand how the materials have
been used.
Within CDER in particular, an internal database of submitted and approved
drugs containing nanoscale materials was created, in order to better understand the
landscape of products that CDER was reviewing (Sadrieh 2014). Some of the
ndings are summarized in Figs. 2 and 3. Most notably, there are tremendous
interests in using nanotechnology, especially in several key areas, including cancer
treatment, pain management, infection, etc. Moreover, more than half of the
nanotechnology-related submissions contain particles with mean particle size
outside the nanoscale range (i.e., 1100 nm) as dened by several institutions
mentioned earlier. Approximately 90 % of the applications involve using particles
with size smaller than 300 nm.
Fig. 2 Nanotechnology-related submissions within CDER database (as of August 2012). Adapted
from slides by Dr. Nakissa Sadrieh, overview of CDER experience with nanotechnology-related
drugs, Advisory Committee for Pharmaceutical Science and Clinical Pharmacology meeting,
August 9, 2012
Fig. 3 Distribution of Mean particle size in CDER database related to nanotechnology.

Adapted from slides by Dr. Nakissa Sadrieh, overview of CDER Experience with
nanotechnology-related drugs, Advisory Committee for Pharmaceutical Science and Clinical
Pharmacology meeting, August 9, 2012
Further effort from FDA includes the formation of CDER Nanotechnology

Working Group, which consists of a multidisciplinary team of scientists from
ofces and divisions across CDER. The working group directly reports to the FDA
Nanotechnology Task Force. The goal of the group is to identify potential risks to
safety, quality, and efcacy from the use of nano-sized materials in drug products,
and to identify areas where CDER may need to develop a new guidance, policy, or
internal procedures to address these risks (i.e., gaps in the current review or reg-
ulatory practices).
In 2013, the working group published the risk assessment results of the use of
nanomaterials in drug products (Cruz et al. 2013). It is found that the current
regulatory review processes indeed can adequately protect the public from potential
risks associated with the use of nanomaterials in drug products. However, there are
areas that could benet from improvement, including:
1. Improvements in analytical methods to characterize nanomaterial and reviewer
training on these methods;
2. Better understanding of how particle size change can affect product perfor-
mance, including product quality;
3. Additional need for clarication of policy in some situations where safety testing is
typically not required or where there may be unintended exposures; and
4. Development of nanotechnology-related educational opportunities for review
staff.
To better understand the type of risk associated with use of nanomaterial from a
regulatory perspective, interested readers are encouraged to read the article pub-
lished by the Nanotechnology Working Group.
4 Challenges Associated with Regulating Nanomaterials

in Drug Product
To date, use of nanomaterials in drug products has been very diverse. Differences
may be found in type, route of administration, intended function, indication,
maturity of the technology, and structural complexity. So far some examples of
nanomaterials in drug products include liposomes, nanoemulsions, microemulsions,
nanosuspensions, micelles, polymer micelles, PEGylated nanoparticles, colloidal
metals, and dendrimers. This is not an exhaustive list, as the eld of nanotech-
nology is still evolving. With the discovery of new materials and new applications
of nanomaterials, the list may expand.
4.1 Characterization
Nanomaterials in general have unique chemical, physical, or biological properties

different from their bulk counterparts. Due to the extremely small size and high
ratio of surface area to volume, they may also have differing magnetic, electric, or
optical activities. Accordingly, the characterization of nanoparticles poses a chal-
lenge to the developers as well as regulators. Two similar materials may have subtle
but signicant differences that determine their behavior and in such instances they
become extremely difcult to characterize. Validated assays are important for
quantifying nanoparticles. Physical characteristics may impact product quality and
performance. With extreme subdivision of particles, it is possible that some part of
the nanoproduct becomes amorphous. That means unforeseen stability and aggre-
gation problems might arise. All the issues are critical for demonstrating control of
a production process and for justifying drug release parameters and bioequivalence
testing approaches. Complexity and heterodispersity of drug products containing
nanomaterials are very difcult to address with current technologies. Additional
standard test methods might ensure appropriate consistency and safety of
nanoparticles for drugs and biologics. Some general considerations in character-
izing nanomaterial quality attributes are discussed with detail below.
4.1.1 Average Particle Size and Size Distribution
Particle size and size distribution is unquestionably the most dening characteristic
of nanomaterial-based drug products. Both size and size distribution of the particles
can have signicantly impact on the pharmacokinetic, biodistribution, and safety.
As discussed earlier, the majority of the application of nanomaterial in drug product
so far has been in the size range of <300 nm. This is not a coincidence. At the
smaller end, nanoparticles with size smaller than 2030 nm are rapidly cleared by
renal excretion after intravenous administration. At the relatively higher end, par-
ticles 200 nm or greater in size are known to be more efciently taken up by the
mononuclear phagocytic system (MPS; also known as reticuloendothelial system),
with cells in the liver, spleen, and bone marrow (Moghimi et al. 2001). At a more
detailed level, nanoparticles of 150300 nm locate primarily in the liver and spleen
(Gaumet et al. 2008), while particles of sizes 200400 nm undergo rapid hepatic
clearance. These form the basis for the enhanced permeability and retention
(EPR) effect (Matsumura and Maeda 1986).
Compared with the average size of the particles (e.g., mean, median, mode), size
distribution is another critical yet less monitor/controlled property. Most of the
engineered nanoparticles exhibit a range of sizes rather than a single size.
Considering that most of the particles follow lognormal size distribution
(Heintzenberg 1994; Smith and Jordan 1964), particles present at the upper end of
the distribution may have completely different in vivo distribution proles.
Therefore, the nanoparticle size and size distribution need to be carefully controlled
during the small-scale preparation and in particular during a larger-scale manu-
facturing process.
Particle size can be measured by various techniques which include
a. Spectroscopy techniques, such as dynamic light scattering (DLS) also known as
Photon Correlation Spectroscopy (PCS), and laser diffraction (LD).
b. Microscopy techniques include scanning electron microscopy (SEM), trans-
mission electron microscopy (TEM), and atomic force microscopy (AFM).
c. Particle tracking techniques.

