Tom's Thesis Submitted

Design, Synthesis and
Application of Enzyme
Responsive Hydrogel
Particles using Peptide
Actuators
A thesis submitted to the University of Manchester for the degree of

Doctor of Philosophy in the Faculty of Engineering and Physical
Sciences
2009
Tom O. McDonald
School of Materials
Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald
Contents
DECLARATION........................................................................................................14
ABSTRACT..............................................................................................................15
ACKNOWLEDGEMENTS...........................................................................................16
1 INTRODUCTION...........................................................................................18
1.1. MOTIVATION OF THE PROJECT...................................................................19

1.2. LAYOUT OF THE THESIS..............................................................................19
2 LITERATURE REVIEW...............................................................................22
2.1. INTRODUCTION...........................................................................................22
2.2. STIMULI RESPONSIVE MATERIALS..............................................................22
2.2.1. Chemical hydrogels...............................................................................22
2.3. BIORESPONSIVE HYDROGELS.....................................................................23
2.3.1. Introduction...........................................................................................23
2.3.2. Actuation based on changes in crosslinking density.............................24
2.3.2.1. Systems incorporating peptide crosslinkers...................................24
2.3.2.2. Non-covalent crosslinking interactions.........................................29
2.3.3. Actuation based on electrostatic interactions.......................................34
2.3.3.1. Electrostatic interactions between charges on the polymer
network..........................................................................................................35
2.3.3.2. Electrostatic interactions between charges present in pendant
actuators .......................................................................................................39
2.3.4. Actuation based on conformational changes........................................42
2.3.5. Summary................................................................................................45
2.4. PEGA.........................................................................................................46
2.4.1. The history of PEGA..............................................................................46
2.4.2. Enzyme catalysed synthesis on PEGA...................................................53
2.4.3. Summary................................................................................................56
2.5. MICROFLUIDIC POLYMERISATION OF PARTICLES.......................................57
2.5.1. Summary................................................................................................67
2.6. AIMS OF THESIS..........................................................................................69
2
3 PEGA POLYMERISATION, CHARACTERISATION AND ENZYME

RESPONSIVE SWELLING THROUGH FUNCTIONALISATION WITH
PEPTIDE ACTUATORS........................................................................................70
3.1. ABSTRACT..................................................................................................70
3.2. INTRODUCTION...........................................................................................70
3.3. EXPERIMENTAL..........................................................................................74
3.3.1. Materials...............................................................................................74
3.3.2. Inverse suspension polymerisation........................................................74
3.3.3. Charged PEGA polymerisation.............................................................74
3.3.4. Microscopy and particle size analysis...................................................75
3.3.5. Solid phase peptide synthesis................................................................76
3.3.5.1. Solid phase synthesis of dipeptides and enzyme treatment...........77
3.3.5.2. Solid phase peptide synthesis of peptide actuators........................78
3.3.6. HPLC.....................................................................................................78
3.3.7. Two-photon microscopy........................................................................78
3.3.8. Determining particle swelling...............................................................79
3.3.9. Assessing accessibility...........................................................................79
3.3.10. Entrapping payload...........................................................................79
3.3.11. Fluorimetry........................................................................................79
3.3.12. Microscopy and determination of swelling.......................................80
3.4. RESULTS AND DISCUSSION.........................................................................81
3.4.1. Production of µPEGA800........................................................................81
3.4.1.1. Particle morphology and size characterisation of µPEGA............82
3.4.2. Production of µPEGA+ and µPEGA-.....................................................83
3.4.3. Further characterisation of µPEGA......................................................88
3.4.3.1. Amine characterisation by two-photon microscopy and enzyme
compatibility..................................................................................................88
3.4.3.2. Comparison of enzymatic hydrolysis within µPEGA800 and
macroparticles................................................................................................90
3.4.4. Functionalisation with peptide actuators..............................................91
3.4.4.1. Enzyme responsive increase in accessibility.................................92
3.4.4.2. Demonstration of the encapsulation of a payload..........................95
3.4.4.3. Enzyme specific release.................................................................96
3
3.4.4.4. Enzyme responsive increase in swelling; effect of ionic strength. 97

3.5. CONCLUSIONS............................................................................................98
4 DESIGNING NEW PEPTIDE ACTUATORS FOR IMPROVED

ENZYME RESPONSIVE BEHAVIOUR..............................................................99
4.1. ABSTRACT..................................................................................................99
4.2. INTRODUCTION...........................................................................................99
4.2.1. Branched peptide actuators...................................................................99
4.3. MATERIALS AND METHODS......................................................................102
4.3.1. Materials.............................................................................................102
4.3.2. Inverse suspension polymerisation and polymer characterisation.....102
4.3.3. Solid phase peptide synthesis..............................................................103
4.3.4. Microscopy and determination of swelling.........................................103
4.3.5. Two-photon microscopy......................................................................103
4.3.6. Entrapping payload.............................................................................104
4.3.7. Release measurements.........................................................................104
4.3.8. HPLC and LCMS.................................................................................104
4.4. RESULTS AND DISCUSSION.......................................................................106
4.4.1. Microparticle characterisation and peptide functionalisation...........106
4.4.2. Actuator design and responsiveness of peptide functionalised particles. .
.............................................................................................................107
4.4.3. Characterisation of enzyme action on peptide actuators....................111
4.4.4. Enzyme triggered release....................................................................113
4.5. CONCLUSIONS..........................................................................................115
5 MICROFLUIDIC PREPARATION OF LOW POLYDISPERSITY PEGA

PARTICLES...........................................................................................................116
5.1. ABSTRACT................................................................................................116
5.2. INTRODUCTION.........................................................................................116
5.3. MATERIALS AND METHODS......................................................................119
5.3.1. Microfluidic system.............................................................................119
5.3.2. Flow focussing setup...........................................................................119
5.3.3. Monomer and continuous phase preparation.....................................120
5.3.4. Particle size measurement...................................................................120
4
5.4. RESULTS AND DISCUSSION.......................................................................121

5.4.1. Microfluidic system.............................................................................121
5.4.1.1. Variation of particle size with flow rate ratios and surfactant
concentration................................................................................................121
5.4.1.2. Effect of increasing total flow rate on particle size.....................126
5.4.1.3. Effect of flow ratios and surfactant concentration on polydispersity
.....................................................................................................127
5.4.1.4. Variation of particle production rates with the conditions assessed..
.....................................................................................................127
5.4.2. Using a simplified microfluidic flow-focussing device to produce
polymer particles.............................................................................................129
5.4.2.1. Orientation of device...................................................................129
5.4.2.2. Optimising conditions with the FF device...................................131
5.4.2.3. Effect of changing oil phase........................................................133
5.4.3. Optimum conditions for particle production.......................................134
5.4.3.1. Particle size distribution...............................................................134
5.4.4. Morphology of particles......................................................................135
5.5. CONCLUSIONS..........................................................................................136
6 CONCLUSIONS............................................................................................137
7 FUTURE WORK...........................................................................................139
8 APPENDICES................................................................................................141
8.1. BACKGROUND..........................................................................................141
8.1.1. Polymers..............................................................................................141
8.1.1.1. Peptides and proteins...................................................................141
8.1.2. Two-photon microscopy......................................................................143
8.1.3. Fluorescence resonance energy transfer.............................................144
8.2. SUPPLEMENTARY DATA............................................................................145
8.2.1. HPLC solvent gradient........................................................................145
8.2.2. Synthesis of activated disulfide-methacrylamide monomer................145
9 REFERENCES...............................................................................................147
5
List of figures
Figure 1-1. The inverse suspension polymerisation of µPEGAs...............................20
Figure 1-2. Enzyme responsive µPEGA....................................................................20
Figure 1-3. Enzyme responsive µPEGA through the incorporation of branched
peptide actuators........................................................................................................21
Figure 1-4. Production of PEGA particles by microfluidic polymerisation..............21
Figure 2-1. Schematic representation of the different categories of bioresponsive
hydrogels....................................................................................................................24
Figure 2-2. Cell responsive synthetic hydrogels........................................................26
Figure 2-3. Antigen responsive hydrogel..................................................................31
Figure 2-4. Displacement-Induced Switching Rates of Bioresponsive Hydrogel
Microlenses................................................................................................................32
Figure 2-5. Novel synthesis of HPMA copolymers containing peptide grafts and
their self assembly into hybrid hydrogels..................................................................34
Figure 2-6. The enzymes and the reactions they catalyse used in glucose responsive
hydrogels....................................................................................................................36
Figure 2-7. Characterization of glucose-sensitive insulin release systems in
simulated in vivo conditions......................................................................................37
Figure 2-8. Enzyme-responsive hydrogel particles for the controlled release of
proteins: Designing peptide actuators to match payload...........................................41
Figure 2-9. Peptide actuator designed for the release of negatively charged protein
molecules...................................................................................................................42
Figure 2-10. Ligand responsive hydrogel that relies on conformational changes.....44
Figure 2-11. Chemical structure of PEGA.................................................................46
Figure 2-12. Inhibition of cruzipain visualized in a fluorescence quenched solid-
phase inhibitor library assay. D-amino acid inhibitors for cruzipain, cathepsin B and
cathepsin L.................................................................................................................48
Figure 2-13. Using two photon microscopy to quantify enzymatic reaction rates on
polymer beads............................................................................................................50
Figure 2-14. A micropatterned hydrogel platform for chemical Synthesis and
biological analysis......................................................................................................52
Figure 2-15. Real-time imaging of protease action on substrates covalently
immobilised to polymer supports..............................................................................53
6
Figure 2-16. Biomimetic synthesis and optimization of cyclic peptide antibiotics.......

...................................................................................................................................55
Figure 2-17. Formation of dispersions using "flow focusing" in microchannels......58
Figure 2-18. An axisymmetric flow-focusing microfluidic device (AFFD).............60
Figure 2-19. Interfacial Polymerization within a Simplified Microfluidic Device:
Capturing Capsules....................................................................................................61
Figure 2-20. Polymer particles with various shapes and morphologies produced in
continuous microfluidic reactors...............................................................................63
Figure 2-21. Continuous microfluidic reactors for polymer particles.......................65
Figure 2-22. A micro-reactor for preparing uniform molecularly imprinted polymer
beads..........................................................................................................................66
Figure 2-23. A predictive approach of the influence of the operating parameters on
the size of polymer particles synthesized in a simplified microfluidic system.........67
Figure 3-1. Schematic of the enzyme responsive swelling of PEGA particles
functionalised with linear peptide actuators. ...........................................................73
Figure 3-2. Monomers used in the synthesis of PEGA and its charged variants.......75
Figure 3-3. Solid phase peptide synthesis scheme.....................................................76
Figure 3-4. Chemical reactions involved in peptide synthesis..................................77
Figure 3-5. Polymerisation of PEGA particles by inverse suspension polymerisation.
...................................................................................................................................82
Figure 3-6. Optical micrograph of microparticles is water........................................83
Figure 3-7. Chemical structure of PEGA and its charged variants.72........................84
Figure 3-8. PEGA+ microparticles.............................................................................85
Figure 3-9. Optical micrographs PEGA-,...................................................................86
Figure 3-10. The uptake of water by the three different types of dry PEGA
microparticles and the effect of ionic strength on the swelling of the three different
types of PEGA microparticles...................................................................................90
Figure 3-12. Comparison of time dependence of enzyme reactions on µPEGA and
commercially available macroparticles.....................................................................91
Figure 3-13. A schematic of enzyme responsive microparticles illustrating both
successful and unsuccessful cleavage of the ECP resulting in a change of
accessibility to a 40 kDa FITC labelled dextran........................................................95
Figure 3-14. Confocal microscopy images of representative microparticles in an
aqueous solution of 40 kDa fluorescently labelled dextran.......................................95
7
Figure 3-15. The pH responsive loading of the 40 kDa FITC labelled dextran (1
mg/ml) into the PEGA particles, gain of images varied............................................97
Figure 4-1. Schematic of enzyme responsive branched peptide actuator................101
Figure 4-2. HPLC quantification of Fmoc removed after each coupling step for
linear and branched peptide actuators......................................................................106
Figure 4-3. pH responsive swelling behaviour of peptide actuators on PEGA
microparticles...........................................................................................................107
Figure 4-4. Enzyme responsive swelling behaviour of peptide actuator
functionalised µPEGA at pH 7................................................................................109
Figure 4-5. HPLC and MS analysis of enzyme hydrolysis of branched peptide
actuators...................................................................................................................110
Figure 4-6. Effect of ionic strength on the maximal enzyme responsive swelling of
branched peptide actuator functionalised µPEGA at pH 7......................................111
Figure 4-7. Comparison of thermolysin action on both linear and branched peptide
actuators on µPEGA................................................................................................112
Figure 4-8. µPEGA particles functionalised with the branched peptide actuator at pH
7...............................................................................................................................114
Figure 5-1. Schematic of microfluidic setup for the synthesis of controlled-size
polymer particles......................................................................................................118
Figure 5-2. Effect of Qc/Qd on mean particle diameter with no surfactant in the
continuous phase,.....................................................................................................122
Figure 5-3. Effect of surfactant concentration on particle size................................124
Figure 5-4. Effect of total flow rate (at constant Q c/Qd) on mean particle diameter.
.................................................................................................................................126
Figure 5-5. Effect of Qc/Qd and surfactant concentration on polydispersity...........128
Figure 5-6. Effect of Qc/Qd and surfactant concentration on particle production rate.
.................................................................................................................................128
Figure 5-7. Schematic representation of the ‘flow-focussing’ device.....................129
Figure 5-8. Effect device orientation on droplet and resulting particle formation.. 131
Figure 5-9. Development of flow-focussing setup..................................................132
Figure 5-10. Optical micrographs of PEGA particles produced using FF device with
silicone oil at a variety of conditions......................................................................133
Figure 5-11. PEGA Particles produced under the optimum conditions..................135
8
Figure 5-12. The typically slightly asymmetric shape of particles produced by

microfluidics each image is of particles produced under different conditions........136
Figure 8-1. The structures of the twenty DNA encoded amino acids. The single letter
abbreviation is indicated with the brackets..............................................................143
Figure 8-2. Jablonski energy diagram showing a comparison of the excitation of a
fluorophore with a single photon (confocal microscopy) and two photons (two-
photon microscopy).................................................................................................144
Figure 8-3. HPLC solvent gradient used in analytical runs.....................................145
Figure 8-4. Synthesis of PDTEMA.130.....................................................................146
Figure 8-5. Characterisation of PDTEMA by NMR................................................146
9
List of tables
Table 2-1. Selected studies of bioresponsive hydrogels based on peptide
crosslinkers................................................................................................................25
Table 2-2. Selected studies of bioresponsive hydrogels based on non-covalent
crosslinking interactions............................................................................................30
Table 2-3. Selected studies of bioresponsive hydrogels based on electrostatic
interactions.................................................................................................................35
Table 3-2. Specificity of the enzymes used towards amino acids (AA) in the
substrate and their molecular weight.........................................................................93
Table 3-1. Values for HPLC relative enzyme cleavage for each ECP......................94
10
List of abbreviations
α-CD α-cyclodextrin
ABP Aminobenzophenone
AFFD Axisymmetric flow-focusing microfluidic device
APS Ammonium persulfate
APTMS 3-aminopropyltrimethoxysilane
BDDA 1,4-butanediol diacrylate
CaM Calmodulin
CDDP Cisplatin
CPGs Controlled pore glasses
CV Coefficient of variation
DCM Dichloromethane
DEAEMA Diethylaminoethyl methacrylate
DIC Differential interference contrast
DIPEA N,N-Diisopropylethylamine
DMAEMA N,N-dimethylaminoethyl methacrylate
DMSO Dimethyl sulfoxide
DOPA L-3,4-dihydroxylphenylalanine
DTT Dithiothreitol
ECM Extracellular matrix
ECP Enzyme cleavable peptide
EG Ethylene glycol
EGDMA Ethyleneglycol dimethacrylate
ESEM Environmental scanning electron microscopy
FF Flow-focussing
FITC Fluorescein isothiocyanate
Fmoc 9-fluorenylmethoxycarbonyl
FRET Fluorescence quenched peptide substrate
GOx Glucose oxidase
HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate
HOBt Hydroxybenzotriazole
HPLC High-performance liquid chromatography
11
HPMC Hydroxypropyl methyl cellulose ether

HPMA N-(2-hydroxypropyl)methacrylamide methacryoyl
ID Internal diameter
IVD Intervertebral discs
LbL Layer-by-layer assembly
LCST Lower-critical solution temperature
µPEGA Microparticular PEGA
MA Methacrylate
MAA Methacrylic acid
MALDI Matrix-assisted laser desorption/ionization
MBAA N,N’-methylenebisacrylamide
MFFD Microfluidic flow-focusing device
MMA Methyl methacrylate
MMP Matrix metalloproteinase
MS Mass spectrometry
NSA N-succinimidylacrylate
PAAc Poly(acrylic acid)
PCP Phosphotpanteheine
PDMA Poly(dimethylsiloxane)
PDTEMA N-[2-(2-pyridyldithio)]ethyl methacrylamide
PEG Poly(ethylene glycol)
PEGA Poly(ethylene glycol)-co-acrylamide
PEGMA Poly(ethylene glycol) monomethacylate
PETA-3 Pentaerythritol triacryalte
PGA Penicillin G amidase
PNIPAAm Poly(N-isopropylacrylamide)
poly(HEMA) poly(2-hydroxyethyl methacrylate)
PLE Porcine liver esterase
PU Polyurethane
PVDF polyvinylidene fluoride
Qc Flow rate of the continuous phase
Qd Flow rate of the dispersed phase
SEM Scanning electron micrscopy
Semi-IPN Semi-interpenetrating network
12
Span 20 Sorbitan monolaurate