d. Analytical ultracentrifugation.
e. Size exclusion chromatography and eld flow fractionation (although they are
closer to separation techniques than analytical techniques).
It should be emphasized that each of the above technique gives very different
size information and it is up to the user to decide which technique is the most
appropriate one for the intended use. First and foremost, each technique has its size
range limit within which it can provide the most accurate values. For instance, laser
diffraction technique is based on analysis of diffraction pattern (at various angles)
where larger particles diffract light at smaller angle and smaller particles diffract
light at larger angle. Due to the limitation of detector at large angles, it may not be
appropriate for accurate analysis of particles smaller than 100 nm. Second, different
techniques provide different types of particle size. For example, light scattering,
laser diffraction, and particle tracking techniques report hydrodynamic radius,
whereas microscopy techniques provide geometric length. For this reason, if the
intent of the analysis is to study the behavior of particles in its native environment
(e.g., in liquid such as in blood circulation), use of microscopy-based techniques
may provide very limited values. Third, some analysis provides size information
based on ensemble values (e.g., DLS and laser diffraction) while others provide
results based on statistical summary of individual particle analysis (e.g., particle
tracking and microscopy techniques), and hence the user needs to decide if the
purpose of the analysis is to look for normality or the abnormity (extreme small or
large) in which case individual particle-based analysis may be more useful.
However, when using analysis based on individual particles the number of samples
need be sufciently large to generate meaningful statistical results. Furthermore,
accuracy and precision of the analysis are highly dependent on the validity of the
hypothesis (e.g., most analysis assume nanoparticle to be spherical) which unfor-
tunately is overlooked in most analysis. As can be seen, there are many challenges
associated with the analysis of particle sizes, and in reality no single particle sizing
technique can provide all the necessary information. For this reason, it is generally a
consensus in the scientic community that orthogonal/complementary methods
should be used. This applies not only to the particle size analysis, but also other
critical product quality attributes.
4.1.2 Morphology (Shape)
It has been widely reported that morphology (shape/aspect ratio) of the nanopar-
ticles has signicant impact on how they interact with cells (Gratton et al. 2008;
Yang et al. 2009). Most of the research so far has been focused on spherical
particles, characterization of nonspherical nanoparticles, in particular to assess how
they interact with the body is expected to be more complex but interesting (Geng
et al. 2007). Due to the extreme small size of nanoparticles, methodologies
available for shape analysis remain scarce. The most direct technique is by
microscopy such as SEM, TEM, and AFM.
4.1.3 Surface Characteristics
Surface properties of nanomaterials play critical role in dening how nanoparticles

interact with the tissue, proteins, cells, and even subcellular components. Important
properties include total surface area, chemical reactivity, presence of functional
ligands, hydrophobicity, roughness, charge, etc. For example, presence of hydro-
philic moiety coating (e.g., PEGylation) on the surface of a nanoparticle can affect
the interaction of engineered nanoparticles with various blood components, via a
process known as opsonization (Owens Iii and Peppas 2006). Additionally,
chemistry of the poly(ethylene glycol) (PEG) linkage, overall PEG length, and
surface density all affect nanoparticle stability (Cedervall et al. 2007; Zhang et al.
2009). Various techniques are available for characterization of these properties, yet
more characterization tools may be needed. For instance, gas adsorption techniques
have been employed to measure specic surface area of materials (using BET
theory). This technique can be applicable for measurement of specic surface area
of nanoparticles. The surface roughness of nanoparticles can be determined using
AFM, while the surface charge can be determined using zeta potential measurement
techniques (detailed discussion in the next section).
4.1.4 Zeta Potential
Zeta potential is electric potential in the interfacial double layer at the location of
the slipping plane versus a point in the bulk fluid away from the interface. In other
words, zeta potential is the potential difference between the dispersion medium and
the stationary layer of fluid attached to the dispersed particle. Zeta potential is
widely used for quantication of the magnitude of the electrical charge at the
double layer. Electrophoresis and laser-doppler anemometry techniques can be used
to measure the zeta potential. The measurements can only be performed for a liquid
formulation, and thus nanoparticles need to be suspended in the liquid prior to
measurement.
4.1.5 Stability (e.g., Agglomeration and Aggregation)
The majority of the nanomaterials used in pharmaceutical drug products are col-
loidal in nature. These include products that are already present in a dispersed state,
such as emulsions, liposomes, micelles, etc., and those that are to be dispersed
(administered) into a continuous phase (aqueous in most of the cases). Accordingly,
in addition to conventional chemical stability considerations, physical stability of
the dispersed systems should also be evaluated.
The physicochemical principles governing the behavior of nanomaterial in colloidal

dispersed systems include thermodynamics, interfacial chemistry, and mass transport
(Burgess 2005). Based on interaction between the dispersed and continuous phases,
colloidal systems are generally classied into three groups: (1) lyophilic or solvent
loving colloids, where the dispersed phase is dissolved in the continuous phase (e.g.,
polymer solution, protein); (2) lyophobic or solvent hating colloids, where the dis-
persed phase is insoluble in the continuous phase (e.g., nanocrystal suspensions,
polymer nanoparticles, emulsions, etc.); and (3) association colloids, where the soluble
dispersed phase molecules spontaneously self-assemble or associate to form
supramolecular structure in the colloidal range (e.g., micelles, and liposomes).
Type of stability assessment should be dependent on the type of colloid. For
example, lyophilic colloids are thermodynamically stable system and change in the
physical stability is very unlikely during storage. However, for lyophobic colloids,
excess surface free energy (as a result of large surface area and high surface tension)
means spontaneous reduction in exposed surface area (or increase in overall size) is a
thermodynamically favorable process. For this reason, aggregation and/or agglomeration
should be monitored and controlled. Classic Derjaguin, Landau, Vervey, and Overbeek
(DLVO) theory can be used as a tool to understand the phenomenon and to
control/mitigate the possible instabilities (e.g., surface modiers, surfactants, size
homogeneity, etc.). From characterization point of view, it is often challenging to discern
aggregates from its original form particles, and it is of critical importance that the chosen
sizing analysis technique is appropriate and validated (refer to particle size
characterization).
4.1.6 Molecular Weight (MW)
Mass spectroscopy, size exclusion, or gel ltration chromatography have been

employed to measure MW. These techniques can separate analytes based on MW,
and therefore, can also be used to differentiate aggregates, if any. Properties of
certain dendrimers depend upon its MW. Also, for colloidal systems to study the
effect of formulation or processing factors, molecular weight can act as a surrogate
marker of their performance. The challenge for characterizing MW is that devel-
opment and validation of chromatographic techniques is quite cumbersome and
challenging. A systematic study was presented recently including design of
experiments for detecting molecular weight using size exclusion chromatography
for a colloidal iron preparation (Shah et al. 2008).
4.1.7 Polymorphism
Polymorphism, changes in crystalline structure or generation of amorphous struc-

ture from a crystalline material might occur during size reduction of crystalline drug
into nanometer range. Thus it is important to evaluate crystallographic properties.
X-ray diffraction and X-ray crystallography have been successfully employed to
study the degree of crystallinity or crystal-to-amorphous transitions. Differential

scanning calorimetry (DSC) has been employed to study the thermal transitions of
free drug and encapsulated drug to evaluate drug-loaded nanoparticles.
Microcalorimetry, thermogravimetry have also been employed to understand
thermal behavior of drugs containing nanomaterials.
Another important polymorphism phenomenon may occur in nanoporous
material where spontaneous phase transformation leads to amorphization. For
example, it is reported that crystalline compounds such as ibuprofen and naph-
thalene becomes amorphous when conned in the nano-sized capillaries of porous
silica (SiO2) (Qian et al. 2011). Using neural scattering technique and X-ray
diffraction, such transformation may be monitored and quantied (Qian et al. 2012).
In general, polymorphism or changes in crystal structure often impacts solubility of
nano-sized drug. Also some changes might be detrimental to their performance, and
thus need to be carefully evaluated by the appropriate techniques.
4.2 Product Quality Control for Nanomaterials
Presence of multicomponent systems and nanoscale structure means more stringent

process control strategy which is needed to ensure that process can produce
products with consistent quality. This may become a sizable obstacle for the
scale-up of drug products containing nanomaterials. Since most nanoparticles are
complex multicomponent products with specic arrangement of components, a full
understanding of the components and their interactions are essential to dening the
key characteristics of the product (i.e., CQAs). Identifying these characteristics
early in development in turn, greatly helps streamline development of larger-scale
manufacturing processes.
At nanoscale dimension, manufacturing of the drug product also faces unique
challenges as compared to conventional pharmaceutical manufacturing processes.
The quality, safety, or efcacy of the drug product containing nanomaterial is
generally sensitive to process conditions and production scales.
Categorically speaking, the methods of nanoparticle preparation can be broadly
divided into two approaches: topdown and bottomup. Topdown approach
generates smaller entities from larger ones, such as grinding of particles using the
milling technique. In contrast, bottomup approach relies on arrangement of smaller
components into more complex assemblies, which often involve polymerization of
monomers or molecular self-assembly (e.g., formation of liposomes) (Lasic 1995).
In either of these approaches, manufacturing involves multistep processes, such as
high-speed homogenization, sonication, milling, extrusion, emulsication,
cross-linking, organic solvent evaporation, centrifugation, ltration, lyophilization,
etc. Use of the same material or even similar processes may not warrant same
quality product. This type of process and scale dependency coupling with inherent
polydispersity of most nano attributes make it a priority to assess both the risk of the
production variability on product quality and the detectability of process failures
starting at early development stage. For example, the degree of the PEG coating on
a particle surface (coating density) is generally related to nanoparticles in vivo
stability (against protein binding). In this case, if inconsistent in vivo performance
occurred for different batches as a result of PEG density variations, assessing the
batch-to-batch consistency using particle size and particle size distribution
(PSD) alone cannot reveal the true source of variation, but rather requires a com-
parison of the material grade on performance (Adiseshaiah et al. 2010).
Control of impurities in pharmaceutical dosage forms is of critical importance to
ensure quality and safety of the product. ICH Q3 document (2006) provides a
general guideline for assessment and control of pharmaceutical impurities. For drug
products containing nanomaterials there may be additional considerations. For
example, in nanomaterial preparations size and shape are commonly heterogeneous
in nature. Therefore, a different shape or size of a nanomaterial could be considered
an impurity if it is impacting the quality, safety, or efcacy of the product, even if it
is the same composition of the intended nanomaterial. Furthermore, empty
nanocarriers or carriers with missing or incomplete surface coatings could also be
considered impurities. As with small molecule drugs, impurities may be introduced
externally (e.g., from processing conditions), or generated internally (e.g., aggre-
gation due to instability during processing, packaging, or storage).
Sterility, in general, is a crucial factor to ensure that the drug product is safe to
use for the patient. Injectable nanoparticles can be sterilized by a number of
techniques including membrane ltration, gamma irradiation, autoclaving, ethylene
oxide sterilization, and high hydrostatic pressure sterilization. Each of these
methods has its unique advantages/disadvantages, and requires careful selection for
use with products containing nanomaterials. For example, size differences between
nanomaterials and microorganisms are marginal, at best, and hence traditional
sterile ltration (0.22 m) may retain nanomaterials in addition to microorganisms.
Various heat treatment techniques and gamma irradiation may induce unwanted
chemical degradation and/or destabilizing delicate structures of certain nanomate-
rials (e.g., liposomes). Aseptic processing is another option if all available steril-
ization techniques cannot meet the requirement.
4.3 In Vitro Performance Assessment
Drug product containing nanomaterials provide many benets compared to tradi-

tional drug products, however, disastrous effects can occur if there is an unantici-
pated change in product quality or performance, especially for parenteral
administered products. Correspondingly, understanding the factors influencing drug
release, from both an in vitro and in vivo perspective, is essential to ensure product
safety, efcacy, and quality.
A commonly encountered challenge in developing an in vitro release testing
method for drug products containing nanomaterials is to ensure that it is relevant to
the actual in vivo release conditions. For example, depending on the drugs in vivo
distribution and release characteristics, such as sustained release, targeting, etc.,

different in vitro release testing method should be developed. For sustained delivery
purposes, a demonstration of slow drug release over time may be sufcient.
However, for targeted drug delivery applications, additional demonstration of the
absence of drug release prior to reaching the target should also be very helpful.
Another challenge in developing an in vitro release testing method for
nanoparticles is how to efciently and accurately separate the released drug content
from the nanocarrier for analysis. Currently used techniques can be broadly divided
into two categories: (1) sample and separation methods (Xiao et al. 2004); and
(2) membrane diffusion methods (such as dialysis sac (Sezer et al. 2004), reverse
dialysis sac (Chidambaram and Burgess 1999; Xu et al. 2012), microdialysis, and
Franz cells). Methods from the rst category may suffer from incomplete drug
release due to sample loss during sampling and erroneous release proles are
frequently reported if the time scale of drug release is close to the sampling
intervals. Due to the presence of separation membranes, diffusion of drug through
the membrane may be erroneously considered as drug release proles. Furthermore,
none of the method from these two categories takes into account the interactions
between nanomaterials and body components (e.g., serum protein, cells, etc.), and
there is a great need to develop more relevant in vitro testing methods.
Another challenge specic to nanocrystalline drugs is that they are often
designed to increase the apparent solubility to enhance dissolution rate. After dis-
solution, solubilized drug may eventually recrystallize under equilibrium condi-
tions. From in vitro testing point of view, the sensitivity of the method at early
dissolution point should be given special consideration (e.g., obtaining dissolution
prole within seconds to minutes, rather than hours).
4.4 Toxicity
In addition to physicochemical characterization, safety and toxicity studies are

imperative for nanoparticles to be used in pharmaceutical arena. Nanoscale mate-
rials may have different toxicology proles than their macro counterparts in some
cases. For instance, nanoparticles may induce free radical reaction in skin or other
organs of the body producing harmful effects (ICTA 2006). Also, certain compo-
nent of the drug products containing nanomaterials may breakdown in the body
which can be detrimental to the health. For this reason, toxicity screenings are often
needed and are generally performed on animals in preclinical settings. Furthermore,
biodistribution (distribution of the drug in various organs of the body) of nano-sized
drugs are generally vastly different from their macro- counterparts, attributed in part
to their dimensions and enhanced permeability. To assess these adverse impacts, in
general validated assays are required to detect and quantify nanoparticles in tissues
and medical products. Unfortunately, given the unique nature of the nanomaterials,
current toxicological screening methodologies may require improvements to
achieve the same level of selectivity and sensitivities. For example, some of the
toxicity screening methods are short-term and hence chronic effects due to pro-
longed exposure may not be accounted for. Also, toxicity screening performed on
single cell type may prove inadequate to assess the impact of exposure to other cell
types and tissues. For these reasons, newer tests might be required as new toxi-
cological risks are identied.
4.5 Environment Impact
When nanomaterials are used in the manufacturing processes of and/or are incor-
porated into the nished drug products, their potential impact on environment may
be different than those from conventional materials (Colvin 2003). As such, envi-
ronmental assessment may be required as part of the submission (see 21 CFR 25.21;
40 CFR 1508.4), unless they qualify for a categorical exclusion. For additional
information concerning environmental health and safety please see: Environmental
Protection Agency (EPA), http://www.epa.gov/ncer/nano/index.html and
Occupational Safety and Health Administration (OSHA) http://www.osha.gov/dsg/
nanotechnology/nanotechnology.html.
5 Conclusions
Combination of nanomaterials with pharmaceutical sciences to produce lifesaving