SPPS Solid-phase peptide synthesis
TEGDMA Tetraethylene glycol dimethacrylate
TEMED N,N,N′,N′- tetramethylethylenediamine
TFA Trifluoroacetic acid
TFP Trifluoperazine ligand
TG Transglutaminase
THF Tetrahydrofuran
TPGDA Tripropyleneglycol diacrylate
TPM Two-photon microscopy
tTG Tissue transglutaminase
13
Declaration
No portion of this work has been submitted in support of an application for another
degree or qualification of this or any other university or institute of learning.
Copyright
(i) The author of this thesis (including any appendices and/or schedules to this
thesis) owns any copyright in it (the “Copyright”) and s/he has given The University
of Manchester the right to use such Copyright for any administrative, promotional,
educational and/or teaching purposes.
(ii) Copies of this thesis, either in full or in extracts, may be made only in
accordance with the regulations of the John Rylands University Library of
Manchester. Details of these regulations may be obtained from the Librarian. This
page must form part of any such copies made.
(iii) The ownership of any patents, designs, trade marks and any and all other
intellectual property rights except for the Copyright (the “Intellectual Property
Rights”) and any reproductions of copyright works, for example graphs and tables
(“Reproductions”), which may be described in this thesis, may not be owned by the
author and may be owned by third parties. Such Intellectual Property Rights and
Reproductions cannot and must not be made available for use without the prior
written permission of the owner(s) of the relevant Intellectual Property Rights
and/or Reproductions.
(iv) Further information on the conditions under which disclosure, publication and
exploitation of this thesis, the Copyright and any Intellectual Property Rights and/or
Reproductions described in it may take place is available from the Head of the
School of Materials.
14
Abstract
Stimuli responsive materials are well documented and function by translating a molecular
event into a macroscopic transition. Polymer hydrogels are a particular type of material that
offers excellent potential for applications within the field of biomaterials. In this thesis an
enzyme responsive polymer hydrogel has been demonstrated that functions through the
incorporation of designed peptide actuators. The experimental findings within this thesis are
separated into three separate chapters.
The first experimental chapter describes the synthesis of poly(ethylene glycol)-co-

acrylamide microparticles (μPEGA) by inverse suspension polymerisation. Particles with
neutral, positive or negative charge were prepared and characterised. Neutral µPEGA were
selected were chosen as the preferred polymer particles. These particles were then utilised as
the basis of the enzyme responsive system. Here linear peptide actuators were incorporated
into the particles. These particles demonstrated an enzyme specific increase in accessibility.
Additionally, functionalised particles displayed pH responsive behaviour allowing for the
physical entrapment of a payload. The release of this payload was only possible when the
functionalised particles were exposed to the target enzyme. However, at physiological ionic
strength no response of the particles was observed due to electrostatic screening.
In order to overcome electrostatic screening, branched peptide actuators were developed and
incorporated into μPEGA. These peptide actuators provided enhanced charge density and
the functionalised particles were able to respond through an increase in swelling to the target
enzyme at physiological ionic strength. Using this system it was possible to selectively
release a macromolecule at physiological ionic strength in response to the target enzyme.
The large size distribution of μPEGA was addressed in the final experimental chapter. Here,
PEGA particles were prepared by a simplified microfluidic setup assembled using a needle
and tubing. The size of the particles produced was determined by the surfactant
concentration and the relative flow rates of the dispersed and continuous phases.
15
Acknowledgements
The last three years of my PhD have been an invaluable time in my life, I feel I have
greatly improved a variety of my skills and abilities as well as maturing as a person.
I have to firstly thank Rein (now Professor Ulijn!) for all his time, comments,
feedback and assistance he has given me throughout my PhD. The opportunities I
have had and a great number of my achievements have only been possible due to
him. I was also very fortunate to have Brian Saunders as my second supervisor, he
has always been available to help me as well as offering me support whenever it
was needed. His constructive criticism regarding my thesis as been invaluable,
thank you Brian!
There were, of course, numerous other individuals who have been very influential in
the completion of this PhD. These people have taught me new skills along with
refining other aspects on my research for which I am very grateful: Paul Thornton,
my mentor at the start of my PhD, without whom I would have been lost. Rob Mart
for his continuous technical advice (no matter how many times I asked...). Last but
not least, Andrew Hirst for his exceptionally useful advice with my writing, along
with his thorough proof reading of this thesis.
All my friends I have made during my studies have made the last three years very
enjoyable and memorable: Richard (our trips to Fab Café), Simon (gym visits and
trading sporting notes), Grace (discussing world politics), Rumana (the cat ‘jokes’),
Alison (all the conference trips), Claire (teaching me so many ‘useful’ French
phrases), Kapil, Riaz (no you can’t take the eighth decimal place into account!)
Louise, Andy T, Bobby T (stay off the T120s...) Nurguse, Andrew, Apurba and
Kate it wouldn’t have been the same without you.
Two technicians have been particularly helpful with my experimental work: Robert
Fernandez for his continuous help with confocal and TPM. Also, Andy Wallwork
for his time and assistance in my attempts to use stereolithography to produce
microfluidic devices.
16
The support I have received from my family (including the regular questioning
asking when I would get a job!) has been very important. I am very grateful for the
encouragement that my parents gave me throughout my time at university.
Finally I have to say many thanks to my girlfriend Jenny Hayes, she has put up with
my last seven years at university! She has also given me enormous moral and
financial support. Hopefully I can repay this in kind during her upcoming teacher
training.
17
1 Introduction
Biomaterials science is an interdisciplinary field that encompasses medicine,

biology, chemistry and materials science. Biomaterials 1 have been defined as
substances other than food or drugs used in therapeutic or diagnostic systems. 2 This
area of science has been applied to the fabrication of medical devices. These include
metallic hip replacements,3 ceramic dental crowns,4 polymeric drug delivery
systems5 and hydrogel based contact lenses.6 Overall, the development of
biomaterials has allowed a great number of diseases to be treated more effectively
and improve the quality of life for patients.7,8 In the majority of cases current
biomaterials are inert i.e. they do not interact with their surroundings. This
characteristic is somewhat limiting when the material is used to replace a function in
the dynamic and changing environment of biological systems. For this reason, there
has been great interest in designed biomaterials that can respond (e.g. swelling, 9
payload release10 and dissolution11) to an environmental stimulus. A particularly
interesting choice of stimulus would be enzymes, nature’s catalysts, because of their
vital importance to living systems. Enzymes drive and control almost every process
that occurs in the human body from respiration to growth. 12 Of special interest is the
role enzymes have within disease processes, these include: the metastasis of
tumours,13 chronic wounds14 and arthritis.15
A common strategy to incorporate responsiveness into a biomaterial is based on

hydrogels. These are water-containing three-dimensional structures composed of
either hydrophilic polymer networks (chemical hydrogels)16 or self-assembled
(macro) molecules.17-19 The former, chemical hydrogels are an area of focus in this
thesis. These crosslinked (either physically of chemically) hydrophilic polymers
give polymer networks that can contain between ten to many thousand times their
dry mass in water.16 This highly hydrated nature of hydrogels presents similar
characteristics to biological tissue. Additionally, the properties of these polymer
18
networks can be altered by changing the polymer’s interactions with neighbouring

chains or the water molecules surrounding the polymer chains.
This thesis consists of two main areas of research, (i) the development of a hydrogel
system amenable to chemical modification thereby incorporating enzyme
responsiveness and (ii), the design and development of enzyme responsive
functionalities.
1.1. Motivation of the project

The motivation of this research was to develop enzyme responsive hydrogel
particles based on peptide actuators. This proof-of-concept research would
demonstrate the potential application of peptide actuators in the enzyme specific
release of a payload. In the future systems such as these might serve a role in the
triggered delivery of drugs.
1.2. Layout of the thesis

Within this thesis the experimental chapters are presented separately, each with its
own introduction, results, discussion, and conclusion sections. By separating the
chapters in this manner it is intended to give the reader a clear view to each section
of research.
The first experimental chapter, Chapter 3 investigates the synthesis and

characterisation of poly(ethylene glycol)-co-acrylamide (PEGA) microparticles
(µPEGA) by way of inverse suspension polymerisation (Figure 1 -1). The resulting
microparticles were the functionalisation with peptide actuators. Finally, the
responsive behaviour of these particles was then determined and exploited for
enzyme specific release of a payload Figure 1 -2.
19
Figure 1-1. The inverse suspension polymerisation of µPEGAs.
Figure 1-2. Enzyme responsive µPEGA. Scheme of enzyme responsiveness of linear peptide
actuators.
Chapter 4 goes on to tackle the limitations of the linear peptide actuators with the
design and development of branched peptide actuators. µPEGA functionalised with
these peptide actuators demonstrate enzyme specific swelling at physiological ionic
strength (Figure 1 -3).
20
Figure 1-3. Enzyme responsive µPEGA through the incorporation of branched peptide
actuators. Schematic of enzyme responsive branched peptide actuator.
The final experimental chapter explores the application of microfluidic devices in

the production of PEGA particles, with the aim of addressing the high polydispersity
of the particles when made by inverse suspension polymerisation (Figure 1 -4).
Figure 1-4. Production of PEGA particles by microfluidic polymerisation. Schematic

representation of microfluidic setup.
21
2 Literature review
2.1. Introduction
This chapter aims to give the reader a good understanding of the literature
predominately from the last ten years in three separate fields: (i) Bioresponsive
hydrogels. (ii) The previous research undertaken on the polymer poly(ethylene
glycol)-co-acrylamide (PEGA), on which most of the experimental work is based.
(iii) The use of microfluidic devices to produce polymer particles.
2.2. Stimuli responsive materials

Stimuli responsive materials are very well documented, 20-25 these materials function
by translating a molecular event into a macroscopic transition. Polymer (chemical)
hydrogels are a particular type of responsive material that offers excellent potential
for applications (e.g. sensing,26 drug delivery27 or tissue engineering28) within the
field of biomaterials.23,29
2.2.1. Chemical hydrogels

Chemical hydrogels are hydrophilic polymers that are highly crosslinked resulting in
insoluble 3-D networks. The first synthetic hydrogels were developed in 1960 by
Wichterle and Lim30 when they produced poly(2-hydroxyethyl methacrylate)
poly(HEMA). They showed that these new hydrophilic networks overcame many
problems associated with polymers that were previously being used in medical
applications. The generation of materials available at that time had poor
biocompatibility and upon implantation caused severe local inflammation. These
chemical hydrogels offered completely new properties due to their high water
content; which was similar to surrounding tissues, gave mechanical properties
comparable to biological materials and allowed the diffusion of metabolites through
the material. At the time this was published Wichterle and Lim suggested that these
polymers could be used to manufacture contact lenses and arteries. 30 Subsequently
there has been great interest in exploiting hydrogels within the field of biomedical
22
applications. Currently the main application of these biomaterials in medicine is as

contact lenses. Indeed, contact lenses are now used by approximately 125 million
people worldwide to augment eye sight.31
Stimuli responsive hydrogels have been shown to respond to numerous stimuli

including pH,32 temperature,33 electric fields,34 ionic strength35 and small
molecules.36,37 It is beyond the scope of this thesis to effectively cover the field of
stimuli responsive hydrogels therefore, further details of these materials can be
found in a number of excellent review articles.21,24,32,38,39
2.3. Bioresponsive Hydrogels1

Bioresponsive hydrogels allow for the potential treatment of diseases by responding
directly to a biological marker for the disease. In the future these systems may
provide more effective medical treatment through highly targeted specific responses.
2.3.1. Introduction
There is a huge range of biological stimuli, these generally fall into two main
categories: small molar mass substances (such as glucose or calcium ions) or
macromolecules (including enzymes and proteins). Through the incorporation of a
moiety capable of recognising a specific biological stimulus and utilising this
recognition event to produce a response it is possible to make a material
‘bioresponsive’. In this thesis the term ‘bioresponsive’ is defined as stimuli
responsive materials that change properties in response to a biological molecular
recognition event.40 These molecular interactions are then translated through
molecular actuation into macroscopic changes in the properties of the material.
This section will focus on different methods used to actuate macroscopic responses
of chemical hydrogels to a biological stimulus. The preparation of these devices will
be covered in detail along with how these systems have been used in the
development of biosensor, drug delivery and tissue scaffold systems. The methods
of actuation within bioresponsive materials can generally be divided into three
1
Short parts of this section have been published by the author in: T.O. McDonald, R.J. Williams,
A.G. Patrick, B.G. Cousins and R.V. Ulijn, Bio-responsive chemically cross-linked and physical
hydrogels. Biomedical applications of electroactive polymer actuators. Wiley. 2009.
23
categories (Figure 2 -5), changes in: (i) cross-linking density, (ii) electrostatic
interactions and (iii) molecular conformation.
Figure 2-5. Schematic representation of the different categories of bioresponsive hydrogels.
2.3.2. Actuation based on changes in crosslinking density

Systems based on actuation through changes in cross-linking density can be
separated into two main categories: covalent crosslinking of polymers by peptides
which are modified by enzymes, or, non-covalent interactions between crosslinking
moieties in which these interactions can be disrupted by competition between free
moieties and those responsible for crosslinking.
2.3.2.1. Systems incorporating peptide crosslinkers

The incorporation of peptide crosslinkers into the hydrogel offers the potential for
enzymes that hydrolyse peptide bonds (proteases) to specifically degrade the
polymer network. There are a number of different approaches by which these
peptides have been integrated into the various polymers to prepare responsive
systems (Table 2 -1).
24
Table 2-1. Selected studies of bioresponsive hydrogels based on peptide crosslinkers.

# Polymer Stimulus Biorecognition Response Ref
41
1 PEG MMP-1 Enzyme hydrolysis of Gel
peptide crosslinks dissolution
42
2 Poly(NIPAM- MMP-13 Enzyme hydrolysis of Gel
co-AAC) peptide crosslinks dissolution
43
3 PEG TG Enzymatic Hydrogel
crosslinking of formation
peptides
11
4 Polyacrylamide Chymotrypsin Enzyme hydrolysis of Hydrogel
peptide crosslinks dissolution
44
5 PEG Factor XIIIa Enzymatic Hydrogel
& MMP crosslinking of formation or
peptides & Enzyme hydrogel
hydrolysis of peptide dissolution
crosslinks
45
6 Poly(HPMA) Calcium ions Enzymatic degradation Release of
of polymer payload
2.3.2.1.1 Enzyme catalysed changes in peptide crosslinker density

The innovative development of systems based on enzyme catalysed changes in
peptide crosslinker density (Table 2 -1, entries 1-5) was by Hubbell and co-
workers.41 In the designing of a cell-responsive hydrogel, in which the hydrated
environment was remodelled by cell-secreted enzymes. The main structural
component of the hydrogel was polyethylene glycol (PEG), offering a hydrophilic
polymer network that resists protein adsorption. Other biological functionalities
were introduced in the form of peptides (Figure 2 -6 A). Four-armed PEG
molecules with vinyl sulfone end groups were first reacted with a low stoichiometric
ratio of mono-cysteine peptide based on RGD (the tripeptide determined to be the
cell-attachment domain in fibronectin).46 This partially derivatised PEG was then
crosslinked by reacting with a bis-cysteine peptide (Ac-GCRD-GPQG↓IWGQ-
DRCG-NH2) (arrow indicates enzyme cleavage site). The flanking of the cysteine
residues with the RD sequence provides water solubility and an optimal pK a for the
formation of cysteine thiolate. This system formed elastic hydrogels under
physiological conditions. Dynamic rheometry (in which the material is subjected to
25
an oscillating strain)47 was used to observe gel formation showing that at higher pH
the gel point occurred more rapidly (Figure 2 -6B).
Figure 2-6. Cell responsive synthetic hydrogels. A:(1) A Michael type addition reaction
between vinyl-sulfone functionalised multiarm PEGs and mono-cysteine adhesion peptides (2)
and bis-cysteine MMP substrate peptides was used to form gels from aqueous solutions in the
presence of cells. These hydrogel networks were designed to respond to local protease activity
at the cell surface (3). B: Dynamic rheometry of a typical gelation reaction, showing the elastic
modulus (G’) and loss modulus (G’’) and the gel point’s sensitivity to pH. C: The swelling and
degradation of the gel network in responsive to incubation with MMP-1. 41
Addition of matrix metalloproteinase (MMP)-1 solution to the gel induced a volume

increase until network dissolution was attained as a result of enzyme catalysed
peptide hydrolysis. Conversely, a hydrogel formed using a peptide crosslinker with a
sequence that the protease does not have specificity for did not show any change in
volume upon MMP-1 treatment (Figure 2 -6 C). MMP sensitive hydrogels allowed
cells to migrate with the hydrogel and in vivo studies showed that extensive
vascularisation only occurred with the MMP sensitive peptide crosslinker.41
26
Furthermore, Healy and co-workers demonstrated a similar system incorporating

protease cleavable crosslinks.42 This system was based NIPAAm and acrylic acid
(AAc) monomers. The peptide crosslinker (QPQG-LAK-NH2) was synthesised
through the acrylation of the amine groups of the lysine residues and the (N-terminal
amine) of glutamine with acryloyl chloride. The resulting hydrogels (by free-radical
polymerisation) were injected through a 2 mm aperture without demonstrating
appreciable macroscopic fracture. Enzyme degradation of the gels occurred when
exposed to MMP-13 with degradation time dependant on cross-linking density. In
this way, degradation times could be modulated, ranging from approximately 100
hours for the lowest crosslinking density up to 300 hours with the highest degree of
crosslinking.42
In another study a system that responded to enzymes by hydrogel formation rather

than degradation was shown by Messersmith and co-workers. 43 The protein
crosslinking enzyme transglutaminase (TG) was used to catalyse reactions between
short peptides conjugated to PEG. This enzyme catalysed an acyl-transfer reaction
between the γ-carboxamide group of protein-bound glutaminyl residues and the ε-
amino group of Lys residues, resulting in the formation of ε-(γ-glutamyl)lysine
isopeptide sidechain bridges. Through the rational design of peptide substrates for
TG rapid formation of a hydrogel was observed when multifunctional PEG
molecules with conjugated peptides were exposed to the enzyme. The incorporation
of the adhesive amino acid L-3,4-dihydroxylphenylalanine (DOPA) was also
demonstrated for the first time with the best substrates for TG crosslinking found to
be DOPA-FKG-NH2 and DOPA-GQQQLG-NH2.43
In 2004 Moore and co-workers published an enzyme responsive hydrogel utilising a

novel conjugation technique.11 Earlier methods based on reacting peptide amine
groups with acryloyl chloride or the Michael addition of cysteines to vinyl sulfones
described by Hubbell had clear limitations. The acrylol chloride reactions lacked
selectivity while Michael additions required a basic residue to be near to the cysteine
(in order to lower the pKa of the thiol giving increased rates of reaction). In the work
presented by Moore and co-workers a scheme that allowed for the selective coupling
of acrylamides to peptide sequences was demonstrated. The disulphide exchange
between an active thiol containing monomer and thiol on a cysteine side chain
27
occurred under acidic conditions and was selective for only thiols. The acidic
environment firstly protonated the pyridyl group providing a good leaving group and
secondly protonated the lysine residues preventing them from performing Michael
addition to the methacrylamide functionality. The activated disulphide monomers
were conjugated to the peptide and these peptide crosslinkers was then
copolymerised with acrylamide by way of UV initiators. With this methodology
poly(acrylamide) hydrogels crosslinked with peptides (CYKC) were prepared that
would dissolve when subjected to chymotrypsin solution. Chymotrypsin solutions
were flowed through microchannels containing hydrogel disks while the sizes of
these disks were monitored. The control sample (containing a peptide for which
chymotrypsin does not have specificity, CSKC) remained unaffected by the enzyme
solution while the test sample containing the enzyme cleavable peptide shrank until
complete dissolution occurred at 20 minutes. 11
More recently, Lutolf and co-workers have further developed the concept of cell
responsive hydrogels with the aim of further mimicking the dynamic remodelling of
the ECM that occurs in vivo.44 This includes the formation of new bonds as well as
degradation. Here, much like Messersmith and co-workers, they made use of a type
of transglutaminase; the crosslinking enzyme factor XIIIa. This enzyme plays a key
role in fibrin clot formation upon tissue damage by catalysing acyl-transfer between
the carboxamide group of a protein-bound glutamine (Q) residue and the ε amino
group in a lysine (K) residue. Multiarm PEG molecules were functionalised with
either Gln in the form of its XIIIa acceptor substrate (NQEQVSPL) or an MMP
cleavable peptide terminated at the C-terminus with Lys. A mixture of these
precursors formed a hydrogel in the presence of factor XIIIa with a gel point time of
about 750 seconds as determined by real-time shear rheometric measurements. The
bioactive binding ligand RGD could also be incorporated in the network by having
the XIIIa acceptor substrate at the C-terminus of RGD. Upon treatment of these
hydrogels with an MMP solution rapid dissolution was observed (within 10 minutes)
dependant on the peptide crosslinker sequence.44 In their most recent publication
they quantitatively incorporated growth factor proteins (via the same XIIIa
crosslinking method) that were released upon cell-derived proteolytic degradation of
the gels.28
28
Messersmith and co-workers again made use of a transglutaminase (TG) enzyme

this time to conjugate peptides to cartilage tissue. Cartilage had previously been
shown to contain a type of TG, tissue transglutaminase (tTG). When cartilage was
incubated with peptides covalently attached to biotin (via a diethylene glycol or PEG
spacer) those that were coupled to the cartilage by the tTG enzyme were detected by
staining with fluorescein anti-biotin antibody. Only peptides to which the enzyme
had specificity (-FKG-NH2 and –GQQQLG-NH2) were found to have coupled to the
cartilage. While peptides in which the asparagine and ornithine residues (analogues
carrying the same charge with different structure) were substituted for glutamine and
lysine respectively were not coupled to the cartilage.48
2.3.2.1.2 Enzyme hydrolysis of hydrogel network

A different approach to using enzymes in a responsive system based on hydrogel
degradation has been described by Kost and co-workers. 49 In that work a calcium
responsive system was prepared through the incorporation of a non-active enzyme
into a polymer matrix consisting of the enzyme’s substrate. The enzyme used was
the starch hydrolysing, α-amylase, while the polymer matrix was a mixture of starch
and hydroxypropyl methyl cellulose ether (HPMC). α-amylase’s are known to
contain at least one atom of calcium firmly and specifically bound to the enzyme
molecule (protein) that is essential for its activity. Therefore, firstly de-activated α-
amylase was prepared by chelating the calcium ions without denaturating the
enzyme. Enzyme containing tablets were then prepared by wet granulation in which
all ingredients were ground, mixed then dried before being pressed into tablets.
Upon incubation of the tablets a concentration dependant release of the model drug
myoglobin was obtained with a constant rate of release over 30 hours.45
2.3.2.2. Non-covalent crosslinking interactions