drug products is a rapidly growing eld. FDA has approved several nanoparticles
for their important therapeutics effect, and is fully prepared to receive and approve
submissions for a variety of products despite the evolving nature of science in the
discovery areas. The main category of submissions either in IND, NDA, ANDA, or
BLA include liposomes, nanoparticles, nanocrystals, micelles, super param-
agemetic iron oxides/colloidal iron products, and dendrimers. There are other
nanotech products besides drugs (e.g., device, veterinary products), but that is
beyond the scope of this chapter. The readers should know that FDA approves drug
products in CDER, not the technology itself. For the approval of nanomaterials, it is
important to understand and control their physical properties and most importantly,
determine what physciochemical properties are critical to perform their special
functions as nanoparticles. Once these critical quality attributes are identied, they
need to be measured and controlled throughout the shelf life of a product to ensure a
consistent performance.
Disclaimer The views expressed in this book chapter are only of authors and do not necessarily
reflect the policy of the agency.
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Index
A B
Active pharmaceutical ingredient, 55, 60, 68, 5,6-benzo-2-methylene-1,3-dioxepane
381, 552, 591 (BMDO), 357
Active targeting, 35, 58, 75, 77, 81, 553, 564, Bilamination, 34
565, 568, 571, 574, 575, 578, 579 Biocompatibility, 21, 65, 68, 100, 112, 187,
Acyclovir, 383, 385, 388, 409, 415, 421, 482, 188, 196, 205, 235, 256, 338, 344346,
484, 488, 493 349, 350, 353, 361, 405, 521, 528, 529,
Adaptive immunity, 226, 531 538, 568, 570, 575
Adsorption, 56, 71, 72, 111, 135, 138, 227, Biocompatible nanoparticles, 355
299, 386, 388, 390, 405, 409, 414, 424, Biocompatible polymers, 21, 344, 538
441, 511, 593 Biodegradable, 21, 100, 110, 205, 294, 338,
Aggregation, 1921, 29, 34, 65, 68, 70, 7173, 346, 349, 364, 369, 508
76, 7880, 98, 105, 110, 148, 164, 235, Biodegradable nanoparticles, 4, 347,
258, 263, 264, 561, 593, 624, 626, 627, 629 528, 538
Alkylcyanoacrylate, 5, 124128, 132, 133, Biodegradable polymers, 99, 102, 110, 112,
138, 140, 142, 143, 145, 147, 148, 211 172, 205, 206, 346, 481, 484, 560
Amelogenin, 166, 167 Biodistribution, 9, 99, 132, 135, 160, 180, 181,
Amphiphilic oligomers, 350 187, 195, 196, 264, 327, 329, 331, 336,
Amphipilic copolymers, 162, 168, 182 371, 382, 417, 439, 516, 522, 526, 533,
Amphotericin B, 43, 65, 329, 385, 405, 407, 536, 541, 578, 593, 594, 602, 604, 606,
481, 482, 485, 486, 492, 493 624, 630
Anaphylatoxin, 512 Biotin, 308
ANDA, 607, 631 Biotin-avidin, 308
Anionic polymerization, 126, 135, 140143, Biotinylated, 136, 137
211, 410 Block Co-Oligomers, 349, 351
Antibodies, 75, 136, 195, 223, 226, 231, 243, Blood-brain barrier, 42, 295, 306, 513, 578,
248, 252, 263, 268, 303, 308, 515, 553, 604
573, 575, 576, 604 Bovine serum albumin (BSA), 21, 75, 114,
Anticancer agents, 35, 291, 334 180, 248, 510, 511
Artifacts, 5, 194, 198, 208, 210, 214, 216, 217, Brain delivery, 41, 308
263
Aspect ratio, 164, 165, 178, 181, 212, 625 C
Autoassembling, 162 -caprolactone (CL), 365
Azobis(isobutyronitrile) (AIBN), 127, 361 CARPA, see complement activation related
4-4-azobis(4-cyanopentanoic acid (ABCPA), pseudoallergy
361 Caveolae, 296, 302

DOI 10.1007/978-3-319-41421-8
636 Index
Caveolae-mediated endocytosis, 234, 291, 292, Dexamethasone, 253, 383, 385, 406, 407, 422,
294, 296, 478 482, 487, 493
Chemokines, 223 Dextran, 127, 135, 259, 410, 528, 532, 559,
Chemical modications, 348, 351, 355, 411, 571
415, 426 Developability, 609
Chemotherapy, 329, 332, 579 Diagnostic, 11
Chitosan, 20, 71, 112, 129, 131, 135, 137, 148, Dialysis, 78, 106, 197, 443445, 447,
210, 228, 247, 406, 409, 412, 415, 420, 449451, 453, 455458, 462, 465469,
483, 495, 532, 534, 536, 559, 571, 576 472, 479, 491, 492
Ciprofloxacin, 383, 400 Dicyclohexylcarbodiimide (DCC), 345, 353,
Cisplatin, 383 354
Clathrin, 234, 254, 292, 295, 297, 305 Dicyclohexyl urea (DCU), 353
Clathrin-mediated endocytosis, 234, 237, 291, Diffusion, 44, 442445, 447461, 463, 474,
292, 294, 295, 299 479
Click chemistry reaction, 349, 358 Dissolution, 173, 442445, 447450, 452455,
Clinical trials, 333, 334, 580, 606, 610, 619 457459, 461463, 465, 473, 479, 492,
Coagulation, 260, 261 494, 513
Colloid(al), 66, 136, 334, 336, 338, 371, 376, Double emulsion, 90, 400
572, 623 Doxorubicin (Dox), 21, 36, 37, 39, 333, 383,
Complement, 222, 225, 228230, 255, 299, 420, 485, 595, 599
305, 442, 508, 509, 512, 531 Drugability, 592, 594
Complement activation related pseudoallergy, Drug delivery, 3, 65, 112, 117, 124, 143, 160,
512 161, 248, 257, 258, 293, 301, 328330,
Complement receptor, 233, 234, 294, 508 332, 334, 335, 338, 362, 386, 506, 507,
Complex, 6, 7, 68, 69, 414 521, 556, 609
Controlled release, 439, 452, 522, 560 Drug delivery systems, 43, 45, 88, 112, 125,
Copolymers, 22, 131, 346, 351, 352, 357, 359, 126, 141, 205, 271, 346, 361, 522, 564,
362365, 367, 369, 370, 374, 375 591, 606
Copper I, 350 Drug encapsulation, 19, 101, 133, 375, 376
Core-shell nanoparticles (CS-NPs), 301, 386, Drug loading, 29, 59, 63, 69, 105, 173,
420 196199, 336, 391, 414, 426, 441, 483,
Corona, 514 485, 495
Covalent bonding, 425 Drug release, 196198, 439, 481, 483, 487,
Cu(I) catalyzed azide-alkyne cycloaddition, 489, 490, 492, 494, 495
349 Drug targeting, 3, 117, 160, 165, 181, 604, 608
Curvature, 180, 229, 262, 509, 530 Dynamic light scattering, 68, 98, 141, 192,
Cryo-TEM, 194 211, 297, 351, 367, 391, 601, 624
Cyclodextrin, 41, 333, 414
Cyclosporine, 385, 388, 406, 414, 483 E
Cytotoxicity, 65, 68, 233, 241, 357, 366, 367, Electron microscopy, 194, 207
422, 486, 488, 493, 514, 523, 524, 527, Electrospinning, 170173
534, 535, 537, 538, 541, 570 Electrostatic interactions, 388, 390, 410
Ellipsoids, 179, 255, 301
D Emulsion, 4, 5, 10, 13, 133, 134, 138, 141, 147
Degradation, 124, 347, 348, 364, 370, 460, 471 Encapsulation, 18, 20, 25, 29, 34, 43, 45
Degradation product, 124, 374, 522524, 528, Endocytic routes, 237, 273
534 Endocytosis, 305, 309
Degradation rate, 124, 347, 348, 364 Endosome, 294, 295, 310312
Dendritic cells (DC), 222224, 228, 238, 242, Endotoxins, 270
244, 245, 506, 529 Entrapment, 386, 422
Dendrimers, 579 Enzymatic degradation, 360, 363, 364, 376
Deoxyribo nucleic acid (DNA), 76, 116, Ethyl ester, 361
239, 241, 246, 248, 357, 359, 513, 527, Excipients, 21, 102, 331, 523, 527, 528, 597,
577 605607, 609
Index 637
European medicines agency, 112, 382, 580, Immunogenicity, 221, 247, 248, 561
591, 596, 601 Industry, 30, 124, 161, 173, 598, 601, 608,
610, 616, 618
F Innate cells, 222224, 231235, 242
Film stretching, 177 Innate immune cells, 222, 223, 233, 235
Flash nanoprecipitation, 30, 31, 34, 46, 5659, Innate immune system, 132, 222, 226, 231
63, 65, 66, 75, 77, 79, 80 Innate immunity, 222, 224
Food and drug administration, 62, 72, 79, 112, Inorganic nanoparticles, 66, 386, 541
188, 332, 344, 575, 601, 617 Interfacial deposition, 18, 20
Fragmentable polymers, 343, 355, 375 Interfacial polycondensation, 123, 125, 143,
150, 151
G Interfacial polymerization, 123, 126, 138,
Gene therapy, 166, 246, 357, 552, 566, 576 142147, 149, 394, 396, 402, 412
Geometry, 58, 159, 161, 162, 164, 181 Interleukin, 223, 224, 226, 511
GFLGKGFG peptide, 359 Internalization, 159, 233236, 247, 255, 259,
Gold nanoparticles, 11, 67, 74, 170, 309, 312, 292, 295, 298, 305, 309, 313, 336, 467,
421423, 573, 615 489, 508, 511, 526, 527, 535, 565
Intracellular delivery, 255, 292, 329
H Intracellular trafcking, 292, 297, 306, 309,
Haemolysis, 529, 530, 533 312, 478, 489
Hemolysis, 44, 229, 230, 249, 253, 255260 Intravenous, 39, 132, 135, 148, 228, 298, 300,
Hierarchical assemblies, 167169 307, 308, 331334, 336, 337, 416, 419,
High density lipoprotein, 505, 510 420, 529533, 539, 566, 599, 602, 609, 624
Hyaluronic acid, 38, 44, 419, 420, 526, 528 injection, 148, 332, 334, 416, 420, 599,
Hydrolysis, 60, 72, 77, 99, 112, 198, 270, 312, 602, 609
347, 348, 352, 353, 359, 362, 371, 375, route, 135, 419, 529, 533
376, 415, 420, 421, 444, 445, 447, 449, Inverse Ouzo effect, 147, 149, 151
483, 524 In vivo, 4, 11, 21, 3544, 46, 61, 65, 72, 73,
Hydrophilic drugs, 21, 29, 45, 111, 115, 116, 100, 124, 131, 135, 141, 147, 152, 160,
388, 405, 409, 410, 412, 413, 441, 487 166, 194, 198, 212, 236, 244, 250252,
Hydrophilic molecules, 1921, 138, 147, 148, 255, 260, 262, 264, 266, 273, 299301,
391, 410, 412, 488 307309, 331, 335338, 367, 371, 382,
Hydrophobic drugs, 18, 20, 34, 55, 71, 197, 385, 390, 405, 415, 421, 440, 475, 486,
386, 389, 405, 406, 408, 410, 412, 414, 489, 492494, 551, 553, 556, 561, 564,
421, 485, 506 567, 568, 575, 578, 598, 599, 606, 624, 629
Hydrophobic interactions, 70, 270, 295, 299, Iron oxide nanoparticles, 11, 424, 555
367, 386, 388, 421 Insulin, 36, 45, 138, 145, 148, 149, 384, 388,
N-(2hydroxypropyl) methacrylamide 390, 402, 405, 409, 411413, 440,
(HPMAm), 561 470473, 481, 488
Hydroxyethyl methacrylate (HEMA), 368, 485 IND, 188, 619, 631
Hydroxypropyl methacrylate (HPMA), 345,
359, 360 J
Hyperbranched poly(ethylene imine), 365, 367 Janus nanoparticles, 168, 180
Jet spinning, 171
I
Ibuprofen, 384, 388, 390, 395, 405, 408, 423, L
452, 482, 492, 493, 628 Layer-by-layer, 12
Imaging, 8, 11, 34, 40, 56, 58, 62, 65, 66, 74, Light scattering, 190, 192195, 207, 364, 556,
77, 163, 192, 194, 207, 212, 215, 239, 245, 573, 601, 625
298, 328, 332, 336338, 552, 554, 556, Lipid-based nanosystems, 564
557, 559, 560, 562, 566, 570, 575, 577, 580 Liposomes, 567, 568, 577, 580
Immune response, 226, 232, 234236, 239, Lithographic methods, 173
240, 242, 247, 248, 250252, 254, 255, 507 L-leucine ethyl ester, 353
638 Index
Lower critical solution temperature, 361 Multifunctional nanoparticles, 9, 12, 55, 58,
L-phenyl alanine methyl ester, 353 65, 81
Lysosome, 295, 310 Multimodal nanoparticles, 62, 193, 552
M N
Macromolecular prodrug, 416, 418, 420, 421, N-acetyltransferase1, 511
422 Nanocapsules, 8, 9, 1820, 28, 29, 32, 41,
Macromolecules, 6, 11, 45, 62, 72, 127, 128, 112114, 123126, 138, 142144,