Systems have also been described based on non-covalent crosslinking interactions.
These typically involve the functionalisation of the monomers with two separate
biological moieties that are able to specifically bind to one another through
molecular recognition. When these functional monomers are copolymerised, the
biological groups non-covalently bind to each other, thereby crosslinking the
polymer. The material therefore responds to the presence of one of the crosslinking
moieties in free solution through a reduction in crosslink density due to the
29
competition between the free moieties and those crosslinking the polymer (Table 2
-2).
Table 2-2. Selected studies of bioresponsive hydrogels based on non-covalent crosslinking

interactions
27
1 Polyacrylamide Antigen Antigen-antibody Reversible
bonding swelling
26
2 Poly(NIPAM- Antigen Biotin-Antibiotin Reversible
co-AAC) binding swelling
50
3 Poly(HPMA) Coiled-coil Self-assembly through Hydrogel
forming coiled-coil formation formation
peptides
An excellent early example of this type of system is the antigen responsive hydrogel
described by Uragami and co-workers.27 In this study a semi-interpenetrating
network (semi-IPN) hydrogel containing both grafted antigens and their
corresponding specific antibody was prepared. Vinyl functionality was introduced to
both of the biological moieties by reacting them with N-succinimidylacrylate (NSA)
(which reacts with the primary amines). The modified goat-anti-rabbit antibody
(GAR) IgG was then copolymerised with acrylamide to give a polymer solution.
Vinyl rabbit IgG (antigen) was mixed with the GAR IgG polymer solution,
acrylamide, a crosslinker N,N’-methylenebisacrylamide (MBAA) and polymerised
with free-radical redox initiators (Figure 2 -7 A). Antigen-antibody binding then
caused the formation of further cross-links within the polymer reducing the swelling
(Figure 2 -7 B). The presence of free antigens in the solution around the hydrogel
created competition between the grafted antigens leading to a decrease in cross-
linking density, thus an increase in swelling of the hydrogel (Figure 2 -7 C). This
increase in swelling was reversible. By removing the hydrogel from the antigen
solution and washing, the polymer would return to approximately its original
volume. This response also corresponds to an increase in permeability of the
polymer and by using the polymer as a membrane, selective permeation of a protein
was shown in response to the specific antibody (Figure 2 -7 D). More recently a
similar design was employed to produce an antigen-responsive membrane that has
30
gating properties (allow for the opening of closing of pores) for selective diffusion
in response to the presence of a free antigen.51
Figure 2-7. Antigen responsive hydrogel. A: Synthesis of the antigen-antibody semi-IPN

hydrogel. B: Diagram of a suggested mechanism for the swelling of an antigen-antibody semi-
IPN hydrogel in response to a free antigen. C: Antigen recognition by antigen-antibody semi-
IPN hydrogel. D: Reversible swelling changes and antigen-responsive permeation profiles. 27
26
Lyon and co-workers have demonstrated an antigen responsive hydrogel, this
system consists of a co-polymer of pNIPAAm and acrylic acid with N,N’-
methylenebisacrylamide as the crosslinker prepared by free radical initiated
precipitation polymerisation. In this process the monomer(s) and initiator are soluble
in the solvent and initiation takes place in solution. The polymer chains then grow
until they reach a critical chain length where they exceed their solubility and
precipitate from solution.52 Lyon and co-workers covalently incorporated a biotin
moiety into the microgels by coupling biotin hydrazide to the carboxyl group of the
acrylic acid using a water-soluble carbodiimide (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide). Aminobenzophenone (ABP) was then also
coupled to the polymer using N,N’-dicyclohexylcarbodiimide in dimethyl sulfoxide
(DMSO). Glass coverslips functionalised with the cationic silane 3-
aminopropyltrimethoxysilane (APTMS) were then placed in an aqueous solution of
biotin/ABP functionalised polymer particles which strongly attached to the glass
surface via Coulombic interactions. These ‘microlens’ covered surfaces were then
31
exposed to a solution of anti-biotin (antibody), leading to antigen-antibody binding

which was followed by photoligation of the antibody to the ABP. This treatment
effectively led to a crosslinking of the surface of the microlenses by antigen-
antibody binding. Exposure of these microlenses to biocytin (antigen) led to
competition in binding with anti-biotin resulting in a decrease in the crosslinking
density of the surface of the microlenses (Figure 2 -8 A). This resulted in an
increase in swelling of the polymer and a corresponding decrease in the microlens
focussing power. This was determined using brightfield transmission and differential
interference contrast (DIC) optical microscopy (Figure 2 -8 B). The response rate of
the hydrogel microlenses were found to be strongly coupled to analyte
concentration.26
Figure 2-8. Displacement-Induced Switching Rates of Bioresponsive Hydrogel Microlenses. A:

The microgels are functionalized with biotin and ABP (top left), are cross-linked by anti-biotin
(top centre) and exposed to a solution of biocytin (top right). B: Response of hydrogel
microlenses to incubation with anti-biotin antibodies. Microscopic images of hydrogel
microlenses (left element in each image) and biotin/ABP functionalized hydrogel microlenses
(right element in each image) are shown, (a) before and (b) after incubation with anti-biotin.
The scale bar is 2 µm.26
Kopeček and co-workers describe a hybrid hydrogel (a hydrogel system consisting
of at least two distinct classes of molecules connected either covalently or non-
covalently) in which the crosslinking was achieved through the self-assembly of
32
coiled-coil forming peptides (Figure 2 -9 A). These peptides were grafted onto a
polymer backbone of N-(2-hydroxypropyl)methacrylamide (HPMA). Coiled-coils
are a native protein conformation that consists of two or more α-helices wound
together to form a super-helix.53 The attraction of incorporating these types of
peptides into a polymer is that the association and disassociation of these coiled-
coils is determined by the primary structure of the amino acid sequence. In this
system polymerisable functionality was imparted into the peptides by capping the N-
termini of the peptide with N-methacyloylglycyl-glycyltryptophan. This offered the
same functionality as HPMA ensuring the compatibility of the comonomers in free
radical copolymerisation. Examination of the self-assembly of these copolymers into
hydrogels was achieved using microrheology and dynamic light scattering. Dynamic
light scattering evaluated the size of nanoparticles formed as association of coiled-
coil forming peptides led to gelation. Analysis of the hydrodynamic radius, Rh
showed that particle size was independent of graft copolymer concentration at low
concentrations but increased dramatically as the concentration was increased above
5.52 mg/ml (Figure 2 -9 B). These results indicated that the self-assembly process
was strongly governed by polymer concentration. Temperature also had an effect on
the self-assembly process, as temperature was increased (Figure 2 -9 C) there is a
gradual increase in the Rh. The increased thermal energy of the system led to an
increase in the probably of a collision of the peptide grafts making the self-assembly
process more effective at higher temperature.50 This system demonstrates that
coiled-coil interactions can be used to induce the self-assembly of polymers, this
offers the potential to use this interaction to crosslink hydrogels and potentially
screen for coiled-coil forming peptides.
33
Figure 2-9. Novel synthesis of HPMA copolymers containing peptide grafts and their self
assembly into hybrid hydrogels. A: the self-assembly of copolymers into hydrogels. B:
Concentration dependence of the hydrodynamic radii Rh of polymer clusters. C: Temperature
dependence of the hydrodynamic radii Rh of polymer clusters.50
2.3.3. Actuation based on electrostatic interactions

A second mechanism to actuate a change in swelling is through electrostatic
repulsion/attraction within the polymeric network. These charges are typically
induced on the polymer chain but it has also been demonstrated that grafting specific
actuators on to the polymer is possible ().
34
Table 2-3. Selected studies of bioresponsive hydrogels based on electrostatic interactions

36
1 Poly(HEMA-co- Glucose Conversion of glucose Increased
DMAEMA) to gluconic acid swelling
54,55
2 Poly(PEGMA-co- Glucose Conversion of glucose Increased
DEAEMA) to gluconic acid swelling
56
3 Poly(AAC) grafted Glucose Conversion of glucose Increased
onto porous to gluconic acid permeabilit
membrane of PVDF y of
membrane
57,58
4 PEGA Protease Enzymatic hydrolysis Increased
of peptide actuators swelling
2.3.3.1. Electrostatic interactions between charges on the polymer network

Actuation based on electrostatic interactions within the polymer network is widely
studied in the context of glucose responsive polymers for the treatment of
diabetes.36,54-56,59-61 Generally glucose oxidase (GOx) is immobilised in the hydrogel,
when glucose is present it is converted to gluconic acid, which lowers the pH within
the microenvironment of the hydrogel. There are two different macroscopic designs
in which this decrease in pH is used to actuate a change in swelling: matrix type
systems where the enzyme and insulin are contained within a bulk polymer, or
membrane type systems where the drug is contained in a reservoir within a
membrane. Kost and co-workers produced an example of the matrix system where
the insulin and enzyme were contained uniformly throughout a hydrogel. The
hydrogel was made from 2-hydroxyethyl methacrylate (HEMA), N,N-
dimethylaminoethyl methacrylate (DMAEMA) with tetraethylene glycol
dimethacrylate (TEGDMA) as a crosslinking agent. The presence of amines and the
low crosslinker concentration (between 0-0.95 %) meant that at low pH the amines
become ionised leading to an increase in swelling (schematic shown in Figure 2
-11).
Figure 2-10. The enzymes and the reactions they catalyse used in glucose responsive hydrogels.
35
GOx, catalase and insulin were incorporated into the hydrogel during the
polymerisation step (free-radical initiation at room temperature). Figure 2 -10
shows the reactions driven by these enzymes, GOx catalyses the reaction of glucose
to gluconic acid forming hydrogen peroxide. A build up of hydrogen peroxide leads
to inhibition of the enzyme, and because oxygen is needed to form gluconic acid a
shortage of oxygen leads to slower swelling rates. For this reason catalase was also
incorporated into this system which serves to convert hydrogen peroxide to water
and oxygen. The effect of crosslinker concentration on the responsive swelling
behaviour was investigated and it was found that polymers prepared without
crosslinker led to the greatest increase in swelling. These polymers did not dissolve
in water even over prolonged periods, Kost and co-workers suggest that this stability
may be due to entanglements and non-covalent interactions between the polymer
chains. Due to the dynamic nature of conditions in vivo non-steady state experiments
investigating swelling changes in response to glucose were examined. The glucose
concentration was switched between a hyperglycaemic blood glucose concentration
and a normal blood glucose concentration, in these experiments they found that
deswelling occurred more rapidly than a further increase in swelling. Analysis of
glucose triggered release of insulin in matrices with varying crosslinker
concentration also showed that the shortest response time and the greatest amount of
insulin was released in matrices without crosslinking agent. The device was
implanted in rats and these in vivo experiments indicated that some of the entrapped
insulin retained its active form and was effective in reducing blood glucose levels.
Additionally, over the 2-3 weeks the matrices were implanted no fibrotic
encapsulation was observed demonstrating the biocompatibility of the devices. 36
36
Figure 2-11. Characterization of glucose-sensitive insulin release systems in simulated in vivo

conditions. Schematic presentation of a matrix system based on poly(HEMA-co-DMAEMA).
(top) unswollen matrix at time t=0, (bottom) swollen matrix.
Peppas and co-workers have previously developed a similar system consisting a co-
polymer of diethylaminoethyl methacrylate (DEAEMA) and poly(ethylene glycol)
monomethacylate (PEGMA) using tetra(ethylene glycol) dimethacryalte (TEGMA)
as the crosslinker. GOx and catalase were given vinyl functionality by reacting with
acryloyl chloride and mixed with the monomer solution prior to UV initiated
polymerisation to give hydrogel films. With this system they demonstrated pulsatile
pH-responsive swelling, but did not show glucose-responsive behaviour. 54 Within
their following publication microparticles with the same chemistry were produced
by inverse suspension polymerisation with redox-initiators. These microparticles
demonstrated rapid swelling/deswelling dynamics in response to changes in pH and
it was determined that faster responses could be obtained from smaller particles.55
37
A membrane type system for insulin release was developed by Liang and co-
workers. Here, poly(acrylic acid) (PAAc) was grafted to a porous membrane of
polyvinylidene fluoride (PVDF), GOx was covalently bound to the PAAc by firstly
activating the carboxyl groups with a water soluble carbodiimide then immersing the
membrane in a aqueous solution of GOx. The PAAc with GOx covalently
immobilised effectively ‘gate’ the pores of the PVDF. At neutral pH and when there
is no glucose in the surrounding environment the pores within the membrane are
‘closed’. When the carboxyl groups present in the PAAc chains are dissociated and
negatively charged, these charges along the polymer chains electrostatically repel
one another forcing the chain to lengthen and extend. When glucose was present it
was oxidised into gluconic acid by the immobilised GOx, this led to a reduction of
pH in the microenvironment of the pores protonating the carboxylate groups on the
grafted PAAc chains. The gates then ‘opened’ because the PAAc chains were
collapsed on the removal of the electrostatic repulsion allowing the insulin to diffuse
out of the membrane. The grafting density of PAAc was varied to find the ideal
value for insulin release, at low values it was found that the PAAc chains were too
short/sparse to effectively close the pores. At higher densities the PAAc became too
long/dense and it was no longer possible for a conformation change to occur. Using
a grafting yield of 1.55% the insulin permeation coefficient after glucose addition
was 9.37 times greater than without glucose.56
Recently there have been fewer publications in which GOx has been utilised to
actuate insulin release (50 % decrease from 2003), it appears that this method has
reached it limits and recent approaches by researchers are based on effective oral
delivery of insulin rather than the development of glucose responsive polymers. An
obvious limitation within GOx hydrogel systems is that the treatment of diabetes is a
continuous long term process; any implanted devices must be able to contain large
quantities of insulin for release over this time frame in addition to maintaining their
dynamic response.
2.3.3.2. Electrostatic interactions between charges present in pendant actuators

The design of actuators in which the molecular actuation involves a change in the
net charge offers the potential to develop more intricate responsive systems. Current
38
approaches utilise enzyme cleavable peptides as the sensing element. Thornton et al

described the development of a functionalised hydrogel that alters its accessibility in
response to an enzyme with a selected specificity. 57,58,62 This was achieved through
modification of poly(ethylene glycol)-co-acrylamide (PEGA) particles by solid
phase peptide synthesis with peptide actuators. Earlier work highlighted tri-peptides
consisting of an enzyme cleavable (di)peptide (ECP) and the charged amino acid
arginine. The presence of these charged groups within the polymer led to
electrostatically induced swelling. Upon cleavage of the ECP by an enzyme with
matching specificity these cationic groups were removed resulting in a decrease in
swelling. The highly hydrated and crosslinked structure of PEGA makes the interior
of the polymer network accessible to macromolecules. The maximum size
(molecular weight) of macromolecule that can diffuse into PEGA is well defined
and termed the molecular cut-off weight. Above this molecular weight molecules are
unable to diffuse into the interior of the polymer.63 An increase in swelling caused a
corresponding increase in this cut-off weight. Through the use of different molecular
weight Fluorescein isothiocyanate (FITC) labelled dextrans it was possible to
monitor the changes in polymer accessibility using two-photon microscopy (this
technique is described in section 8.1.2). If a dextran with a molecular weight greater
than the cut-off for the polymer could diffuse into the interior of the particles it
indicated there was an increase in accessibility (due to charge induced swelling). It
was found that enzymes with the correct specificity for the ECP (thus removing the
charged groups) led to a decrease in accessibility and diameter of the particles. 62
Thornton et al went on to develop this system of hydrogel particles functionalised
with peptides for the controlled release of entrapped payloads. Here, the peptide
actuator consisted of two opposing charged amino acids separated by an uncharged
ECP, corresponding to the sequence Fmoc-D-(ECP)-R-PEGA (Figure 2 -12 A).
This zwitterionic peptide had a net neutral charge but upon enzyme cleavage of the
ECP only the cationic amino acid arginyl and half the ECP remained covalently
attached to the hydrogel. With pKa of the amino group being 7.5, at pH values below
this the magnitude of response was greater due to the ionisation of amine group.
This enzyme responsive increase in swelling (a maximum increase in volume of 100
%) corresponded to increase in accessibility which was used to release an entrapped
payload (Figure 2 -12 B). Approximately 50 % of the payload being released in 30
minutes in response to an enzyme with the correct specificity. 57 This type of release
39
system has a number of advantages over other release methods: the payload
molecule does not need to be covalently modified, enzymatic cleavage of the ECP
results in a tunable number of payload molecules being released and the payload
loading method is relatively mild (a pH switch). In a recent publication 58 Thornton
et al modified the system to release proteins with different charges at physiological
pH (avidin pI= 10.0 and albumin pI= 4.7). This was achieved by tailoring the design
of the peptide actuator to give a net charge after ECP cleavage that was matched to
the charge on the protein. It was possible to release the positivity charged avidin
with the conventional peptide actuator (Fmoc-D-(ECP)-R-PEGA) due to the
electrostatic repulsion between the actuators and the protein as observed by two-
photon microscopy (Figure 2 -13). In order to release albumin a new actuator was
designed (Fmoc-R-R-(ECP)-D-D-PEGA), again, this actuator had a net overall
neutral charge but upon enzyme specific hydrolysis of the ECP a net negative charge
remained coupled to the polymer (two negative carboxyl groups on the aspartic acid
one positive amine group). This allowed for release of the payload (albumin) due to
electrostatic repulsion. Analysis of the proteins released from the particles showed
that although some proteolysis did occur it was not a major concern. Another
limitation of this approach was the release of Fmoc-peptide fragments upon
enzymatic hydrolysis.
40
Figure 2-12. Enzyme-responsive hydrogel particles for the controlled release of proteins:
Designing peptide actuators to match payload. A: The peptide designed for the release of
positively charged proteins was comprised of Fmoc–D–AA–R, where the amide bond between
the two alanine residues is particularly liable to cleavage by our target enzyme. B: Generation
of positive charges by enzymatic cleavage of the bond between alanine residues allows protein
molecules to diffuse through the polymer pores for payload release. 58
However, the main limitation of this concept was its inability to respond at
physiological ionic strength. When, counter-ions present in the solution screened
the electrostatic interactions between the peptide actuators, this led to an
insignificant release rate.58
Figure 2-13. Peptide actuator designed for the release of negatively charged protein molecules.
A: Two N-terminal arginine units are separated from two aspartic acid groups by two alanine
residues. A net negative charge remains on the particle following enzymatic hydrolysis. B:
Exclusion of albumin from the negatively charged swollen particle occurs following hydrolysis
of the bond between alanine residues.58
2.3.4. Actuation based on conformational changes

The third mechanism of actuation is based on changes in conformation of natural
proteins. These systems incorporate a natural protein into the hydrogel that
undergoes a conformation change and thus alters the characteristics of the material.
41
The use of proteins as actuators is a new development in bioresponsive hydrogels. 64

65
An example of this system was developed by Mrksich and co-workers . In that
study the functional nature of the hydrogel was conferred to the system through the
introduction of the protein calmodulin (CaM). CaM is a 16.5-kDa protein with two
distinct conformational states. In the presence of calcium ions, CaM has an
extended, dumbbell-shaped conformation where the distance between the ends of the
protein is approximately 50 Å (extended CaM). This calcium-bound extended CaM
undergoes a transition from an extended dumbbell to a collapsed conformation
(collapsed CaM) upon the binding of ligands (with a distance between the protein
ends of approximately 15 Å). An engineered version of the CaM protein was
prepared in which the tyrosine residues at the ends of protein are replaced with
cysteine residues. This modification meant it was possible to selectively react the
acrylate end groups on the four-armed PEG molecules to the protein through a
Michael-type addition. This formed a water soluble conjugate. The success of this
reaction was shown by MALDI-TOF MS. A hydrogel was then formed by mixing
the conjugate with dithiothreitol (DTT) at room temperature crosslinking the
remaining acrylate groups to give a solid hydrogel. The hydrogel showed a
macroscopic decrease in volume when exposed to the trifluoperazine ligand (TFP).
TFP binds specifically to CaM, causing the CaM to undergo a conformation change
from extended to collapsed (Figure 2 -14 A & B). This decrease in volume could be
reversed by chelating the calcium ions thus removing the calcium bound ligand.
Numerous cycles between and extended and collapsed material were possible
demonstrating the reversible nature of the hydrogel (Figure 2 -14 C). More recently
this concept has been developed to incorporate a photochemical assembly allowing
spatial control of the location of the dynamic proteins.66
42
Figure 2-14. Ligand responsive hydrogel that relies on conformational changes. A: The two
conformational states of CaM, an extended conformation in the presence of calcium ions (left),
and a collapsed conformation upon binding to a ligand (right). B: A hydrogel with CaM in a
ligand-free state (left) and the same gel with CaM in a ligand-bound state (right) (scale bars: 1
mm). C: Hydrogels were exposed to TFP ligand, and the volume was measured at various
intervals for 2 h. The gel was then washed repeatedly and incubated in a calcium-containing
buffer to restore the extended CaM conformation. 65
43
2.3.5. Summary
Bioresponsive hydrogels are a relatively new area of research but there have already
been a range of different successful approaches to the design of these materials.
These systems present methods of: detecting biological compounds, controlling cell
modelling of scaffolds and the targeted/controlled release of active agents for
disease specific treatment. However, there are two main obstacles that need to be
overcome for many of these systems to provide actual medical usage: Few of these
systems offer true reversible responses, rather than one thermodynamically favoured
direction. Secondly, the high degrees of complexity within some of these materials
makes acquiring approval to use within the body difficult as it is essential to
understand what happens to all compounds once within the body.40 That said, the
developments within this field help outline the design rules for future researchers to
continue to progress and refine the concepts. In the future, research built upon this
groundwork should help to provide more effective treatments within the medical
field.
44
2.4. PEGA
2.4.1. The history of PEGA

In 1992, Meldal developed PEGA, a copolymer of poly(ethylene glycol) (PEG) and
acrylamide (Figure 2 -15).67 This material was a highly polar solid support for solid
phase peptide synthesis particular continuous flow peptide synthesis. PEGA offered
a number advantageous properties: It was transparent with no absorbance in the
aromatic region allowing for easy spectrophotometric monitoring of the reaction
process. It had a highly branched polymer network with swelling in both organic and
polar solvents. Finally, the resin was highly polar assisting peptide solvation. 67 This
section covers developments within the field of PEGA detailing methods and
techniques from the literature that have arisen since the first publication of the
material.
Figure 2-15. Chemical structure of PEGA.
PEGA provided an important new property to the field of solid phase peptide
synthesis (SPPS)68 in its compatibility with both organic and aqueous solvents. This
property allowed peptides to be incorporated onto the polymer which could then be
45
placed in an aqueous environment. The open structure of the polymer allowed

enzymes to diffuse into the interior of the polymer particles where they catalysed
reactions.69,70 This development inspired a number of publications in which
techniques used in SPPS such as split and mix synthesis were exploited to prepare
libraries of peptides. These peptide libraries were then exposed to selected enzymes
and their effect determined. An early example of this approach was demonstrated by
Meldal and co-workers. Here, they prepared a ‘one bead, two compounds’ (Figure 2
-16) library to screen for inhibitors for proteolytic enzymes that are essential for
parasite development. Two different peptides were incorporated into individual
particles (referred to as beads within SPPS literature) by firstly temporarily
protecting a fraction of the amine groups in the PEGA particles with
hydroxymethylbenzoic acid, while using the remaining amines to synthesise the
fluorescence quenched peptide substrate (FRET). These substrates allow for the
detection of enzyme action; upon hydrolysis of the substrate the fluorescence
quencher is cleaved resulting in a fluorescent molecule. The hydroxyl function on
the PEGA particles was then esterificated and a second peptide (to screen for
inhibition) prepared by split synthesis from the ester bond to the hydroxymethyl
benzamide. These particles were then exposed to the enzyme solution (cruzipain)
and hydrolysis of the fluorescence quenched peptide substrate resulted in highly
fluorescent particles. The fluorescence was only seen on the surface of the particles
indicating that the 57 kDa enzyme was not able to diffuse inside into the polymer
network. The darkest particles were manually collected and the peptides sequenced
by Edman degradation-gas phase sequencing. This method yielded a first generation
of effective cruzipain inhibitors.70
46
Figure 2-16. Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor

library assay. D-amino acid inhibitors for cruzipain, cathepsin B and cathepsin L. The strategy
used in the synthesis of the ‘one bead, two compounds’ libraries. 70
The mesh size of the polymer matrix in PEGA is controlled by the length (molecular
weight) of the PEG chains cross-linking the polymer. Most earlier work on had been
carried out on PEGA1900 (subscript refers to the molecular weight of the PEG in
g/mol), it was found that enzymes up to 50 kDa could diffuse into the polymer
network.69,71 Meldal and co-workers then went on to address this limitation of the
earlier PEGA resin by preparing PEGA cross-linked with PEG with molecular
weights of 4000, 6000, 8000. Libraries of fluorescence quenched peptide substrates
(FRET) (described in more detail in section 8.1.3) were then prepared on these
resins and incubated with the enzyme, MMP-9 which has active forms of 67-83
kDa. Particles that appeared bright were manually isolated and sequenced to identify
substrates for MMP-9, effectively identifying MMP-9 substrates. 72 This method of
fluorescence-quenched peptide substrates was later used to screen for substrates the
cysteine protease, Papain. Here, the limited loading of the PEGA 4000 was doubled
through the incorporation of a K-K dipeptide as the first functionalisation step on the
resin.73
Interest in utilising PEGA in the preparation of peptide libraries for screening with
enzymes soon increased. This led to a drive to further understand the polymer’s
structure/property relationship with enzyme catalysed reactions. Bradley and co-
workers63 presented a paper in which confocal Raman microscopy was used to
47
investigate enzyme accessibility. Three different supports for SPPS; TentaGel,

PEGA1900 and controlled pore glasses (CPGs) were functionalised with a peptide
substrate terminated with 4-cyanobenzamide and incubated with five different
enzymes with a range of molecular weights. The library of particles were then
investigated by confocal Raman microscopy to ascertain if the peptide sequence was
hydrolysed by monitoring the stretching frequency of the cyano group. TentaGel
was not accessible to any of the enzymes, indeed, there was no detectable effect on
the peptide with even the smallest enzyme (MMP-12, 22 kDa). PEGA 1900 displayed
peptide cleavage with all enzymes below 35 kDa but not enzymes above 42.5 kDa.
Finally, beaded CPG showed complete cleavage of the peptide for all enzymes
tested. This was because the pores in the CPG used in the study were 100 nm in
diameter which was much larger than the hydrodynamic radius of the enzymes. This
study provided a well defined accessibility for PEGA to enzymes from which the
research community to work.63
An alternative approach to that used by Meldal and co-workers to increase the

accessibility of PEGA was described by the groups of Flitsch and Gardossi in a
series of papers.74-77 Here, rather than increasing the length of the cross-linking PEG
chains they substituted some of the acrylamide in the polymerisation mixture with a
permanently charged acrylamide based monomer. These PEGA+ and PEGA-
particles demonstrated greater swelling than the neutral PEGA 1900, although as the
ionic strength of aqueous solution was increased the swelling of the charged PEGAs
reduced to the same value as that of PEGA1900. The swelling behaviour of the
charged polymers was due to the electrostatic repulsion between adjacent polymer
chains, this resulted in a increase in pore size as determined by an increase in
enzyme accessibility. The substrate for the enzyme used (penicillin G amidase,
PGA, 88 kDa) was N-phenylacetylated L-Phe, and was only cleaved to low
conversions (10 %) when coupled onto PEGA1900 and PEGA- however, for PEGA+
much greater conversions of 50 % were observed. 74 In their following paper these
electrostatic effects were further investigated with hydrolytic yields as high as 80 %
being obtained by increasing the amount of positive charges in the polymer network.
These electrostatically attracted the negatively charged enzyme (PGA, pI = 5.2-5.4)
thus favouring the accessibility of the bulky enzyme.75 Additionally, Gardossi and
co-workers showed that PEGA+ retained the most enzyme within the particles and
48
that PEGA- retained the least.77 In a final publication on this topic, they made use of
the improved yields of PGA catalysed reaction on PEGA + to demonstrate a
hydrazide enzyme cleavable linker.76 Overall, the introduction of permanent charges
to the polymer network offers an interesting method for the adjustment of the
polymers properties and interaction with surrounding proteins.
Another analytical technique was introduced into the area of enzyme catalysed
reactions on PEGA in 2003 by Flitsch and co-workers in the form of two photon
microscopy (TPM) (described in section 8.1.2).78 This technique allows the
production of images detailing the spatial resolution of fluorophores within polymer
particles. PEGA1900 was functionalised with Fmoc-F-F and exposed to the protease
thermolysin for different lengths of time. Where hydrolysis had occurred free amine
groups were present, these were chemically acylated with dansyl chloride (Figure 2
-17 A). These areas then showed as fluorescent, revealing where enzyme hydrolysis
has occurred. Thermolysin treated particles initially displayed a bright ring on the
outside of particle, which then expanded into the particle until after approximately
45 minutes when the whole particle was fluorescent (Figure 2 -17 B). This indicated
that the enzymatic action was limited by diffusion of the enzyme into in the polymer
particle. Indeed, it was determined that for the enzyme to diffuse the distance from
the outside of the particle to the centre (100 µm) in an aqueous environment would
take approximately one minute.79 The use of TPM presented an effective method to
determine spatial and temporal resolution of enzyme hydrolysis within polymer
particles.
Figure 2-17. Using two photon microscopy to quantify enzymatic reaction rates on polymer
beads. A: Thermolysin catalysed hydrolysis of solid supported Fmoc–Phe–Phe. B: Thermolysin
catalysed hydrolysis of PEGA1900 bound dipeptide 1 as examined by TPM. From left to right the
images represent 5, 10, 20, 45, 60, 90, 120 and 240 min.
49
In 2003 a new development in the use of combinatorial libraries on PEGA was

developed. With the establishment of large libraries of PEGA-bound peptide for
screening, a faster and effective method was needed to separate particles with ‘hits’
from particles that were ‘non-hits’. Previously, this process had been carried out
manually on PEGA. Meldal and co-workers prepared a ‘one bead, two compound’
combinatorial library; one compound consisted of a fluorescence quenched peptide
substrate (FRET) while the second compound was a randomly synthesised peptide
(by split and mix synthesis) to screen for inhibition of their test enzymes,
metalloproteinases (MMPs). These two compounds then competed for binding with
the same enzyme and if the FRET substrate was not cleaved this indicated inhibition
of the enzyme by the library compound. Once incubated with the enzyme the
particles were analysed with an instrument developed originally developed for high
throughput screening and sorting of different transgenic-fluorescent tagged
organisms. It was further adapted for the purpose of sorting labelled particles. This
device allowed ‘hits’, dark particles, to be separated from ‘non-hits’ in a very fast
and reliable manner. Using this approach ten dark particles were selected, sequenced
and resynthesised to provide very potent inhibitor activity towards a number of
MMPs.80
In 2006 Ulijn and co-workers described a different morphology of PEGA in the

form a micropatterned PEGA surface.81 Within this work three different techniques
were utilised to prepare a micropatterned surface: photolithography, capillary force
lithography (using a patterned stamp) and spotting with a manual microarray (Figure
2 -18). These patterned surfaces maintained the same chemistry as PEGA particles
allowing for functionalisation with peptides by SPPS. Using this methodology
PEGA surfaces were prepared that could direct cell adhesion (using the peptide
RGD) or screen for protease specificity with FRET peptide substrates.81
50
Figure 2-18. A micropatterned hydrogel platform for chemical Synthesis and biological
analysis. Optical micrographs of fibroblasts cultured in serum-containing media for 48 h on
(functionalized) PEGA-patterned surfaces. (a) Unmodified PEGA surface; fibroblast cells
strictly adhere and spread only on the glass surroundings, and not to the PEGA surface. (b)
RGE-modified control surface that resists cell adhesion. (c) RGD-modified PEGA surface that
promotes cell adhesion.81
A recent development comes from the Halling and Flitsch groups. 82 Within this
work they made use of TPM to demonstrate real-time spatially resolved
measurement of enzyme activity on polymer particles. Aminocoumarin-carboxylic
acid (a fluorescent derivative used extensively in biological assays) was coupled
onto PEGA1900 via a hexa-glycine linker. A peptide or amino acid was then coupled
to the aminocumarin resulting in quenching of its fluorescence (Figure 2 -19 A).
When Bz-R-OH was coupled onto the aminocumarin and these particles were
treated with trypsin fluorescence appeared as an annular ring around the outside of
the particle which then gradually progressed inwards. This indicated that enzyme
hydrolysis was initially confined to the outside of the particles gradually diffusing
towards the centre of the particle (Figure 2 -19 B). This was in agreement with the
non-real-time studies using TPM that were previously carried out by Flitsch and co-
51
workers.79 Indeed, it took over three hours for the fluorescence values of the centre
of the particle to match those of the outside, this delay was likely a result of the
electrostatic repulsion between the positively charged trypsin (pI = 8.69) and the
positively charged arginine covalently attached to the aminocumarin. When a
different enzyme (Subtilisin Carlsberg) was incubated with the functionalised
particles (Bz-R-OH was replaced with Z-G-G-L-OH) different behaviour was
observed; there were no defined rings noted and increase in fluorescence upon
enzyme action was relatively homogeneous across the particles (Figure 2 -19 C).
This was presumably due to the smaller size of the Subtilisin Carlsberg (although
this is not detailed within the paper) resulting in fast enzyme diffusion that was not
rate limiting. In summary, TPM provides a useful tool for the real-time analyse of
enzyme hydrolysis although it not shown whether the aminocumarin itself effects
enzyme rate or hydrolysis yield.82
Figure 2-19. Real-time imaging of protease action on substrates covalently immobilised to

polymer supports. A: Schematic of coupling and enzymatic reaction processes on PEGA 1900
particles. B & C: Two photon cross-section images of PEGA1900 particles treated with B; trypsin
and C; Subtilisin Carlsberg respectively.
2.4.2. Enzyme catalysed synthesis on PEGA

A different area of research on PEGA was the synthesis of peptides on solid support
by enzymatic means, the first example of this was published by Flitsch and co-
workers.83 In this research, PEGA particles functionalised with phenylalanine were
52
prepared using solid phase methodology, enzymatically catalysed amide bond

formation then occurred upon treatment of these particles with an excess of amino
acids (with their amines protected with Fmoc) in the presence of thermolysin. High
yields were found for more hydrophobic amino acids while more polar residues led
to lower yields. The authors suggested that amide formation was driven by three
main factors: the large excess of substrates, the removal of ionisation (amines)
within the solid support and the improved solvation of the hydrophobic (Fmoc
protected) acyl donors within the PEGA. This publication provided a method for
enzymatic synthesis of peptides allowing for high enantioselectivity and without the
need of side chain protection that is required in conventional chemical synthesis. 83
Within a following publication Flitsch et al84 went on to develop this method to
obtain L,L and L,D diastereoisomers of dipeptides and L-amino acids in good yields
starting from enantiomeric mixtures of amino acids using the enantioselectivity of
the enzyme (thermolysin) catalysed reactions.84 Recently, Flitsch et al85 investigated
the main factors controlling the enzymatic synthesis of peptides. From the three
main factors described in their first publication it was determined that reduction in
the unfavourable hydrophobic hydration of the Fmoc group within the solid support
compared with the free amino acid in solution was the most important driving force
in the enzyme catalysed synthesis.85
A separate publication was also produced documenting enzymatic synthesis on

PEGA by another group of researchers. Burkart and co-workers published
describing a biomimetic approach to the synthesis of a cyclic peptide antibiotic, 86 a
scheme is shown in Figure 2 -20 A. A peptidic linkage was first synthesised on the
resin by traditional chemical means, this was structurally homologous to the tether
found naturally (phosphotpanteheine, PCP). A linear decapeptide, the substrate for
the enzymatic cyclisation was constructed from the linker (via an ester bond) via
conventional SPPS. Incubation of these functionalised PEGA particles with TycC
TE (the isolated C-terminal thioesterase domain excised from the larger synthetase
protein found naturally) resulted in the release of the cyclised product (tyrocidine A,
a cationic peptide antimicrobial). By employing SPPS to produce a library of
decapeptides in which the fourth amino acid (D-phenylalanine) was substituted for
either natural or non-natural amino acids an insight of the enzymology of TycC TE
was obtained (Figure 2 -20 B). Furthermore, the effective substrates for the
53
enzymatic cyclisation were investigated as artificial analogies of the cyclic peptide

antibiotic.86 This paper demonstrated the enzymatic synthesis of cyclic peptides on
solid supports (PEGA) and how this methodology may be exploited to investigate
the specificity of the enzyme. This allowed for the production chemical analogies of
the peptide offering the possibility of finding peptides with greater therapeutic
effect.
Figure 2-20. Biomimetic synthesis and optimization of cyclic peptide antibiotics. A: Natural
versus biomimetic macrocycle synthesis. The enzymatic assembly line involved in biosynthesis
of the cyclic cationic antimicrobial peptide, tyrocidine A. A carrier protein (PCP) in each
module is loaded with a phosphopantetheine prosthetic group (red). The individual modules
contain domains that load amino-acid building blocks onto the thioester tether and condense
via successive peptide bond giving the terminal PCP domain loaded with a linear decapeptide.
The terminal thioesterase domain (TE) catalyses head-to-tail cyclisation. In the biomimetic
synthetic strategy, a linker (red) that mimics phosphopantetheine was chemically synthesized
onto a solid-phase resin. Solid-phase peptide synthesis is used to construct a tethered linear
peptide, which can then serve as a substrate for cyclisation by the TE domain excised from the
synthetase proteins. B: Addition of the TE catalyses formation of the cyclisation product or the
hydrolysis product.
2.4.3. Summary
PEGA, a material initially developed as a new resin for use in continuous SPPS,
offers a number of unique properties that make it highly suitable for the preparation
54
of peptides. These peptides covalently attached to solid supports can then be

exposed to enzymes in aqueous solutions. This concept has been used to screen for
protease inhibitors and determine protease specificity. Effective methods have been
shown that have allowed enzyme activity within the hydrated polymer to be better
understood, as well as providing useful tools for future researchers. Additionally,
PEGA polymers have been created that contain permanent charges that give
increased yields for reactions catalysed by enzymes with the opposing charge.
Protease responsive PEGA particles have been demonstrate through the
incorporation of charged peptide actuators (described in detail in section 2.3.3.2)
Finally, PEGA has been used to demonstrate enzymatic synthesis of peptides on
solid supports and employed to use a biomimetic approach for the partially
enzymatic synthesis of cyclic peptides allowing for the screening of effective
substrates and production of analogies.
55
2.5. Microfluidic polymerisation of particles