130, 148, 152, 179, 291, 292, 295, 349, 145151, 206, 215, 216, 226, 248, 255,
388, 389, 405, 408, 409, 412, 466, 491, 273, 298, 382, 386, 388, 411, 412, 440,
523, 528, 579 482, 484, 526, 532
Macropinocytosis, 233, 234, 237, 291, 292, Nanocrystals, 11, 67, 575, 591, 594, 595, 598,
294, 296, 302, 305, 309, 312, 478 599, 608, 610, 631
Magnetic resonance imaging, 11, 245, 555, 557 Nanobrils, 166, 168
Manufacturing methods, 102, 109, 113, 161, Nanofluidics, 170, 175, 176
174, 578, 610, 617, 618, 624, 628, 631 Nanogel, 6, 7, 9, 10, 382, 386, 389, 410, 411,
Market, 417, 551, 591, 592, 594, 602, 467, 469, 477, 484, 487, 493, 495
605610, 619, 621 Nanoprecipitation, 6, 10, 12, 1724, 2835, 41,
Mass spectrometry, 190, 196, 197, 250, 264, 45, 46, 5581, 161, 164, 206, 212, 363,
265, 269, 506, 515 364, 370, 374, 387, 389, 392, 394396,
Methods, 1, 46, 8, 10, 12, 13, 55, 77, 80, 81, 398, 401403, 405, 407, 408, 412415,
8890, 92, 95, 96, 98, 99, 102, 105, 106, 419, 423, 426, 561, 601, 609
109, 111113, 116, 123125, 127, 132, Nanospheres, 9, 18, 20, 44, 88, 97, 98, 112,
138, 139, 142, 143, 145, 147, 148, 150, 123127, 133, 138141, 143, 144, 167,
152, 159, 161, 162, 166, 170, 173175, 206, 260, 306, 308, 370, 373, 374, 382,
182, 191, 194, 197, 198, 207, 209, 230, 386, 440, 531, 538, 540, 573
231, 235, 236, 238, 241, 243245, 249, Nanoribbons, 162, 167, 171
250, 252, 255, 256, 262264, 267273, Nanotoxicology, 522
328, 332, 336338, 352, 359, 370, 375, Nanotube, 166, 168, 180, 509, 512, 514, 576,
376, 381, 382, 386, 387, 389391, 615
405407, 409, 410, 413, 414, 418, 420, Nanovaccines, 229, 236, 247, 249, 251, 273
422, 426, 439441, 489496, 506, 514, Nasal delivery, 41, 45, 538
515, 570, 573, 578, 601, 608, 610, Natural polymers, 106, 348, 349, 351, 353
623625, 628631 NDA, 606, 607, 618, 619, 620, 621, 631
Micelles, 6, 7, 9, 57, 68, 7173, 79, 162, 163, Needles, 69, 171, 178, 180, 207, 247
206, 307, 310, 312, 351, 367, 375, 389, Negative staining, 194, 208210
398, 408, 424, 443, 531, 533, 554, 561, N-methacryloyl-glycylphenylalanylleucyl
568570, 575, 576, 623, 626, 627, 631 glycyl-doxorubicin (MA-GFLG-Dox), 359
Microbubbles, 92, 555, 556, 579, 580 Na-(methacryloyl)-threonine, 361
Microemulsion, 10, 147, 149, 404, 413, 422 N,N-dimethylaminoethyl methacrylate
Microfluidic(s), 5, 30, 31, 33, 46, 57, 95, 175, (DMAEMA), 357
176, 179, 245, 259, 426, 609 Non-spherical nanoparticles, 13, 159, 162, 169,
Milling, 397, 592, 608, 610, 628 170, 179, 212, 625
Miniemulsion, 5, 10, 126, 130, 138142, Nuclear magnetic resonance, 190, 353, 491
145147 Nucleation, 6, 7, 19, 33, 34, 55, 57, 58, 62, 66,
Molding techniques, 173 68, 74, 139, 142144, 147, 512
Mononuclear phagocytic system, 293, 506, Nucleic acids, 6, 11, 112, 195, 205, 246, 291,
529, 624 292, 296, 311, 329, 331, 388, 390, 409,
Morphology, 96, 101, 103, 159, 160, 194, 206, 411, 413, 553, 576
207, 211, 213, 228, 244, 259, 263, 297,
338, 353, 465, 525, 539, 625 O
Mucosal administration, 247, 251, 331, 336, Oblate, 164166, 177, 180, 255
524 Ocular delivery, 43, 44, 540
Index 639
Opsonization, 195, 225, 228, 235, 264, 293, Poly(alkylcyanoacrylate) (PACA), 4, 43, 136,
299, 301, 508, 529, 564, 565, 593, 602, 626 152, 211, 299, 390, 405, 409, 410, 414
Optical imaging, 11, 239, 554556 Poly(benzyl malate-co-malic acid)
Oral delivery, 414, 488, 535 (PMLABe80H20), 374
Ouzo effect, 138, 143, 144, 147, 149151 Poly(benzyl malate) (PMLABe), 345, 346, 373
Poly(e-caprolactone) (PCL), 171, 206, 363,
P 364, 466, 481, 530, 568
PAGE, see Polyacrylamide gel electrophoresis Polycondensation, 125, 143, 150, 151, 356,
Paclitaxel, 21, 36, 38, 39, 60, 61, 63, 64, 94, 364
95, 116, 147, 307, 329, 331333, 384, 385, Poly(dimethylamino ethyl methacrylate)
388, 394, 405, 406, 414, 415, 418420, (PDMAEMA), 348, 357
424426, 441, 447, 481, 482, 485, 486, Polyelectrolyte complex (PEC), 382, 386, 389,
492, 493, 551, 596, 609 481, 484
Particle replication in non wetting template, Poly(ethylene glycol) (PEG), 22, 23, 31, 40,
170, 173, 174 63, 64, 71, 72, 9395, 127, 128, 130, 140,
Particle size, 18, 25, 2730, 33, 34, 57, 6264, 150, 165, 176, 188, 195, 228, 229, 248,
66, 67, 69, 70, 71, 74, 7681, 89, 9395, 258, 264, 272, 357, 507, 526530, 539,
97, 98, 100, 105, 107109, 113115, 142, 541, 568, 599, 626
187, 193, 194, 207, 214, 235, 248, 249, Poly(ethylene imine) (PEI), 148, 311, 357, 365
251, 254, 298, 301303, 353, 368, 391, Poly(g-benzyl-L-glutamate) (PBLG) g=gamma
462, 465, 483385, 495, 505, 507, 509, symbol, 10, 164, 165, 212
510, 513, 524, 538, 578, 594, 598, Poly(c-glutamic acid) (PGGA), 388
601603, 621625, 627, 629 Poly(glycolide) (PGA), 99, 568
Patchy nanoparticles, 168, 169 Poly(hexyl malate-co-malic acid)
Patent, 88, 109, 173, 592, 610, 618 (PMLAHe90H10), 374
PCR, seePolymerase chain reaction Poly(hexyl malate) (PMLAHe), 374
Personalized medicine, 334, 336 Poly(isopropylacrylamide) (PIPAAN), 361
PET, 552, 555, 556, 558, 559, 568 Poly(lactide-co-glycolide) (PLGA), 18, 38, 93,
Phagocytosis, 222, 233235, 237, 250, 259, 172, 176, 206, 210, 299, 405, 483, 526, 560
291294, 296303, 309, 313, 524, 529 Poly(lactide) (PLA), 5, 18, 59, 95, 299, 405,
Pharmacodynamic (PD), 494, 506, 605 524, 526, 539, 560
Pharmacokinetics (PK), 8, 9, 61, 105, 159, 178, Poly(malic acid) (PMLA), 351
181, 182, 198, 439441, 481, 493, 494, Polymerase chain reaction, 76, 240, 243, 246,
506, 565, 577, 578, 594, 598, 599, 603, 250
605, 624 Polymerization, 4, 5, 10, 13, 76, 88, 116,
Phosphate buffered solution (PBS), 23, 193, 124150, 152, 199, 206, 211, 295, 298,
197, 257, 353355, 360, 363, 366, 367, 302, 344, 345, 355, 356, 359363,
371, 375, 394, 443445, 447451, 453, 365370, 372376, 389, 392, 394404,
455, 456, 458, 462, 463, 465, 467469, 410, 412415, 421
472475, 479, 489, 492, 569, 570, 575 Polymer(s), 313, 1821, 2434, 41, 43, 44,
Photolithography, 173 46, 61, 63, 64, 66, 7074, 79, 88117,
Photosensitizer, 68, 561, 562 124127, 130, 133, 134, 139, 142, 143,
Physico-chemical characterization, 159, 187, 145, 147, 148, 150, 160, 161, 166,
188, 249, 251, 601, 630 169178, 188191, 195199, 205207,
Pinocytosis, 233, 234, 294, 295, 508 209, 212217, 222, 226, 228, 232, 235,
Plasma proteins, 193, 197, 222, 225, 227, 257, 241, 245, 254, 258, 259, 265, 292,
259, 264, 265, 299, 336, 506508, 512, 299301, 307, 311, 328339, 344349,
514, 565 351, 353357, 359, 361, 364, 365,
PLGA particles, 20, 27, 28, 4042, 44, 45, 106, 367372, 374376, 382, 386, 387,
115, 208, 210, 211, 214, 304, 307, 308, 389391, 405408, 412, 414416, 420,
370372, 407, 409411, 414, 422, 491, 421, 424426, 439441, 442, 444, 445,
527, 535539, 604 447, 449, 452, 455457, 461, 463, 465,
Polyacrylamide gel electrophoresis, 265, 514 479, 481, 483, 485, 487, 489, 495, 506,
640 Index
508, 513, 521530, 532542, 552554, studies, 359, 360, 376

559562, 564, 568, 571, 576, 578, 579, Reactive oxygen species (ROS), 238, 239, 249,
592597, 599, 603, 605607, 610, 616, 258, 272, 346, 513, 539, 561, 562
621, 623, 627 Receptor-mediated endocytosis, 295, 303
Polymer solution, 6, 10, 12, 33, 101, 112, 171, Regulations, 239, 243, 246, 331, 577, 591, 601,
172, 174, 175, 163, 426, 627 602, 605607, 618, 619
Polymersome, 9, 10, 206, 210, 215, 216, 382, Regulatory, 4, 102, 188, 197, 223, 224, 227,
387, 389 243, 328, 405, 541, 591, 601, 602,
Poly(methyl malate-co-malic acid) 605607, 616619, 623
(PMLAMexHy), 352 Release prole, 101, 197, 439, 440, 481487,
Poly(methyl malate) (PMLAMe), 352 489, 492, 494, 495, 596, 598600, 630
Polymolecularity index, 352, 353 Reticuloendothelial system, 21, 40, 293, 344,
Poly(N-(2-hydroxypropyl) methacrylamide) 564, 603, 624
(PHPMAm), 421 Reversible Addition Fragmentation Chain
Polyplexes, 311, 312, 366 Transfer, 356, 359, 376
Polysaccharide, 39, 127132, 134, 136, 206, Ring-opening polymerization, 343, 356, 362,
235, 267, 300, 305, 347, 348, 408, 411, 363, 365370, 372376
522, 524, 532, 534, 540, 541, 559 Risk assessement, 528, 605607, 617, 623
Poly(vinyl alcohol) (PVA), 23, 27, 38, 93, 100, Rods, 162165, 177, 179181, 309, 526
168, 172, 210, 352, 527
Precipitation, 5, 6, 10, 12, 19, 25, 28, 32, 34, S
56, 58, 59, 63, 6871, 78, 80, 81, 96, 198, Safety, 9, 12, 30, 44, 46, 104, 124, 143, 191,
350, 355, 395, 407, 8488, 492 195, 222, 245, 259, 260, 264, 265, 273,
Preparation methods, 95, 169, 370, 423 327, 334, 336339, 391, 405, 516, 522,
Print, 8, 10 534, 536, 538, 539, 541, 552, 559, 575,
PRINT(TM) method, 170, 173, 174 576, 578, 602, 605607, 610, 616624,
Prodrug, 5964, 78, 234, 382, 388, 414423, 628631
425, 426, 559, 560, 562, 568570 Scale-up, 90, 92, 96, 106, 110, 116, 159, 162,
c-propargyl-L-glutamate N-carboxyanhydride 166, 190, 579, 601, 608, 609, 619, 628
(PLG-NCA), 348, 363 Scaning electron microscopy (SEM), 18, 19,
Protein corona, 229, 257, 264, 265, 507509, 25, 68, 79, 165, 169, 171, 175, 176, 192,
513516 194, 207, 211217, 237, 249, 250, 259,
Proteins, 6, 11, 29, 40, 61, 7173, 7577, 81, 263, 273, 624, 626
111, 112, 116, 124, 132, 135, 136, 162, Self-assembly(ing), 68, 12, 5558, 66, 73, 81,
163, 166, 168, 173, 178180, 193, 162, 166, 168, 169, 182, 206, 419421, 628
195197, 222, 225229, 231, 233, 235, Shape, 8, 159182, 194, 207, 212, 215, 227,
243245, 247249, 255, 257261, 228, 235, 249, 254, 255, 266, 301, 309,
263269, 273, 292296, 298300, 306, 312, 335, 374, 382, 512, 523, 524, 526,
312, 331, 332, 336, 348, 363, 388, 390, 528, 532, 573, 601, 602, 625, 626, 629
391, 405, 409, 410, 421, 465, 477, 487, Shape controlled nanoparticles, 159182
506516, 522, 524, 528, 529, 531, 538, Silica nanoparticles, 169, 181, 257, 259, 305,
565, 596, 627, 629, 630 510, 511, 530
Pulmonary delivery, 336, 537 Single emulsion, 110113, 116
SiRNA, 55, 68, 69, 70, 79, 148, 149, 234, 246,
Q 247, 250, 329, 331, 333, 334, 388, 403,
Quality control, 195, 199, 268, 338, 628 405, 409, 411413, 418, 423425,
Quantum dots, 11, 312, 512, 570, 575 474478, 484, 488, 489, 492, 493, 494,
496, 577, 606
R Sirolimus, 385, 388, 398, 405, 407, 425, 462,
Radical polymerization, 127132, 134138, 482, 483, 486, 487, 491, 492, 592
140142, 343, 356, 361, 376, 402, 410, Site-specic drug delivery, 344, 348
415, 421 Size, 5, 6, 8, 18, 19, 2546, 55, 6264, 66,
RAFT 6780, 91117, 134136, 139, 141152,
polymerization, 356, 359, 360 160, 170, 173, 175, 176, 179, 192200,
Index 641
211, 214, 217, 228, 234, 235, 250, 254, TEM, see transmission electron microscopy
261, 272, 294, 297299, 303, 305, 338, Theragnostic, 62, 65, 66, 81
353, 355, 370, 382, 391, 441480, Theranostic, 327, 336, 337, 551580
483485, 489, 495, 505514, 523, 524, Therapeutic activity, 329, 331, 334, 336, 337,
528, 529, 531, 532, 535, 537, 538, 573, 339, 385
598, 601, 624 Therapy, 11, 35, 68, 77, 166, 229, 246, 266,
Size exclusion chromatography (SEC), 191, 357, 552, 563, 566, 568, 572, 573, 576,
192, 249, 250, 353, 355, 359, 367, 371, 578, 579
374, 514, 625, 627 Thermo-responsive diblock copolymer
Small molecules, 55, 64, 68, 74, 75, 76, 81, nanoparticles, 511
388, 416, 629 Toxicity, 3941, 67, 68, 102, 105, 112, 165,
Sol-gel, 387, 389, 392, 395, 397 182, 195, 222, 228, 238, 249, 250, 253,
Solvent displacement, 18, 87, 88, 90, 109, 110, 260, 266, 271, 272, 292, 336, 349, 353,
116, 396, 399, 414 357, 359, 385, 417, 485496, 506516,
Solvent evaporation, 5, 30, 87, 88, 97, 98, 522524, 528, 533, 535, 537, 539541,
101105, 110, 112, 114116, 167, 170, 564, 565, 567, 571, 572, 574, 575, 578,
367, 370, 374, 394, 398, 402, 414, 415, 579, 595, 607, 630, 631
419, 422, 628 Transmission electron microscopy, 25, 74, 163,
SPECT, 552, 555, 556, 558, 559, 568 167, 169, 171, 192, 194, 207, 208,
Spherical particles, 8, 192, 601 210217, 249, 272, 471, 601, 626
SPION, 424, 571, 576 Treatment, 9, 11, 3943, 60, 188, 242, 261,
Stannous octanoate, 365, 367, 369, 370 291, 329, 332, 334, 336, 338, 425, 476,
Static Light Scattering, 192194, 351 521, 532, 539, 552, 554, 556, 558, 561,
Sterilization, 105, 249, 270273, 422, 578, 629 564580, 609, 629
Supersaturation, 19, 33, 5760, 62, 6870, 81,
143, 462 U
Surface functionalization, 12, 75, 228, 387 Ultrasound imaging, 11, 555
Surface properties, 21, 135, 136, 141, 152, 178,
182, 187, 228, 230, 294, 297299, 302, V
305, 346, 507, 510, 521, 523, 526528, Vaccines, 111, 222, 232, 236, 241, 242, 247,
602, 626 251, 255, 411, 531
Synthetic polymers, 112, 235, 292, 343, 348,
354356, 376 X
Systemic activity, 328, 331 X-ray imaging, 554
T Y
Target cells, 35, 136, 141, 160, 229, 232, 240, Yields, 67, 163, 173, 176, 192
241, 242, 246, 292, 329, 331, 335,
336338, 409, 464, 482, 489, 602 Z
Targeting ligands, 39, 55, 7477, 81, 188, 192, Zeta potential, 25, 31, 3638, 43, 71, 97, 134,
195, 196, 306, 307, 513, 539, 552, 564, 136, 142, 195, 199, 251, 257, 297, 305,
580, 604 507, 508, 510, 603, 626
Target tissue, 329, 439, 561, 604