Polymerisation processes in which a monomer solution is dispersed within another
immiscible solvent may overcome many problems that occur with other
polymerisation techniques such as autoacceleration and heat transfer limitations. 87
There are numerous techniques (such as suspension polymerisation 88 and emulsion
polymerisation89) used to form discrete polymer particles through the formation of a
monomer emulsion however, these are “top-down” approaches where the mixing of
the two liquids occurs as a bulk process. This leads to little control over the
formation of individual droplet dimensions and therefore typically results in
particles with a relatively broad size distribution. Ideally a “bottom-up” approach to
emulsification in which the formation of each individual droplet is controlled and
defined would be ideal. One strategy to achieve these objectives is based on
microfluidic devices. A early example of the controlled formation of liquid
dispersions using a microfluidic flow-focusing device (MFFD) was shown by Stone
and co-workers.90 Here, a pressure gradient along the long axis of the device forced
the two immiscible liquids through the orifice of the MFFD. The continuous phase
(supplied by the two outside channels) surrounded the dispersed phase (flowing
through the central channel) (Figure 2 -21 A) causing the inner flow to become
unstable. This flow then broke in the orifice to give discrete droplets which then
entered the outlet channel (Figure 2 -21 B & C). The planar microchannel design
was made by soft lithography in poly(dimethylsiloxane) (PDMS) and the smallest
droplets produced were much smaller than the orifice radius. Ultimately the flow
ratios of the water and oil through the device determined the size of droplets sizes
formed.
56
Figure 2-21. Formation of dispersions using "flow focusing" in microchannels. A: Flow-

focusing geometry implemented in a microfluidic device. An orifice is placed downstream of
three coaxial inlet streams. Water flows in the central channel and oil flows in the two outer
channels. B & C: Droplets are formed at the orifice and move into the outlet channel 90
In 2005 the idea of combining microfluidic droplet formation with a polymerisation

process was utilised to produce polymer particles.91 By dissolving the monomer in
the dispersed phase uniform monomer droplets were produced, a method of
initiation was then included to give polymer particles. These systems typically use
UV radiation to initiate polymerisation although interfacial polymerisation and
thermally initiation have also been used.92
Whitesides and co-workers produced a MFFD in either PDMS or polyurethane, this

allowed both water-in-oil and oil-in-water dispersion to be produced.91 They found
that it was possible to also produce uniform non-spherical particles by restricting the
dimensions of the outlet channel. If one of the dimensions is less than that of the
diameter of a regular droplet then disks were formed, by restricting both the height
and the width of the channel polymer ellipsoids or rods were produced. A number of
different monomer were used and with a single MFFD device they produced up to
57
250 polymer particles per second with a polydispersity (defined as the standard
deviation in the particle diameter divided by the mean particle diameter) of 1.5 %.91
At the same time Whitesides and co-workers also published a variation of the flow-
focusing microfluidic device in the form of an axisymmetric flow-focusing
microfluidic device (AFFD) (Figure 2 -22 A).93 This device was fabricated from
PDMS but rather than using photolithography to create microchannels, glass
microfibres and polyethylene tubing were used to template the design (Figure 2 -22
B). The main advantage of an axisymmetric design over the conventional setup was
that AFFD confines droplets to the central axis of the channel, protecting droplets
from shear or damage resulting from adhesion or wetting of the walls of the outlet
channel. Polymer membranes of Nylon-6,6 enclosing aqueous solutions of either
ions or superparamagnetic particles were produced by interfacial polymerisation.
The polydispersity or coefficient of variation (CV) of these microcapsules was
approximately 5 %. The orientation of the device was found to be very important
due to the relative densities of the two liquids, the dispersed phase (water) had a
higher density than the continuous phase (hexadecane). This would lead to the
droplets settling against the floor of the channel where they formed a high volume
fraction emulsion, this altered the flow of the continuous phase providing a higher
resistance to flow in the outlet channel, increasing the pressure in the orifice region.
Through the vertical orientation of the device these problems were avoided (as the
flow and gravity were in the same orientation) (Figure 2 -22 C). The interfacial
polymerisation could be quenched by flowing the nylon coated droplets into a
beaker of dodecan-1-ol in hexadecane (Figure 2 -22 D).93
58
Figure 2-22. An axisymmetric flow-focusing microfluidic device (AFFD) A: An axisymmetrical

flow focussing channel, composed of a cylindrical tube with a narrow cross-section halfway
along its length. The narrow region serves as the orifice where fluid is focused and breaks into
aqueous droplets. B: A scheme depicting the fabrication process of an AFFD. C: a) The
diameter of droplets at various flow rates of continuous phase. (b & c) images showing droplets
created in the AFFD oriented (b) horizontally and (c) vertically. D: a) A collection of nylon-6,6-
coated aqueous droplets. b) A coated-droplet containing 50 nm diameter magnetic particles in
an applied magnetic field.93
McQuade and co-workers described a simplified microfluidic device that produced

capsules by interfacial polymerisation without the need for any form of
microfabrication.92 This setup required only needles and tubing (Figure 2 -23 A);
the continuous phase (30 % w/v aqueous solution of glycerol) flowed through the
tubing and the dispersed phase (3:1 cyclohexane/chloroform with 2 % Tween 80
(surfactant)) was introduced via a 30 gauge needle inserted through the wall of the
tubing into the middle of the channel (Figure 2 -23 B). Analogous to Whiteside and
co-workers’ axisymmetric device, droplets produced with the needle and tubing
were entirely surrounded by continuous phase (coaxial flow) with the additional
advantage that any blocking of the device was very simple to overcome by replacing
the tubing and/or needle. Oil filled polyamide capsules were produced by
introducing polyethyeneimine (PEI) to the continuous phase and sebacoyl and
59
trimesoyl chloride to the dispersed phase. By varying the continuous flow rate
capsules between 313 - 865 µm in diameter with a coefficient of variation (CV) of
3.3 - 8.6 % were obtained (Figure 2 -23 C).92
Figure 2-23. Interfacial Polymerization within a Simplified Microfluidic Device: Capturing

Capsules. A: Photograph of fluidic device including needle and dye-filled organic droplets
dispersed in the continuous aqueous phase. B: Schematic of fluidic device. C: Light microscope
images of capsules in water formed with constant organic flow rate (0.141 mL min-1) and
increasing aqueous flow rate (clockwise). 92
Kumacheva and co-workers published two papers on the use of microfluidic reactors
for the production of polymer particles. 94,95 The first paper demonstrated a novel
approach to the continuous production of core-shell droplets and polymer capsules.
In this work, the microfluidic reactor was fabricated in polyurethane by soft-
lithography to give a MFFD with five separate channels approaching the orifice.
This design allowed three immiscible liquids to be supplied to the flow-focussing
region; the outer channels contained an aqueous solution containing surfactant
60
(sodium dodecyl sulphate) while the middle channels contained the oil phase
(silicone oil) and the monomer phase (tripropyleneglycol diacrylate (TPGDA) or
ethyleneglycol dimethacrylate (EGDMA)) flowed through the centre channel.
Photoinitiator was contained in the monomer phase and SPAN 80 in the oil phase.
Upon a pressure gradient along the long axis of the axis of the MFFD the three
liquids were forced through the orifice and the continuous water phase surrounded
the monomer-oil thread which adopted a circular cross-section (Figure 2 -24 A). To
minimise interfacial tension this coaxial jet broke into segments of with a spherical
shape. These monomer droplets were then photopolymerised in the wavy channel
(this shape maximises the curing time with minimum size) of the microfluidic
reactor. By controlling the flow rates of the three phases the diameter of the cores,
the size of the core-shell droplets and the thickness of the shells were controlled.
Generally if the ratio of flow rates of outer to inner phases was increased the size of
droplets was reduced due to the increased shear stress imposed on the undulated jet
of the dispersant liquid. If the flow rate of the aqueous phase was increased smaller
droplets with thinner cores were formed (with thicker shells). While at higher oil
phase flow rates the diameter of the cores increased and the shell thickness
decreased. It was also demonstrated that it was possible to control the number of
cores within the droplets. This was achieved by varying the value of interfacial
capillary wavelength and shifting the length and phases of capillary waves
(undulations). The oil cores could be removed after the photopolymerisation of the
monomer by using acetone to obtain particles with different shapes (Figure 2 -24
B). Overall productivity of the microfluidic reactor was 200 to 1000 s -1 with
particles polydispersity not exceeding 2.5%.94
61
Figure 2-24. Polymer particles with various shapes and morphologies produced in continuous
microfluidic reactors. A: (a) Schematic of production of droplets in MFFD by laminar co-flow
of silicone oil (A), monomer (B), and aqueous (C) phases. (b) Schematic of the wavy channel
used for photopolymerization of monomer in core-shell droplets. (c) Optical microscopy image
of core-shell droplets. The scale bar is 200 µm. (d) Photograph of a PU microfluidic system. The
arrow is pointing to the orifice. B: (a-e) Scanning electron microscopy images of polymer
microparticles obtained by polymerizing monomer in droplets, after removing a silicon oil core.
(Inset) Cross section of the core-shell particle. (f) Cross section of a polymer particle with three
cores obtained by polymerizing core-shell droplets with three cores. Scale bar is 40 µm.94
A second paper by the Kumacheva group95 that was published at the same time was
a comprehensive paper detailing the use of MFFD to produce polymer particles
(Figure 2 -25 A). As in the Whitesides and co-workers MFFD publication, these
devices were produced using soft lithography in either poly(dimethylsiloxane)
(PDMS) or polyurethane (PU). Here, four different multifunctional acrylate
monomers were emulsified. Three major regimes in the formation of monomer
62
droplets were found; at low flow rates for the aqueous continuous phase (Qc) and
low flow ratios of continuous to dispersed phases (Q c/Qd) the device operated in a
dripping regime with the monomeric thread breaking into monomer droplets behind
the orifice with droplet size significantly larger than the orifice size. The second
flow regime observed was at moderate values of Q c and Qc/Qd in which the
monomer droplets were generated by the breaking up of the monomer thread in or
behind the orifice, upon releasing a droplet the monomeric thread retracted break
upstream. The final flow regime was jetting mode and this was observed at high
flow rates and high Qc/Qd, the monomeric thread remained behind the orifice
breaking into droplets (Figure 2 -25 B). Under certain conditions the formation of
smaller satellite droplets were observed along with the main droplet population. The
size of the droplets produced was shown to be governed by the properties of the
monomer liquid (viscosity and interfacial tension) and the flow rates of the
continuous and dispersed phases. Therefore, the conditions with which monomer
droplets with a very narrow size distribution varied for each monomer and by
varying these conditions along with the design of the MFFD particularly the size of
the orifice droplets (Figure 2 -25 C) as small as 18 µm were formed. By adding
photoinitiator to the monomer liquid and exposing the monomer droplets to UV light
after the orifice polymer particles were formed, additionally, it was demonstrated
that by changing the dimensions of the channel in which polymerisation took place
different shape particles could be obtained. These included discs of various
morphologies if the height of the channel was less that the initial diameter of the
monomer droplet or rods if both the height and width of the channel were less than
the diameter (Figure 2 -25 D). This paper provided an in-depth study of the use of
MFFDs to produce monodisperse polymer particles.95
63
Figure 2-25. Continuous microfluidic reactors for polymer particles. A:(a) Schematic of droplet
formation in the microfluidic flow-focusing device. (b) Wavy channel for the
photopolymerization of monomer droplets. B: Breakup of the monomer thread in 2 wt %
aqueous SDS solution. (a) Regime 1, (b) Regime 2, (c) Regime 3. C: SEM image of polymer
particles. Scale bar is 100 µm. D: Schematic (a-c) and optical microscopy (a’-c’) images of
polymer particles with different shapes: microspheres (a, a’), disks (b, b’), (c, c’) and rods of
obtained via photopolymerization of droplets. Scale bar is 50 µm.95
An alternative microfluidic reactor has been described by Goddard et al,96 this

system utilises a 35 µm diameter hole through which the dispersed phase was
introduced into spiral channel where photopolymerisation took place (Figure 2 -26
A & B). This device was fashioned from a polycarbonate sheet using a precision
milling machine. Using this device it was possible to produce poly(methacrylate)
(pMA) particles with a low CV (typically around 2%) in a range of diameters
between 10-115 µm (Figure 2 -26 C). Additionally, it was possible to produce
molecularly imprinted particles through the incorporation of propranolol into the
monomer solution prior to polymerisation, these particles showed the same uptake
of propranolol as particles produced by conventional suspension polymerisations.96
64
Figure 2-26. A micro-reactor for preparing uniform molecularly imprinted polymer beads A:
Schematic of the spiral micro-flow-reactor showing the overall layout and the sample and oil
inlet points. B: Detail of the tapered region of the reactor where the monomer is introduced into
the flowing continuous phase. Monomer is extruded into the flowing oil, where it breaks off into
droplets. These are swept into the spiral polymerisation reactor where they are polymerised by
UV light. C: SEM of typical particles produced in oil using the micro-reactor. 96
A different route to prepare near monodisperse particles was demonstrated by

Hadziioannou and co-workers,97 this was an axisymmetrical needle/tubing device
that could be prepared without the need for any microfabrication techniques (Figure
2 -27 A). Within conventional planar microfluidic devices surface modification is
usually required to prevent an inverse emulsion. The axisymmetrical setup overcame
this requirement by avoiding direct contact of the dispersed phase with the channel
walls.93 A simple T-junction and needle were used to introduce the dispersed phase
into the continuous phase, both removing the need for microfabrication and avoiding
the clogging problems that microchannels often suffer from. By varying the flow
rates and solvent viscosities they were able to produce poly(methylmethacylate)
(poly(MMA)) particles with a narrow distribution between 150-550 μm in diameter
(Figure 2 -27 B & C).97
65
Figure 2-27. A predictive approach of the influence of the operating parameters on the size of
polymer particles synthesized in a simplified microfluidic system. A: Schematic of the
microfluidic system for the synthesis of controlled-size polymer particles. The dispersed phase
is injected via a thin needle positioned along the main axis. B: Effect of flow rate ratio of
continuous and dispersed phases for two different continuous phase flow rates on average
diameter, Dp, of polymer particles. C: Effect of flow ratio and continuous phase viscosity on the
average diameter of polymer particles.97
2.5.1. Summary
Microfluidic polymerisation techniques offer a highly controlled approach to the
preparation of polymer particles. There are a number of different configurations
available that allow particles to be produced with a very narrow size distribution
(generally with polydispersities less than 2.5 %). Additionally, by introducing
further co-flowing solvents it is possible to make particles with very well defined
morphologies. However, there are a number of limitations relating to the preparation
of polymer particles based on microfluidics. These include: the overall production
rate of particles is low. Microfluidic devices cannot be ‘scaled up’ (although this
66
might be addressed though the combination of many microfluidic reactors in

parallel). Additionally, microfabrication techniques are required to construct many
microfluidic devices (those fabricated by soft lithography) making these approaches
inaccessible for many researchers.
Overall, microfluidics presents a versatile method for the small scale production of
polymer particles with very narrow size distribution and desired morphology.
67
2.6. Aims of thesis

The overall objective of this thesis was to synthesise and characterise enzyme
responsive hydrogel particles functionalisation with peptide actuators. These
functionalised particles were then to be applied to the triggered release of a
macromolecule payload.
Firstly, PEGA microparticles (µPEGA) were to be produced and characterised with

the aim of establishing the optimum support of the enzyme responsive system.
Previous approaches to modifying the properties of PEGA such as the introduction
of charged monomers74,75 were to be assessed for µPEGA. Particles are then to be
modified with peptide actuators57,58 in order to demonstrate enzyme responsive
release of an entrapped payload.
Peptide actuators with enhanced functionality were to be designed and incorporated

into µPEGA, the enzyme responsive behaviour of this system could then be
characterised and utilised for the triggered release of an entrapped macromolecule.
Finally, based on the literature, a simplified microfluidic device 97 was to be used

produced and used to prepare PEGA particles with the smallest diameter possible
and a very narrow size distribution.
68
3 PEGA polymerisation, characterisation and enzyme
responsive swelling through functionalisation with

peptide actuators2
3.1. Abstract
Within this chapter the polymerisation of PEGA hydrogel microparticles (µPEGA)
with either neutral, positive or negative charge were demonstrated. The swelling of
these different particles was characterised and neutral µPEGA were selected for
further investigation. These particles had a mean diameter of 16 µm (similar to that
of biological cells) and were compatible with different enzymes. Furthermore, it was
demonstrated that enzyme catalysed reactions occur faster with these microparticles
than with commercially available macrobeads which are typically 200-400 µm in
diameter. μPEGA was then functionalised with peptide actuators. These particles
demonstrated an enzyme specific increase in accessibility allowing fluorescently
labelled dextran to diffuse into the particles. The pH responsive nature of the linear
peptide actuators was shown and utilised for the physical entrapment of a
macromolecule payload. Release of this payload was only possible when the
functionalised particles were exposed to the target enzyme. However, at
physiological ionic strength no response of the particles was observed due to
electrostatic screening.
3.2. Introduction
PEGA is a polymer hydrogel that was developed as a resin for solid phase peptide
synthesis (SPPS). A particularly attractive property of this material is its
compatibility with both aqueous and organic solvents. This allows for easy chemical
modification through SPPS67 as well as compatibility with aqueous solutions
containing biomolecules. There has been thorough research into using enzymes,
2
Published in part as: McDonald, T. O., Christensen, S., and Ulijn, R. V., Making peg-based
microparticles for applications in biology and medicine. Mater. Res. Soc. Symp. Proc 1008E (T05),
18 (2007).
69
specifically proteases to catalyse reactions on peptides synthesised on PEGA. 63,72,74

Several workers have shown that PEGA is accessible to small enzymes and that
enzyme activity can occur inside the resin.63,69,73,98 PEGA polymers possess a highly
crosslinked network which gives the material a molecular cut-off weight, above
which molecules are not able to diffuse in or out of the polymer. 63 Further details on
the development and further investigation of enzyme reactions on PEGA polymers
are outlined in section 2.4 within the literature review.
Currently, PEGA particles are commercially available between 200 - 400 µm in

diameter, for convenience in solid phase chemistry applications. 72 Depending on the
biomedical applications smaller size ranges are desirable (such as injected into tissue
(<200 μm), inhaled (<100 μm) or released into circulation (<10 μm). 99 Smaller
particles also offer an increase the rate of response due to their greater surface area
to volume ratio.55 Additionally, microparticles allow for possible use in automated
analysis of ‘on-bead’ libraries using a cell sorter. Therefore, PEGA microparticles
were prepared that are smaller in size, thereby enhancing responses, but still
conveniently handled for solid phase synthesis and analysis by fluorescence
microscopy.100 Work carried out in the author’s MSc project investigated the effect
of stirring speed and surfactant concentration on mean particle size.101
Numerous stimuli have been exploited in the development of responsive materials in

a biomedical context including pH,102 temperature,9 ionic strength35 and small
molecules (e.g. glucose).36 Bioresponsive materials21 are stimuli-responsive surfaces,
self-assembled structures or chemically crosslinked polymers that change their
properties in response to biochemical recognition events, offering potential
applications in biosensing,103 tissue regeneration28 and controlled release.54 The
responsiveness of these materials is determined by a biorecognition moiety that,
upon a recognition event, actuates structural changes in the material. In systems
established using hydrogels there are three main categories of molecular actuation
11,27,28,41
based on changes in: (i) crosslinking density, (ii) electrostatic
interactions36,56,57 or (iii) molecular conformation changes.65,66 These systems have
been discussed in detail in section 2.3.
There has been significant interest in exploiting enzyme catalysed reactions as the
stimulus to trigger molecular actuation in polymer hydrogels. 8,51,104,105 Enzymes
70
generally function under mild conditions and possess a high degree of selectivity.
Also, they are vital to the function of all living systems, catalysing and controlling
healthy and diseased biological processes. In particular proteases (enzymes that
hydrolyse peptide bonds) have been shown to be specific markers in many disease
states including cancers13 and chronic wounds.106
Most enzyme responsive systems that have been described make use of enzyme
cleavable linkers which covalently attached the drug to a polymer whereby enzyme
action detaches the drug.107-109 An alternative approach through the development of
57,58
peptide actuators has been shown by Ulijn and co-workers. These actuators
constructed by SPPS on PEGA, have the ability to release entrapped payloads in
response to a specific enzyme by exploiting changes in electrostatic interactions
resulting in an increase in the swelling of the polymer (shown schematically in
Figure 3 -28). This method of release offers advantages over pro-drug approaches:
There is no need to covalently modify the drug molecules and drug release is not
directly governed by enzyme kinetics, i.e. instead of the enzymatic hydrolysis of one
bond corresponding to the release of one drug molecule. This system allows for a
variable number of payload molecules to be released upon enzymatically induced
swelling. Using designed peptides allows the possibility to develop a range of
different actuators using the varied functionalities that are offered by nature’s
building blocks, for example the benefit of designing the actuator to match payload
charge has previously been shown (described within section 2.3.3.2).58
71
Figure 3-28. Schematic of the enzyme responsive swelling of PEGA particles functionalised with
linear peptide actuators. The linear peptide actuator is made up of 2 parts, a sensor and an
actuator. The actuator consists of the 2 oppositely charged amino acids (a zwitterion) and the
sensor is the amino acids between the charged residues. The sensor is hydrolysed by an enzyme
with the correct specificity; therefore it is termed the enzyme cleavable peptide or ECP.
Therefore, the aims of this chapter were to: (i) Produce and characterise PEGA
microparticles (µPEGA) and to investigate the effect of changing the chemical
structure of PEGA on its swelling behaviour. (ii) Study the compatibility of µPEGA
with enzyme activity using TPM. (iii) To establish the ideal experimental conditions
to produce PEGA particles that offer the suitable properties (loading and particle
size distribution) for use in the enzyme responsive release systems. (iv) Incorporate
peptide actuators on µPEGA, (v) characterise the enzyme specific changes in
accessibility of the functionalised particles, (vi) demonstrate the possibility of
loading these particles with a payload and (vii) use enzymes to trigger specific
release of the payload.
72
3.3. Experimental
3.3.1. Materials
All chemicals were used as supplied from Sigma with the exception of amino acids
(Bachem) and O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) (AGTC Bioproducts Ltd). The enzymes used were
thermolysin (EC 3.4.24.27), 36.5 U/mg and chymotrypsin (EC 3.4.21.1), 60 U/mg
(both were supplied by Sigma).
3.3.2. Inverse suspension polymerisation