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Polymer Nanoparticles For Nanomedicines

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ChristineVauthier GillesPonchel

ISBN 978-3-319-41419-5 ISBN 978-3-319-41421-8 (eBook)

Springer International Publishing Switzerland 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Polymers are macromolecules composed of many repeated subunits of different

therapeutic aim. The surface properties of the polymer when formulated as

influence the nanoparticle biodistribution. A special chapter describes the concep-

Part I Methods for the Manufacturing of Nanoparticles: Principles

Part II Characterization of Polymer Nanoparticles

8 Imaging Polymer Nanoparticles by Means of Transmission

Part III Turning Polymer Nanoparticle Technologies

Part IV From Lab to Prescription Desk

19 Regulatory Perspective on the Development of Polymer

About the Editors

Christine Vauthier received her Ph.D. in polymer

Gilles Ponchel is full Professor at the University of

Sergio Alcal-Alcal Laboratorio de Posgrado en Tecnologa Farmacutica,

Jeffrey D. Clogston Nanotechnology Characterization Laboratory, Cancer

Adela Lopez de Cerain Department of Pharmacology and Toxicology, University

Christina Tang Department of Chemical and Biological Engineering, Princeton

c-CDC6 c-cyclodextrine modied with carbon chain in C6

APS Ammonium persulfate

EEM EmulsicationEvaporation Method

HLB Hydrophilic-lipophilic balance

LLC Lewis lung carcinoma

PECs Peritoneal exudate cell macrophages

PMLAHe90H10 Poly(hexyl malate-co-malic acid)

ROS Reactive Oxygen Species

TNF Tumor necrosis factor

In the 1970s, polymer nanoparticles were found to be suitable materials thanks to

Springer International Publishing Switzerland 2016 3

regulatory registration and fulll stringent specications. Besides, they must

2 Development of Methods of Preparation

In another series of methods, nanoparticles are prepared directly from polymer

(a) Amphiphilic Hydrophobic drugs entrapped in the

Fig. 1 Preparation of nanoparticles by nucleation of particles thanks to self-assembling of soluble

3 Producing Polymer Nanoparticles with Different

A broad range of methods of preparation of polymer nanoparticles was requested to

Matrix-like type nanoparcles Reservoir type nanoparcles

nanospheres core-corona aqueous reservoir

nanogel polyelectrolyte oil reservoir

polyelectrolyte lipid core

Fig. 2 Different types of polymer nanoparticles showing the structures

Nanosphere Core-corona nanosphere Oil-containing nanocapsule Water-containing nanocapsule

Nanosphere Nanosphere Nanogel Nanosphere Nanogel Nanogel

Oil-containing Oil-containing Oil-containing Micelle-polyelectrolyte Micelle-

Fig. 3 Summary of general principles of methods of preparation of nanoparticles from

(a) (b) (c)

Table 1 Functionalities that may be associated with polymer nanoparticles designed as

seen as an opportunity to elaborate personalized therapeutic protocols (Mura and

based on the use of emulsions. Methods based on polymerization are described in

Andrieux K, Couvreur P (2009) Polyalkylcyanoacrylate nanoparticles for delivery of drugs across

Fessi H, Puisieux F, Devissaguet JP, Ammoury N, Benita S (1989) Nanocapsule formation by

Karim Miladi, Sana Sfar, Hatem Fessi and Abdelhamid Elaissari

Keywords Supersaturation Nucleation Encapsulation Hydrophilic

K. Miladi H. Fessi (&) A. Elaissari (&)

Springer International Publishing Switzerland 2016 17

Nanoprecipitation is also called solvent displacement or interfacial deposition. It is

Fig. 1 a Scanning electron microscopy (SEM) micrographs of PLGAPEG nanoparticles (Anand

molecules. Figure 1a, b show scanning electron microscopy (SEM) images of

2.1 Mechanism of Particle Formation by Nanoprecipitation

Nanoprecipitation is a simple and reproducible technique that produces particles with