μPEGA was prepared by inverse suspension polymerisation. A stainless steel baffle-
less reactor (250 ml) stirred with an anchor-style agitator was used for the
polymerisation reactions. 3.14 g (3.5 mmol) of the PEGA800 macromonomers (a 2:1
(molar) mixture of acrylamide-PEG-acrylamide and amino-PEG-acrylamide) was
dissolved in 10 ml of distilled water along with 0.156 g (2.2 mmol) of acrylamide
(the chemical structures of the monomers are shown in Figure 3 -29). This solution
was then purged for 30 minutes with N 2 gas. 50 ml of Isopar M (isoparaffin) was
added to the reactor and was also purged for 30 minutes. The reactor was heated to
80°C. After 20 minutes of purging 0.16 ml (1.0 mmol) of N,N,N′,N′-
tetramethylethylenediamine (TEMED) was added to the oil phase, and 0.156 g (2.2
mmol) of acrylamide to the dissolved macromonomer solution. 0.164 g (0.47 mmol)
of Span 20 (sorbitan monolaurate) was dissolved in the oil, which was stirred at 500
rpm for 30 seconds to ensure the surfactant was fully dispersed in the oil phase.
0.070 g (0.30 mmol) of ammonium persulfate (APS) was dissolved in the
macromonomer solution, which was added to the oil phase in the reactor which was
stirred at 2000 rpm for a further 30 minutes. The particles were washed with (3 x 50
ml) dichloromethane (DCM), (3 x 50 ml) Tetrahydrofuran (THF), (3 x 50 ml)
methanol and (4 x 50 ml) distilled water (retained by centrifugation at each step).
3.3.3. Charged PEGA polymerisation

Particles that incorporate permanent charges were prepared by substituting the
acrylamide with either (3-trimethylammonium chloride) propyl acrylamide to
produce positive particles or 1,1-dimethyl-2-(sulphonate) ethyl acrylamide to give
negative particles (the chemical structures of these monomers are shown in Figure 3
73
-29). All remaining aspects of the polymerisation were the same as with the inverse
suspension of neutral particles
Figure 3-29. Monomers used in the synthesis of PEGA and its charged variants. From the top
down: amino-PEG-acrylamide, acrylamide-PEG-acrylamide, acrylamide, (3-
trimethylammonium chloride) propyl acrylamide and 1,1-dimethyl-2-(sulphonate) ethyl
acrylamide.
3.3.4. Microscopy and particle size analysis

An optical microscope was used to acquire images of the particles and
environmental scanning electron microscopy (ESEM) images were taken on a FEI
Quanta 200 ESEM, using ESEM low vac mode at 10.0 KV. Size distribution
analysis of the microparticles was carried out using a Malvern Mastersizer particle
size analyser. Deionised water was used to fill the small volume dispersion unit and
the dispersion control unit set to 1500 rpm, the polymer particles were added until
an obscuration value of above 10% was obtained. Mastersizer Microplus software
version 2.18 was used to analyse the results and plot distribution graphs.
3.3.5. Solid phase peptide synthesis

Peptides can be prepared synthetically in good yields, solid phase peptide synthesis
(SPPS)68 is a popular synthetic method used to achieve this. Here, amine
74
functionalised crosslinked polymers (resins) are reacted with amino acids (with
protected their amines protected) resulting in amide bond formation (shown
schematically in Figure 3 -30, while the chemical reactions used in this work can be
seen in Figure 3 -31). The removal of the protecting group allows for this process to
be repeated in a step-wise process to build up the desired peptide.
Figure 3-30. Solid phase peptide synthesis scheme. A: A scheme of the step by step process in
the synthesis of a peptide from protected amino acids. B: The key, (left) amines are present in
PEGA particles, (right) Fmoc protected amino acid.
75
Figure 3-31. Chemical reactions involved in peptide synthesis. A: Formation of an amide bond.
Firstly, the carboxyl of the amino acid/peptide reacts with O-(Benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HBTU) in the presence of N,N-
diisopropylethylamine resulting in an activated ester. The amine of the free amino acid then
reacts with the ester resulting in amide bond formation. B: The deprotection of the Fmoc
protected amine group with piperidine.
3.3.5.1. Solid phase synthesis of dipeptides and enzyme treatment

To quantify the loading of the microparticles Fmoc Amino acids (3 equiv.) were
coupled to PEGA particles using DIC (di-isopropyl carbodiimide) (6 equiv.) and
HOBt (hydroxybenzotriazole) (6 equiv.) in DMF. The first coupling was performed
for 3 hours and the second overnight. A roller mixer at room temperature was used
to agitate the solutions. Between steps the resin was washed extensively using 5 ml
volumes of 5 x MeOH, 5 x 50 : 50 (v/v) DMF : MeOH and 5 x DMF. A 20 %
76
solution of piperidine in DMF was used for Fmoc deprotection for 2 hours. Enzyme
reactions: 1.5 mg of thermolysin per 1 ml 0.01 M phosphate buffer solution of pH
7.5, reactions were at room temperature on a roller mixer. To determine how quickly
the enzyme catalysed hydrolysis has occurred, a thermolysin solution was added to
the Fmoc-A-A modified particles the reaction was then stopped with 0.1%
trifluoroacetic acid (TFA) in deionised water at seven different times and analysed
with high performance liquid chromatography (HPLC) to quantify any cleaved
residues.
3.3.5.2. Solid phase peptide synthesis of peptide actuators

Peptide actuators (Fmoc-DAAR-PEGA) were prepared by solid phase peptide
synthesis using Fmoc protected amino acids. 8 equivalents of the amino acid and 7.8
equivalents of HBTU were dissolved in 2 ml of N,N-Dimethylformamide (DMF). 16
equivalents of N,N-Diisopropylethylamine (DIPEA) was added to this solution prior
to its addition to the PEGA particles. The coupling reaction was left for 16 hours,
and the Kaiser test26 was used to ensure complete coupling. Deprotection was
achieved using 20 % piperidine in DMF for 2 hours. This cycle was repeated to
build up the required peptide sequence with thorough washing between steps (5 x 5
ml methanol, 5 x 5 ml 1:1 methanol:DMF, 5 x 5 ml DMF). A solution 95 %
Trifluoroacetic acid (TFA) 5 % water was used to remove the side chain protecting
groups.
1.1.1. HPLC
HPLC experiments were undertaken on a Dionex HPLC (P680 pump, ASI-100
Automated sample injector, Nucleosil 100-5-C18 column with a UVD170U
detector), using a solvent ramp of 20 % ACN and 80 % water to 80 % ACN 20 %
water over 30 minutes (0.1 % TFA was present in both phases) shown in Figure 8
-66. Chromeleon 6.60 software was used for analysis.
3.3.6. Two-photon microscopy

The distribution of amine groups and homogeneity of enzyme hydrolysis were
assessed with TPM. Firstly, three differently modified batches of particles were
prepared; unmodified, Fmoc-A-A (prepared with the previously described SPPS
method) and Fmoc-A-A treated with thermolysin solution. 0.1 g of these particles
77
were then placed in a filter column and rinsed with DMF (3 x 5 ml). 0.036 g (0.133
mmol) of dansyl chloride was dissolved in 2 ml of DMF and 25 μl (0.143 mmol)
DIPEA was then added to this solution. This coupling solution was then added to the
PEGA particles and put on the blood rotator in the dark for 2 hours. The polymer
was then rinsed with DMF (3 x 5 ml), ethanol (3 x 5 ml) and water (3 x 5 ml). TPM
was used to take visual cross-sections of the particles to determine the distribution of
free amine groups. A Ti:Sapphire laser was tuned to 770 nm and fluorescence from
the sample was filtered using a 525/550 nm filter.
3.3.7. Determining particle swelling

The neutral, positive and negative PEGA microparticles were swollen in water or
buffer 0.1 M (pH 7.5) and centrifuged at 3000 rpm. 0.50 g of the hydrated polymers
were weighed out and placed in a Gallenkamp vacuum oven at room temperature at
100 mbar. Every three hours the polymers were weighed, and when no further
weight loss was recorded this mass was taken to be the dry mass.
1.1.2. Assessing accessibility

To analyse the change in accessibility of the particles a 2 mg/ml solution of 40 kDa
fluorescently labelled dextran was prepared in water. Approximately 50 mg of
PEGA particles were added to these solutions. 10 minutes was left to allow diffusion
into the hydrogel prior to imaging. The confocal microscope used was a Lecia sp2
AOBS. The laser was an Argon laser tuned to 488 nm, the fluorescence from the
sample was filtered to leave 490-550 nm.
1.1.3. Entrapping payload

The particles (0.1 g) were loaded with a 40 kDa FITC Dextran using the following
method; 2 ml of solution of FITC dextran in 0.01 M HCl (10 mg/ml) was added to
the particles for 30 minutes (pH 2.5). After this time 0.4 ml of 0.05 M NaOH was
added dropwise (pH 7). The particles were then filtered and washed (2 x 5 ml water,
2 x 5 ml 0.18 M buffer, 2 x 5 ml methanol).
1.1.4. Fluorimetry
The release of entrapped dextran was determined using the following method;
loaded particles were treated in solution for 80 minutes, these samples were
centrifuged and the supernatant taken off. A Jasco FP – 6500 Spectrofluorometer
78
using an excitation wavelength of 490 mn was used to obtain the fluorescent

intensity at 519 nm. Treated particles were filtered and imaged using fluorescence
microscopy.
3.3.8. Microscopy and determination of swelling

Swelling measurements were obtained using a Zeiss Imager A1 microscope, a
Leistungselektronik mbq 52 AC power source (Jena, Germany) equipped with a
Canon Powershot G6 camera. For pH responsive swelling the diameter of a
minimum of 300 particles was measure and the mean volume calculated. For
enzyme responsive swelling individual particles were observed throughout the time
course and ImageJ 1.38x analysis software was used to determine the change in
particle diameter. An average V/Vo was determined from at least six representative
particles, where V is the volume of the particle at time t and Vo is the volume of the
particle immediately after exposure to enzyme solution. Enzyme solutions were
made up at a concentration of 1 mg/ml and buffers were prepared by using the
appropriate amounts of sodium phosphate dibasic heptahydrate and sodium
phosphate monobasic.
79
3.4. Results and Discussion
3.4.1. Production of µPEGA800

PEGA microparticles (µPEGA) were prepared by inverse suspension polymerisation
using thermal initiators.67 The water soluble monomers were dispersed along with
the initiator in the oil phase, which contained the co-initiator and the surfactant
(Figure 3 -32). Upon stirring monomer droplets were formed and undergo
continuous break-up and coalescence, this leads to a dynamic equilibrium being
established giving a stationary mean droplet size. 110 The size and the distribution of
the particles formed were controlled by the imposed shear field within the reactor
and the stabilisation effect of the surfactant. Currently PEGA particles are
commercially available in size ranges above 150 μm for ease of handling in SPPS.
Depending on the biomedical applications smaller size ranges are desirable (such as
injected into tissue (<200 μm), inhaled (<100 μm) or released into circulation (<10
μm).99 Smaller particles also offer the potential to give faster response times due to
their greater surface area to volume ratio (as shown by Peppas and co-workers). 55 In
previous work, the stirring speed and the surfactant concentration were varied in
order to find the optimum conditions to produce microparticles. 101 There are three
monomers used in the preparation of PEGA; aminoacrylamido PEG, bisacrylamido
PEG and acrylamide. The amino functionalised monomer imparts the amine
functional group or ‘handle’ on a flexible chain allowing easy modification by solid
phase peptide synthesis (SPPS). The bisacrylamide PEG was the crosslinking agent
in the polymerisation while the acrylamide acts as a spacer to separate the PEG
based monomers. The PEG macro-monomers used had an average molecular weight
of 800 g/mole, the molar mass of the PEG chain in the resulting cross-linked
polyacrylamide-graft-PEG co-polymer. This structure leads to PEGA’s well defined
63
molecular weight cut-off, with molecules above this unable to diffuse in or out of
the polymer matrix.
80
Figure 3-32. Polymerisation of PEGA particles by inverse suspension polymerisation. The

monomers; acrylamide, aminoacrylamido PEG and bisacrylamido PEG along with the initiator
APS were dissolved in water and dispersed in an oil phase containing surfactant and co-
initiator. Upon stirring, monomer droplets are formed and undergo continuous break-up and
coalescence and a dynamic equilibrium is established giving a stationary mean particle size.
3.4.1.1. Particle morphology and size characterisation of µPEGA

In order to produce μPEGA with the desired size distribution a stirring speed 2000
rpm was employed with 3.28 mg/ml of surfactant in the oil phase. These particles
had a mean diameter of 15 µm. Further investigation was carried out on these
particles, including analysis of the size distribution, determining the loading capacity
and its homogeneity throughout the particles as well as their compatibility with
enzymes. Study of the particles by optical microscopy (Figure 3 -33 A) showed that
the microparticles are separate spherical entities, with no obvious surface detail or
texture on the sub-micron scale between the range of about 5-25 µm. The data
provided by particle size analysis (Figure 3 -33 B) gave a mean particle diameter of
15 µm with a polydispersity ((standard deviation/mean particle diameter) x 100) of
43 %. Images of the particles obtained by environmental scanning electron
microscopy (ESEM) (Figure 3 -33 C & B) concurred with the optical microscopy.
The ESEM images showed that the majority of particles have diameters between 1-
81
10 µm. This difference in size measurement is due to the low pressure within the
ESEM chamber resulting in removal of water from the particles (hence reducing the
particle diameters). Individual spherical PEGA microparticles with a mean diameter
of 15 µm and polydispersity of 43 % were produced as a result of the combination of
shear forces and stabilisation effect of surfactant on the droplets within the reactor.
Figure 3-33. A: Optical micrograph of microparticles is water. B: Size distribution of

microparticles in water obtained by particle size analyser. C and D: ESEM micrographs of
PEGA microparticles. (Scale bar in all figures is 20 µm).
3.4.2. Production of µPEGA+ and µPEGA-

Other researchers74-77 have shown that by modifying the chemical structure of PEGA
to include permanent charges within the acrylamide backbone improved swelling,
accessibility and yields in enzyme catalysed reactions. This change in properties is
due primarily to the electrostatic repulsion between neighbouring charges in the
polymer structure but also offers the possibility to use electrostatic interactions
between the enzyme and the polymer. By substituting the acrylamide in PEGA with
an acrylamide-based monomer carrying a permanent charge it maybe possible to
introduce a charge into the backbone of the polymer. Two different monomers were
82
used; (3-trimethylammonium chloride) propyl to produce positively charged PEGA

(PEGA+) and 1,1-dimethyl-2-(sulphonate) ethyl to give negatively charged PEGA
(PEGA-) (Figure 3 -34). To determine whether the conditions used to polymerise
the neutral PEGA800 microparticles would successfully produce charged particles,
positive and negative PEGA polymers were prepared and analysed for mean
diameter and size distribution.
Figure 3-34. Chemical structure of PEGA and its charged variants.75
3.4.2.1.1 Particle morphology and size characterisation of µPEGA+

The inverse suspension polymerisation of the PEGA monomers with the acrylamide
substituted with (3-trimethylammonium chloride) propyl acrylamide produced
PEGA+ particles.74 Optical and ESEM microscopy images (Figure 3 -35) show the
positively charged particles produced are highly aggregated with a narrow size
distribution. Based on optical micrograph analysis the particle diameter were
approximately 10 μm, whilst analysis of the ESEM images indicates the particles
had a diameter of approximately 8 μm. Much like the neutral PEGA 800 particles this
was because the polymer is not fully hydrated within the ESEM chamber. When the
83
pressure within the ESEM chamber was further lowered there was an appearance of
a textured surface, this was water being drawn out of the hydrogel onto the surface
prior to evaporating (Figure 3 -35 D). The positively charged particles have no
apparent surface features or texture on the sub-micron level. The aggregation of the
particles meant that determining a size distribution using the particle size analyser
did not yield any quantative information. The PEGA+ particles had an aggregated
structure which may have be due to incomplete polymerisation during stirring
leading to coalescence of the partially polymerised monomer droplets upon settling.
It is possible that the negatively charged initiator (ammonium persulfate) cancelled
the electrostatic repulsion between the positively charged polymerising monomer
droplets. This may have removed the electrostatic stabilisation within the system
allowing the partially polymerised particles to aggregate during the sticky period of
the reaction.111
Figure 3-35. PEGA+ microparticles. A & B: ESEM micrograph of positively charged PEGA
particles (scale bar in A is 50 μm and in B is 10 μm). C: Optical micrograph of positively
charged PEGA particles (scale bar is 15 μm). D: ESEM micrograph of positively charge PEGA
particles, arrows indicate examples of water droplet forming on the surface as the pressure is
reduced (scale bar is 5 μm).
84
3.4.2.1.2 Particle morphology and size characterisation of µPEGA-

By carrying out the inverse suspension polymerisation reaction with 1,1-dimethyl-2-
(sulphonate) ethyl acrylamide replacing the acrylamide (with all other conditions
kept the same) PEGA- was prepared.74 Optical microscopy of these negative
microparticles (Figure 3 -36) produced images similar to μPEGA although with a
slightly larger and more polydisperse. Unlike the PEGA+ there was no aggregation
visible. Data produced from the particle size analyser agrees with this comparison (a
mean diameter of 20 μm with a polydispersity of 59 % compared to 15 μm and 43 %
respectively for μPEGA). It is likely that the different between PEGA 800 and PEGA-
is due to batch-to-batch variation. PEGA- particles were produced with morphology
similar to that of neutral PEGA.
Figure 3-36. Right: Optical micrographs PEGA-, scale bar is 20 μm. Left: Size distribution of
PEGA-.
3.4.2.1.3 Comparison of swelling the different types of PEGA

The swelling properties of a hydrogel are dependent on the ability of the material to
absorb water. This ability is determined of a number of structural factors such as:
crosslinking density, hydrophilicity of the polymer and the number of
polar/ionisable groups. Greater swelling may be advantageous as in certain cases,
greater swelling of the network results in increased mesh size of the polymer and
therefore accessibility. The uptake of water and 0.1 M buffer (pH 7.4) into the
particles was determined for dry particles from each of the three different PEGA
polymers. This was achieved by measuring the dry and hydrated masses of the
85
particles, providing information about polymer’s interactions with water

(hydrophilicity) and neighbouring polymer chains (Figure 3 -37).
Figure 3-37. The uptake of water by the three different types of dry PEGA microparticles and
the effect of ionic strength on the swelling of the three different types of PEGA microparticles.
Blue; uptake of water, red; uptake of 0.1 M buffer.
The positively charged particles gave the highest values for uptake of water because
of the electrostatic repulsion between chains resulted in an osmotic driving force
drawing more water into the polymer. The swelling of negative PEGA was much
less than seen for PEGA+, this is likely due to interactions between the amines
present (positively charged at pH below 7.5 in a test solution (water) which has a pH
of 6.5) and the negatively charged acrylamide. This finding is in agreement with
published work in which it was shown that PEGA - swelled less than PEGA+.75
Increasing the ionic concentration of the uptake solution resulted in a decrease in
swelling in all three polymers. This observation was a result of the dissolved ions
associating with the oppositely charged permanent groups with the effect of
screening the charges reducing the effect of electrostatic repulsion. This reduction
was least pronounced with PEGA800 this is because this polymer has the lowest
density of charges (only the protonated pendant amines). Indeed, the charge
densities of within the three different polymers were calculated to be 0.46, 0.37 and
1.2 mmol/g for PEGA-, µPEGA and PEGA+ respectively. A similar trend was found
86
by other researchers on PEGA1900, and charged PEGA macroparticles.75 Although

they found that increasing the ionic strength with neutral PEGA system showed ‘no
measureable effect’ on swelling. This difference may be the result of the
experiments being carried out on PEGA1900 whereas this work was using PEGA 800
which has double the loading and therefore charged amines. Overall, the charged
PEGAs particularly PEGA+ had greater swelling in water than µPEGA. Increasing
the ionic strength of the solution absorbed by the particles reduced the swelling of
all three polymers.
3.4.3. Further characterisation of µPEGA

µPEGA was selected as the most promising polymer for the incorporation of the
enzyme responsive functionalisation. PEGA+ and PEGA- offered greater swelling
and have been demonstrated in the literature to have higher accessibilities than
neutral μPEGA. An important criterion for the material selection is accessibility; it
must high enough to allow the target enzyme to diffuse into the particles while being
low enough to entrap the payload molecules. Neutral µPEGA offered this, as well a
simpler base to understand the effect of peptide actuator design of particle swelling.
Therefore, further characterisation was carried out on µPEGA.
3.4.3.1. Amine characterisation by two-photon microscopy and enzyme

compatibility
The ease with which PEGA could be chemically modified with amino acids or
peptides by SPPS and compatibility with aqueous environments makes it a
particularly interesting material. This capacity for functionalisation is determined by
the loading; the amount of amines per unit mass of polymer, usually quoted in
mmol/g. In order to access this property, µPEGA was functionalised with an Fmoc
protected amino acid using standard DIC/HOBT chemistry was undertaken. Within
SPPS, piperidine is used to remove the Fmoc protecting group. These Fmoc groups
were quantified by HPLC against Fmoc standards and the loading was found to be
0.37 mmol/g of (dry) µPEGA. This is in close agreement with the declared loading
of commercially available PEGA800 macrobeads (0.4 mmol/g). Furthermore, the
distribution of the amine groups was analysed by TPM. 79 Here, the amine groups
87
within the particles were reacted dansyl and the spacial distribution of the
fluorophore determined. In TPM the sample is irradiated with a laser with a
wavelength approximately twice that of the excitation length wavelength of the
fluorophore. This means that excitation can only occur when two photons are
absorbed simultaneously. This is extremely unlikely and therefore only occurs at
very high photon density, the focal point of the laser beam. This method of imaging
is well established for the characterisation of PEGA particles as the long wavelength
of the infrared excitation laser gives good penetration into solid objects allowing for
optical cross-sections within the z plane of the particles to be obtained.79,82 The TPM
analysis of the µPEGA are shown in Figure 3 -38. Here, an optical cross-section
obtained was at the equatorial plane (the widest point) and fluorescence indicates
where amines were present. Firstly, unmodified µPEGA were treated with dansyl
chloride and imaged. From Figure 3 -38 A it is clear that the free amine groups are
homogeneously distributed throughout the particles. Other microparticles were
functionalised with the protected dipeptide; Fmoc-AA and some then treated with
dansyl chloride and imaged (Figure 3 -38 B), it can be observed that 90 % of the
amine groups have been functionalised with the protected dipeptide.
The enzyme compatibility of µPEGA was then assessed. Here, Fmoc-AA

functionalised particles were also exposed to a solution of the enzyme thermolysin
for 12 hours before being reacted with dansyl chloride and imaged. Thermolysin, a
protease which has been used previously on PEGA particles was used as the model
62,63,75,85
enzyme. Thermolysin is a proteolytic enzyme with a well-known specificity
for hydrophobic amino acids in the P’1 and little specificity for the P1 position. 112
Additionally, thermolysin is known to be active on peptides linked to PEGA
particles.62,79. Figure 3 -38 C shows that ~70 % of the original number of amine
groups available (due to the cleavage of the peptide bond) and that enzyme action
occurs evenly throughout the interior of the particles. This result agrees with HPLC
results, in that >100 minutes the enzyme cleaves a maximum of around 70 % of the
peptide bonds.
88
Figure 3-38. Assessment of the distribution of the amines within μPEGA by TPM. A: Schematic
of the process used to determine the homogeneity of free amine groups and enzyme activity
using dansyl labelling. Three different batches of particles were prepared: a) unmodified and
labelled with dansyl-chloride, (b) functionalised with Fmoc-AA then labelled with dansyl-
chloride and (c) functionalised with Fmoc-AA then treated with thermolysin and labelled with
dansyl-chloride (scale bar is 15 μm). B: Two photon micrographs (TPM) of representative
microparticles for each of the different treatments. C: Cross-section of the intensity of
fluorescence in each of the 3 microparticles
3.4.3.2. Comparison of enzymatic hydrolysis within µPEGA800 and macroparticles

Enzyme hydrolysis is the process that initiates a responsive change in particle
swelling (when peptide actuator functionality has been incorporated). Therefore, the
rate at which enzyme hydrolysis occurs within the PEGA particles was the main
factor determining the response time of these functionalised particles. In order to
assess how quickly the enzyme catalysed hydrolysis occurs, a thermolysin solution
was added to Fmoc-AA modified particles. The reaction was then stopped with
89
0.1% TFA at different times and analysed with HPLC to quantify any cleaved
residues. The Figure 3 -39 shows that a maximum hydrolysis of the dipeptide was
attained after approximately 110 minutes for µPEGA. This experiment was repeated
on larger (200-400 µm diameter) commercially available PEGA 800 macroparticles,
where maximum hydrolysis was found after 150 minutes with an initial rate three
times slower than the enzyme reaction on the microparticles. Other workers79 have
also shown that on larger commercially available particles maximum hydrolysis was
obtained after a much longer time, typically ~ 4 hours.
Figure 3-39. Comparison of time dependence of enzyme reactions on ♦ (blue): µPEGA and ■
(red): commercially available macroparticles (curves are guides to the eye).
1.1.5. Functionalisation with peptide actuators

Enzyme responsiveness was incorporated into PEGA microparticles through SPPS.
The peptide actuator functionality is shown schematically in Figure 3 -28. These
peptide actuators are made up of four amino acids and which can be divided into two
sections based on their role; in the centre of the peptide actuator is an uncharged
dipeptide (shown in green) this is the sensing element the enzyme cleavable peptide
(ECP), while at each end of the peptide actuator is an oppositely charged amino acid
(making the peptide actuator a zwitterion). If a protease with specificity for the ECP
90
is present it hydrolyses the amide bond in the ECP and only the positively charged
half of the peptide actuator remains covalently attached to the polymer.
Additionally, dependant on the pH of the surrounding solution (if below pH 7.5) the
amine on the remaining half of the ECP may become protonated and therefore
positively charged. Within the polymer network these neighbouring cationic
fragments of the peptide actuators electrostatically repel one another resulting in an
increase in the swelling of the polymer particles. These peptide actuators have
already been shown on macroparticles (this is covered in more detail in the
preceding literature review, within section 2.3.3.2). By making use of the faster rates
of complete hydrolysis observed on microparticles it should be possible to
demonstrate a faster enzyme responsive system.
1.1.5.1. Enzyme responsive increase in accessibility

Enzymes are highly specific and in order to determine whether this specificity could
be used to for selective release, the change in accessibility of the microparticles was
measured after exposure to three different enzymes. µPEGA was firstly
functionalised with peptide actuator with three different ECPs, these were AA, GG
and FF. The change in accessibility upon enzyme treatment was then determined by
immersing the particles in a solution containing FITC labelled dextrans of known
molecular weight, then using confocal microscopy to visualise the location of
fluorescence. Confocal microscopy allows fluorescent cross-sectional images of the
particles to be obtained. This technique can have problems with quenching occurring
in the centre of hydrogel particles 79 however, because of the small size of the
microparticles quenching was not observed and the technique is suitable for this
purpose.
The three proteolytic enzymes tested were Thermolysin, Chymotrypsin and Elastase.
Each of these enzymes has a different specificity (i.e. the composition of the
peptides that they will hydrolyse), details of these specificities and the molecular
weight of the enzymes is shown in Table 3 -4. Thermolysin, a thermostable
metalloproteinase produced in the culture broth of Bacillus thermoproteolyticus113
has a well-known specificity for hydrophobic amino acids in the P’1 and little
specificity for the P1 position.112 Chymotrypsin, an enzyme found in the digestive
systems of mammals was chosen due to its complementary specificity towards
substrates with aromatic groups or to a lesser extent, hydrophobic amino acids with
91
bulky side chains in the P1 position with little specificity for the amino acid in
P’1.114 Finally, Elastase was included due to its clinically relevance in its
involvement in breaking down the extracellular matrix (ECM); it preferentially
cleaves peptide bonds between amino acids with small uncharged side chains
(alanine or valine in the P1).114
Table 3-4. Specificity of the enzymes used towards amino acids (AA) in the substrate and their
molecular weight.
Enzyme Thermolysin Chymotrypsin Elastase
Specificit Hydrophobic AA in Aromatic/Hydrophobic Small uncharged
y P’1 AA in P1 AA in P1 and P’1
Molecular
Weight 35 25 25
(kDa)
Table 3 -5 shows the relative cleavages of each ECP for each enzyme, in which
100% cleavage was taken as the maximum peak area detected on HPLC for that
ECP (therefore discounting the dipeptides at sites inaccessible to the enzymes). As
expected, thermolysin was found to give the highest cleavages for both the AA and
FF enzyme cleavable peptides due to its specificity for hydrophobic residues. Some
hydrolysis also occurred for ECP the composed of GG indicating that the enzyme is
somewhat unspecific. Chymotrypsin would be expected to cleave the FF ECP more
than the other ECPs, however, the comparatively high cleavage of the peptide
actuator with GG as the ECP was not expected (due to the hydrophilic nature of
glycine). Standard chymotrypsin is known to contain trypsin impurities, as it is
derived from the proteolytic cleavage of chymotrypsinogen by trypsin. 115 Trypsin
has been shown to cleave peptides when hydrophobic residues or lysine or arginine
are in the P’1 position (except when praline is in the P1),116 therefore, the higher
observed value of hydrolysis for the GG ECP may be due to some cleavage
occurring between the ECP and arginine. Elastase was observed to give high
percentage cleavages for the AA ECP due to its preference for cleaving peptide
bonds between amino acids with small uncharged side chains.
Table 3-5. Values for HPLC relative enzyme cleavage for each ECP.
ECP Thermolysin Chymotrypsin Elastase
92
Ala-Ala (AA) 100% 4% 99%

Gly-Gly (GG) 9% 20% 6%
Phe-Phe (FF) 100% 23% 14%
When the peptide actuator was intact (the peptide actuators were zwitterions) the
accessibility was determined to be less than 40 kDa. The 40 kDa fluorescently
labelled dextran was not able enter the particles. This was because the net charge of
the peptide actuators was neutral. However, if the ECP was cleaved then there was a
switch from uncharged to positively charged resulting in an increase in swelling of
the particles and therefore mesh size of the network. This made the polymer network
more accessible allowing the 40 kDa dextran to diffuse into the microparticles. This
process is shown schematically in Figure 3 -40. The confocal micrographs of
representative microparticles observed in the enzyme specificity investigation are
provided in Figure 3 -41. It can noted that a sufficient increase in accessibility to
allow the FITC dextran to enter the particles is only seen for thermolysin treatment
of AA and FF, while elastase only initiated a response with AA as the ECP. HPLC
was used to provide quantitative information about the hydrolysis of the peptide
actuators (Table 3 -5) showing that there was still some hydrolysis of the peptide
actuators for all ECPs. However, the hydrolysis (up to 23 %) was insufficient to
trigger an increase in accessibility.
Figure 3-40. A schematic of enzyme responsive microparticles illustrating both successful (top)
and unsuccessful (bottom) cleavage of the ECP resulting in a change of accessibility to a 40 kDa
FITC labelled dextran.
93
Figure 3-41. Confocal microscopy images of representative microparticles in an aqueous

solution of 40 kDa fluorescently labelled dextran. Scale bar is 20 µm.
1.1.5.2. Demonstration of the encapsulation of a payload

In order to be able to release a payload it must first be encapsulated. To achieve this
the pH responsive nature of the peptide actuators was utilised. The charges within
the peptide actuator are due to the acidic and basic amino acid side groups. By
lowering the pH below the pKa of the carboxylate side group it becomes protonated,
changing the net charge of the peptide actuator from neutral to positive. This results
in electrostatically induced swelling leading to an increase in accessibility. Through
the use of confocal microscopy to observe the spatial confinement of a FITC
labelled 40 kDa dextran the loading and encapsulation process was demonstrated
(Figure 3 -42). At pH 7 fluorescence was not observed within the particles
indicating that the dextran was not able to diffuse into the polymer. Upon lowering
the pH to 3 the 40 kDa FITC dextran was able to diffuse into the particles. The
apparent decrease in the fluorescence at pH 3 is due to the pH dependence of the
fluorophore efficiency. By returning the pH to around pH 7 by the addition of base
the dextran remained in the particles. After repeated washing with water,
fluorescence remained inside the particles.
94
Figure 3-42. The pH responsive loading of the 40 kDa FITC labelled dextran (1 mg/ml) into the
PEGA particles, gain of images varied. (A) Particles in a solution of the dextran in water. (B)
Particles in a solution of dextran in dilute HCL at pH 3, purple colour represents detector
saturation. (C) Particles after being lowered to pH 3, neutralised to pH 7 and washed several
times with water. Scale bar is 15 µm.
1.1.5.3. Enzyme specific release

Utilising the method described in section 1.1.5.2 the peptide actuator functionalised
particles were loaded with 40 kDa FITC dextran and washed. The particles were
then treated with either thermolysin or chymotrypsin in water. Thermolysin has
specificity for the ECP (AA) in the peptide sequence therefore it should cleave the
sequence triggering an increase in swelling and thus release. The fluorescent
intensity of the solution surrounding the particles was then measured (after 85
minutes), determining whether the FITC labelled dextran has been released. The
result showed there was a dramatic difference between the amount of release for
thermolysin and chymotrypsin with approximately 15 times more release occurs
when treated with thermolysin compared to chymotrypsin. This is due to the specific
enzymatically hydrolysis of the ECP that only occurs with thermolysin.
1.1.5.4. Enzyme responsive increase in swelling; effect of ionic strength

The responsive nature of the functionalised particles could also be assessed by
monitoring the change in size of individual particles when treated with different
enzymes. Figure 3 -43 shows that when the particles were treated with thermolysin
dissolved in water there is an increase in volume of ≈ 60 % after 15 minutes.
Additionally, we observe the specificity of the response as chymotrypsin did not
trigger an increase in swelling. However, if the linear peptide actuator functionalised
µPEGA was treated with thermolysin in a solution at ionic strength of 0.18 M no
change in swelling was observed. This was a result of the electrostatic screening of
the charges on the peptide actuators by the ions within the solution. This finding has
95
also been observed on peptide actuator-functionalised PEGA macroparticles by

other researchers.58
Figure 3-43. Change in volume of individual particles over time with different treatments: ♦
(blue): Thermolysin in water. ■ (red): Chymotrypsin in water. ▲ (green): thermolysin in buffer
at an ionic strength of 0.18 M.
96
3.5. Conclusions
Three different types of PEGA microparticles have been prepared. PEGA hydrogels
incorporating charges within the polymeric backbone displayed increased swelling
in water due to the electrostatic repulsion resulting in a greater uptake of water.
Within a buffer solution 0.1 M all PEGA hydrogels had similar swelling ratios due
to the screening of some of the longer range electrostatic interactions within the
polymers. Neutral µPEGA was chosen for further investigation these cell-sized
particles had a mean diameter of 15 µm and a polydispersity of 43 %. The
distribution of amines was homogeneous throughout the particles. It was shown that
enzyme action on coupled peptides was also homogeneous. These microparticles
gave rise to faster enzymatic hydrolysis than commercially available large PEGA
particles (macroparticles) due to the greater surface area to volume ratio of smaller
particles. When µPEGA was functionalised with peptides actuators an enzyme
specific response through the increase in the accessibility was observed. The pH
responsiveness of these particles has been demonstrated by the loading of the
particles with a fluorescently labelled macromolecule. Enzyme specific release of
this payload was possible. However, at physiological ionic strength no enzyme
triggered swelling of the functionalised particles was observed. This limitation will
be addressed in the following chapter with the design of branched peptide actuators.
97
4 Designing new peptide actuators for improved enzyme
responsive behaviour3
4.1. Abstract
This chapter describes the functionalised μPEGA with branched peptide actuators.
These peptide actuators provided enhanced charge density and responded through an
increase in swelling to the target enzyme at physiological ionic strength. Analysis of
enzymatic activity revealed that the target enzyme (thermolysin) could access the
core of particles when linear peptides are used, while access was restricted to the
surface when using branched actuators due to electrostatic interactions. These
responsive μPEGA particles were then loaded with a fluorescent labelled dextran by
application of a sequential pH change. The macromolecule payload could be
selectively released at physiological ionic strength when exposed to the target
enzyme.
4.2. Introduction
1.1.6. Branched peptide actuators

As demonstrated at the end of Chapter 3 and earlier systems from the Ulijn and co-
workers,58 responsive systems based on electrostatic actuation using peptide
actuators are inherently sensitive to the ionic strength of the solution. This limitation
meant that they were unable to function in solutions with physiological ionic
strength due to electrostatic screening.58 In this chapter this problem is addressed
through the design of branched peptide actuators with enhanced charge density.
The inability of the linear peptide actuator to respond at physiological ionic strength
was related to the electrostatic screening of neighbouring charges. 58 For charge
induced swelling to occur the distance between the charges must be less than the
3
This chapter has been published as: Mcdonald, T.O., Qu, H., Saunders, B.R. and Ulijn, R.V.,
Branched Peptide Actuators for Enzyme Responsive Hydrogel Particles. Soft Matter. (2009).
98
Debye length. At physiological ionic strength (0.15 M) the Debye length is 0.8 nm.
By increasing the space occupied by the actuator we aimed to reduce the distance
between neighbouring charges to less than 0.8 nm. The approach used to was to
incorporate the di-amino functionalised amino acid lysine (K) to act as a branch
point thus causing the peptide actuator to occupy more space (Figure 4 -44). Each
arm consists of three parts: i) a di-glycine (G) spacer to enable enzyme access, ii)
oppositely charged actuation amino acids and which are iii) separated by an enzyme
cleavable peptide (ECP) sequence which serves as the sensing element. By matching
the ECP to the specificity of a target protease, the material may be programmed to
respond exclusively to a target enzyme. Enzymatic hydrolysis of the ECP leads to
release of anionic fragments and conversion of the branched zwitterionic peptide
actuator to cationic groups remaining covalently bonded to the polymer (Figure 4
-44). These neighbouring cationic groups induce swelling and an increase in the
mesh size within the polymer which can be exploited in triggered release of pre-
entrapped payload molecules.
The objectives within this chapter were to: (i) synthesise and characterise branched
peptide actuators on µPEGA, (ii) investigate the enzyme response behaviour of the
functionalised particles and (iii) utilise these particles for triggered enzyme of a
payload at physiological ionic strength.
99
Figure 4-44. Schematic of enzyme responsive branched peptide actuator. Left; peptide
actuator, Right; upon cleavage of peptide by an enzyme only the cationic groups remain
attached. These charged peptide fragments then electrostatically repel one another leading to
an increase in swelling of the polymer.
100
4.3. Materials and methods
4.3.1. Materials
All chemicals were used as supplied from Sigma with the exception of amino acids
(Bachem), PEGA macromonomers (Versamatrix), Isopar M (Multisol) and N,N,N′,N
′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)
(AGTC Bioproducts Ltd). The enzymes used were thermolysin (EC 3.4.24.27), 36.5
U/mg and chymotrypsin (EC 3.4.21.1), 60 U/mg.
4.3.2. Inverse suspension polymerisation and polymer characterisation

A stainless steel baffleless reactor (250 ml) stirred with an anchor-style agitator was
used for the polymerisation reaction. 3.14 g (3.4 mmol) of the PEGA 800
macromonomers (2:1 ratio of acrylamide-PEG-acrylamide to amino-PEG-
acrylamide) and 0.156 g (2.2 mmol) of acrylamide were dissolved in 10 ml of
distilled water and purged for 30 minutes with N gas. 50 ml of Isopar M
2
(isoparaffin) was added to the reactor and was also purged for 30 minutes. The
reactor was heated to 70°C. After 20 minutes of purging 0.16 ml (1.0 mmol) of
N,N,N′,N′- tetramethylethylenediamine (TEMED) was added to the oil phase. 0.164
g (0.47 mmol) of Span 20 (sorbitan monolaurate) was dissolved in the oil, which
was stirred at 500 rpm for 30 seconds to ensure the surfactant was fully dispersed in
the oil phase. 0.070 g (0.30 mmol) of ammonium persulfate (APS) was dissolved in
the macromonomer solution, which was added to the oil phase in the reactor and
stirred at 2000 rpm for a further 30 minutes. The particles were washed with (3 x 50
ml) dichloromethane (DCM), (3 x 50 ml) Tetrahydrofuran (THF), (3 x 50 ml)
methanol and (4 x 50 ml) distilled water. Particle size distribution and mean particle
diameter were determined using a Malvern Mastersizer particle size analyser,
Mastersizer Microplus software version 2.18 was used to analyse the results.
Environmental scanning electron microscopy (ESEM) images were taken on a FEI

Quanta 200 ESEM, using low vac mode at 10.0 KV.
4.3.3. Solid phase peptide synthesis

Peptide actuators (linear: Fmoc-DAAR-PEGA) and branched: (Fmoc-DAARGG)2-
K-PEGA) were prepared by solid phase peptide synthesis using Fmoc protected
101
amino acids. 8 equivalents of the amino acid and 7.8 equivalents of HBTU were
dissolved in 2 ml of N,N-Dimethylformamide (DMF). 16 equivalents of N,N-
Diisopropylethylamine (DIPEA) was added to this solution prior to its addition to
0.1 g of µPEGA. Coupling was left for 16 hours, and Kaiser test 117 was used to
check for complete coupling. Deprotection was achieved using 20 % piperidine in
DMF for 2 hours. This procedure was repeated to build up the peptide sequence with
thorough washing between steps (5 x 5 ml methanol, 5 x 5 ml 1:1 methanol:DMF, 5
x 5 ml DMF). A solution of 95 % Trifluoroacetic acid (TFA) and 5 % water was
used to remove the amino acid side chain protecting groups.
4.3.4. Microscopy and determination of swelling

Swelling measurements and fluorescent images were obtained using a Zeiss Imager
A1 microscope, a Leistungselektronik mbq 52 AC power source (Jena, Germany)
equipped with an HBO 50 mercury lamp and Canon Powershot G6 camera. A Zeiss
filter set 09 (excitation 450 - 490 nm, emission 515 + nm) was used to visualise
FITC. For pH responsive swelling peptide actuator functionalised PEGA particles
were immersed in either water (CHROMASOLV plus for HPLC) or 0.01 M HCl
(pH 2.5) and images obtained using the microscope. ImageJ 1.38x analysis software
was used to determine the change in particle diameter. For pH responsive swelling
the diameter of a minimum of 300 particles was measure and the mean volume
calculated. For enzyme responsive swelling individual particles were observed
throughout the timecourse and an average V/V o was determined from at least six
representative particles, where V is the volume of the particle at time t and V o is the
volume of the particle immediately after exposure to enzyme solution. Enzyme
solutions were made up at a concentration of 1 mg/ml and buffers were prepared by
using the appropriate amounts of sodium phosphate dibasic heptahydrate and
sodium phosphate monobasic.

µPEGA functionalised with either linear or branched peptide actuators was exposed
to an aqueous thermolysin solution (1 mg/ml) for 40 minutes. The particles were
then washed with (5 x 1 ml) Acetonitrile (AcN):H 2O (50:50) containing 0.1 % TFA
and then (5 x 1 ml) DMF. 6 equivalents of dansyl-Cl were dissolved in 2 ml DMF
along with 10 equivalents DIPEA this solution was added to the particles and
102
incubated in the dark at room temperature for 2 hours. The dansyl-labelled particles
were washed with (3 x 1 ml) DMF and (3 x 1 ml) water. Two-photon microscopy
images of the particles in water were obtained on a Leica TCS SP2 inverted
microscope. ImageJ software was used to produce the surface plots of intensity. The
intensities at the centre, the outside (1 µm from the edge of the particle) and the
average intensity across the particle were determined for 15 particles functionalised
with either the linear or branched peptide actuators.
4.3.6. Entrapping payload

The particles (0.1 g) were loaded with a 40 kDa FITC Dextran using the following
method; 2 ml of solution of FITC dextran in 0.01 M HCl (10 mg/ml) was added to
the particles for 30 minutes (pH 2.5). After this time 0.4 ml of 0.05 M NaOH was
added dropwise (pH 7). The particles were then filtered and washed (2 x 5 ml water,
2 x 5 ml 0.18 M buffer, 2 x 5 ml methanol).
4.3.7. Release measurements

The release of entrapped dextran was determined using the following method;
loaded particles were treated in solution for 80 minutes, these samples were
centrifuged and the supernatant was removed. A Jasco FP – 6500
Spectrofluorometer using an excitation wavelength of 490 nm was used to obtain the
fluorescent intensity at 519 nm. Treated particles were filtered and imaged using
fluorescence microscopy.
4.3.8. HPLC and LCMS

HPLC experiments were undertaken on a Dionex HPLC (P680 pump, ASI-100
Automated sample injector, Nucleosil 100-5-C18 column with a UVD170U
detector), using a solvent ramp of 20 % ACN and 80 % water to 80 % ACN 20 %
water over 30 minutes (0.1 % TFA was present in both phases). Chromeleon 6.60
software was used for analysis. All LCMS analyses were carried out on a reverse-
phase Luna C18(2), 250 x 2mm, 5 micron column (Phenomenex). The LC-MS
instrument was an Agilent 1100 Series HPLC, coupled to an Agilent 1956B Mass
Detector. The solvent ramp of 90 % water 10 % ACN to 15 % water 85 % AcN over
14 minutes was used in all analyses; the flow rate was set at 1.0 mL min -1. Mass
detection was set to analyse in SCAN mode with electrospray ionisation.
103
4.4. Results and discussion
4.4.1. Microparticle characterisation and peptide functionalisation

µPEGA was produced (described in detail in Chapter 3) and peptide
functionalisation was achieved through solid phase peptide synthesis using Fmoc
protected amino acids. Use of the Kaiser test and the quantification of Fmoc
removed at each deprotection step indicated high yield peptide formation with over
90 % of the initial loading achieved for the final step (Figure 4 -45).
Figure 4-45. HPLC quantification of Fmoc removed after each coupling step for linear and
branched peptide actuators.
The charged structure of the peptide actuators can be indirectly determined by

examining the pH responsive swelling of the peptide actuators. The charged state of
these peptide actuators is pH dependant, as the amino acid side chains have pK a
values of 4.4 and 12.0 for aspartic acid (D) and arginine (R) respectively. Therefore,
if the pH was lowered below 4.4, the previously anionic carboxylate groups became
protonated giving the peptide actuators a net positive charge. Electrostatic repulsion
between neighbouring positively charged actuators led to an increase in swelling of
the particles. This can be seen in Figure 4 -46 where at pH 2.5 there was a 30 %
increase in volume for particles functionalised with linear peptide actuator while a
57 % increase in volume was noted with branched peptide actuators. This difference
can be attributed to the higher density present in the branched actuator.
104
Figure 4-46. pH responsive swelling behaviour of peptide actuators on PEGA microparticles

(average of at a minimum of 300 particles). Student’s t-test shows a difference is significant at
98 %.
4.4.2. Actuator design and responsiveness of peptide functionalised particles

The responsive increase in particle swelling is dictated by the extent of electrostatic
repulsion between neighbouring charged groups. Figure 4 -47 A shows the enzyme
responsive swelling of PEGA microparticles functionalised with linear peptide
actuators in water. The distance between charged groups in linear actuators (Fmoc-
DAAR-PEGA) is greater than the Debye length at 0.15 M (0.8 nm) and therefore did
not give rise to sufficient electrostatic repulsion to cause swelling. The branched
design of the new actuator occupies more space than the linear actuator resulting in
the cationic groups being closer together. Figure 4 -47 B shows that when the
particles functionalised with the branched actuator are treated with thermolysin
(from Bacillus thermoproteolyticus rokko E.C. 3.4.24.27) at an ionic strength of
0.18 M an increase in volume is observed reaching a maximum of ~1.3 V/V o after
20 minutes. This response is exclusively observed upon treatment with thermolysin.
This enzyme has a relatively broad specificity preferring hydrophobic residues in the
P1’ position.112 While treatment with α-chymotrypsin from bovine pancreas (E.C.
232.671.2), which has a preference for large hydrophobic residues in the P1
105
position,114 did not give rise to a change in swelling (visualised in Figure 4 -47 C).
Indeed, HPLC and LCMS analysis of the solutions obtained after enzyme treatment
shows that ECP cleavage occurs exclusively with thermolysin (Figure 4 -48 A).
Furthermore, LCMS data after enzyme treatment shows two main peaks consisting
of fragments of the desired peptide sequence (Figure 4 -48 B & C). Therefore,
functionalisation of µPEGA with the branched peptide actuator has achieved
enzyme specific response at physiological ionic strength.
Next, the ionic strength dependence of the branched actuators (Figure 4 -49) was
investigated by determining the final increase in swelling. It was found that, due to
the enzyme triggered increased charge density maximum swelling (~1.3 V/V o) was
observed at and above physiological ionic strength (0.15 M)118,119 up to 0.3 M. This
corresponds to a Debye length of 0.55 nm. Presumably, at higher ionic strengths the
mobile ions in solution begin to screen the static peptide charges effectively
resulting in a decrease in the amplitude of the response. This is most pronounced at
an ionic strength of about 0.45 M where V/V o ≈ 1.15. As expected, chymotrypsin
did not initiate a response at any ionic strength tested. To conclude this section, the
branched peptide actuator functionalised particles demonstrate specific enzyme
responsive at physiological ionic strength and above.
106
Figure 4-47. Enzyme responsive swelling behaviour of peptide actuator functionalised µPEGA
at pH 7. A: Swelling of particles functionalised with the linear peptide actuator; ●:
Thermolysin in water, ¯ : Thermolysin at ionic strength 0.18 M. B: Swelling of particles
functionalised with the branched peptide actuator; ¯: Thermolysin at ionic strength 0.18 M, r:
Thermolysin at ionic strength 0.76 M, ¢: Chymotrypsin in water. C: Swelling of individual
particles treated with either Thermolysin or Chymotrypsin at 0.18 M ionic strength (circle
shows original size, scale bars are 15µm).
107
Figure 4-48. HPLC and MS analysis of enzyme hydrolysis of branched peptide actuators. A:
HPLC traces for thermolysin (blue) and chymotrypsin (pink) treated particles modified with
branched peptide actuators. Absorbance at 254 nm. The ratio of peak areas is 3:1 (1 st peak : 2nd
peak). B: Mass spectra for thermolysin treated particles modified with branched peptide
actuators peak at 22.1 minutes. C: Mass spectra for thermolysin treated particles modified with
branched peptide actuators peak at 24.8 minutes. D: Key of peptide fragments found.
Figure 4-49. Effect of ionic strength on the maximal enzyme responsive swelling of branched
peptide actuator functionalised µPEGA at pH 7 (after 40 minutes) ¯ :Thermolysin , r: No
enzyme, ¢: Chymotrypsin. Dashed line indicates physiological ionic strength.
108
4.4.3. Characterisation of enzyme action on peptide actuators

Enzymatic hydrolysis of the peptide actuators produces polymer-bond peptide
fragments with free amines (see Figure 4 -44). The distribution of amines can be
examined through labelling with dansyl which allows the location of enzyme
hydrolysis within the particles to be investigated (as also previously described in
section 3.4.3.1). TPM was used to assess the distribution of fluorophores (dansyl)
within PEGA particles functionalised with either linear or branched peptide
actuators after 40 minutes thermolysin treatment. As seen in Figure 4 -50 A & B
enzyme cleavage of the linear peptide actuators on PEGA particles was
homogeneous on the micrometer scale, while branched peptide actuator
functionalised particles had greater cleavage in the outer regions of the particles. The
pI of thermolysin is 4.97120 and the protein was therefore negatively charged at
neutral pH. It is likely that electrostatic attraction between the cationic fragments on
the particle formed as a result the enzymatic hydrolysis of the ECP and the anionic
enzyme lead to enzyme retention. It is expected that this interaction would to be
stronger for branched actuators (double the positive charge). Therefore, enzyme
diffusion may be slower in particles containing branched actuators (i.e. enzymes are
held in place after enzymatic hydrolysis). Indeed, similar electrostatic retention has
been observed previously.58 Linear peptide actuators have been shown in water to
produce a maximal V/Vo of 1.6 (Figure 4 -47 A) while the maximal swelling for the
branched peptide actuators observed was 1.35 (Figure 4 -47 B). The heterogeneous
enzymatic cleavage apparent for the branched peptide actuators explains the reduced
maximum swelling observed when compared to the linear peptide actuator.
109
Figure 4-50. Comparison of thermolysin action on both linear and branched peptide actuators
on µPEGA. A: Two-photon micrographs of representative particles labelled with dansyl (scale
bar is 15 μm). B: Surface plot of two-photon images indicating qualitatively the fluorescent
intensities of the particles. C: Quantification of fluorescent intensities of dansyl labelled,
enzyme treated particles functionalised with either linear or branched peptide actuators.
4.4.4. Enzyme triggered release

The applicability of branched peptide actuator functionalised particles to
enzymatically triggered release a payload was assessed. Firstly, a payload was
entrapped within the particles. This was achieved by utilising the pH responsiveness
of the peptide actuators.58 Here, fluorescein isothiocyanate (FITC) dextran (40 kDa)
was the payload macromolecule. By lowering the pH of the payload solution to pH
2.5 the aspartic acid residue side chain is protonated (while the R side chain remains
110
protonated) making the net charge of the peptide actuator positive, resulting in an
increase in the swelling (Figure 4 -46 B) and thus mesh size of the polymer
microparticles. The pH switch therefore allowed the 40 kDa dextran to diffuse into
the particle. By returning the system to pH 7 the D side chain carboxyl group again
becomes ionised. In this case the net charge of the peptide actuator becomes zero
and the repulsive forces are removed. The polymer particle deswells and entraps the
dextran. Figure 4 -51 A shows a fluorescent micrograph of these particles after
washing demonstrating that the FITC labelled dextran was entrapped within the
particles. In Figure 4 -51 B & C the particles are treated with buffer and
chymotrypsin respectively (both at 0.18 M ionic strength), because no actuation had
occurred the majority of the FITC dextran remained entrapped with some leakage
apparent. It is likely that some of the dextran was not completely entrapped within
the particles resulting in leakage.
Figure 4-51. µPEGA particles functionalised with the branched peptide actuator at pH 7 (scale
bars are 100 μm). A: After loading with FITC labelled 40 kDa dextran and washing. B: After
loading and 80 min treatment with Thermolysin. C: After loading and 80 min treatment with
Chymotrypsin. D : After loading and 80 min treatment with buffer E: mmoles of FITC labelled
dextran released per g of particles after 80 min treatment. (all experiments were carried out at
an ionic strength of 0.18 M)
111
When the particles were treated with thermolysin (Figure 4 -51 D) the increase in
swelling (and corresponding increased pore size) allowed the dextran to diffuse out
of the particles, resulting in a reduction of fluorescence of the particles. By
measuring the fluoroscence of the solution surrounding the particles it was possible
to measure the amount of FITC dextran released (Figure 4 -51 E). When the
particles were exposed to either buffer alone or chymotrypsin a small amount of
leakage was observed (~ 0.02 mg dextran/mg particles). While treatment with
thermolysin resulted in a 350 % increase in dextran release (Figure 4 -51 E). Hence
the enzyme triggered increase in swelling and accessibility was responsible for the
greater release of the entrapped macromolecules.
To summarise, by exploiting the enzyme responsive swelling it was possible to

release an entrapped macromolecule in the presence of the target enzyme.
Electrostatically induced swelling occurs upon cleavage of the ECP within the
peptide actuator at physiological ionic strength.
4.5. Conclusions
Branched peptide actuators have been synthesised and incorporated into μPEGA.
These functionalised particles were capable of specifically responding to selected
enzymes at a variety of ionic strengths. Through the physical entrapment of a
macromolecule payload within the functionalised particles it was possible to obtain
payload release at physiological ionic strength in response to target enzyme. This
system may have applications in a number of areas including drug delivery and
automated bio-sensing of ‘on-bead’ libraries.
112
5 Microfluidic preparation of low polydispersity PEGA
particles
1.2. Abstract
This chapter details the preparation of PEGA particles by a simplified microfluidic
setup assembled using a needle and tubing. The size of the particles produced was
determined by the surfactant concentration and the relative flow rates of the
dispersed and continuous phases. The minimum particle size obtained was limited
by the diameter of the needle (335 μm). The development of a flow-focussing setup
allowed for the production of smaller particles down to 100 μm in diameter.
Optimum conditions were determined allowing for the production of 4000 particles
a minute with a mean diameter of 160 μm.
1.3. Introduction
This chapter describes the development of microfluidic devices for the preparation
of near monodisperse4 polymer particles. Monodisperse particles present a number
of advantages over polydisperse samples: the sample of particles is well defined, any
reaction occurring on or in the particles occurs at the same rate. Additionally, any
change in swelling or size is easy to determine without the need for a large sample
size.
Microfluidic systems offer the potential to produce particles with a very narrow
distribution because the size each monomer droplet formed is determined by the
conditions at the mixing point. As each droplet is formed under constant conditions
the size of the droplets are the same. The introduction of a polymerisation method,
normally UV initiation results in the production of polymer particles. In these
systems there are a number of variables that control the mixing process and therefore
4
According to the standards of the National Institute of Standards and Technology (NIST): “a
particle distribution may be considered monodisperse if at least 90% of the distribution lies within
5% of the median size” (Particle Size Characterization, Special Publication 960–961, January 2001).
113
ultimately determined particle size. The following work aims to find the optimum
conditions to produce near monodisperse µPEGA. These particles would be better
defined and more homogenous than the samples prepared by inverse suspension
polymerisation in Chapter 3. The very narrow size distribution would offer the
potential for rapid screening with an automated cell sorter. Using this technique it
could be possible to rapidly screen for enzyme action on µPEGA using peptide
libraries.72,73,80
The polymerisation of monodisperse particles using microfluidic devices was first

demonstrated in 2005 by Whitesides and co-workers,91 since then there has been
great interest and developments in the use for microfluidics for the preparation of
polymer particles.92,93,95-97,121 These systems conveniently allow for the size of the
droplets (and therefore particles) to be controlled by the flow rates of the continuous
and dispersed phases. However, in order to prepare most microfluidic devices
microfabrication techniques are needed; typically, soft-lithography is used to form
planar microchannels.91,95,122 There are a number of limitations associated with these
devices: Usually a clean room is required for device production. Surface treatment
of the channel walls can be needed to prevent an inverse emulsion forming.
Additionally it is often easy for the microchannels to become blocked by solid
polymer. Simplified microfluidics setups have been shown that do not require no
specialist fabrications techniques. These typically make use of needles and/or tubing
to create near monodisperse droplets.92,97 These publications have been covered in
more detail in the preceding literature review (Section 2.5). The research in this
section is based on a paper by Hadziioannou and co-workers in which the dispersed
phase is introduced to the continuous phase via a needle positioned along the main
axis of the tubing.97 The axisymmetric setup of the device prevents the need of
surface modification because the dispersed phase droplets do not come into contact
with the channel walls.93 While any blockages in the tubing due to polymer build up
can simply be rectified through a fast and low cost replacement of that section of
tubing. In this chapter a microfluidic device as described by Hadziioannou (shown
schematically in Figure 5 -52) was used to produce PEGA particles.
114
Figure 5-52. Schematic of microfluidic setup for the synthesis of controlled-size polymer
particles. The dispersed phase (containing the monomer) and the continuous phase are
delivered by syringe pumps. The dispersed phase is introduced to the co-flowing continuous
phase the centre of the PTFE tubing by means of a 26 gauge needle.
The objectives of this chapter were to: (i) Optimise the conditions to manufacture
particles with the smallest diameter possible with a very narrow size distribution. (ii)
Develop strategies to produce smaller particles, through the use of a simplified flow-
focusing setup.
115
1.4. Materials and methods
1.4.1. Microfluidic system

Figure 5 -52 shows a schematic of the microfluidic system. A stainless steel needle
blunt tip needle (26 gauge) internal diameter (ID) 240 μm was inserted into a T-
junction (Swaglok T-junction SS-100-3). The needle tips exits the T-junction in the
centre of the polytetrafluoroethylene (PTFE) tubing. Two syringe pumps (New Era
Pump Systems Inc. NE-1000) were used to deliver the continuous and dispersed
phases at a specific flow rate. The two phases were carried in PTFE tubing with an
internal diameter (ID) of 1.6 mm. The dispersed phase was injected through the
needle while the continuous phase was injected perpendicular to the main axis of the
T-junction. The outlet tubing was also PTFE with ID 1.6 mm tubing with a length of
120 cm. The exit of the needle was located in the centre of the outlet tubing. The
outlet tubing entered a UV box where it was exposed to UV light (320-390 nm
wavelength) from a Dymax model 5000 Flood using a Dymax 400 W power source.
Residence time within the UV box was 30-240 seconds depending on the flow rates.
Particles were collected at the end of the outlet in a water bath.
1.4.2. Flow focussing setup

A modified T-junction was used to produce smaller droplets. Here, a 26 G needle
was inserted though a Fisher Tubing connector T connector Nylon 1/16in Masterflex
entering a 21 G needle. The internal needle ends approximately 2/3 of the way down
the external needle (Figure 5 -58). Two syringe pumps were used to deliver the
continuous and dispersed phases at a specific flow rate. The two phases were carried
in PTFE tubing with an ID of 1.6 mm. The dispersed phase was injected through the
central needle while the continuous phase was injected perpendicular to the main
axis of the T-junction and flowed through the outer needle. The outlet tubing was
also PTFE ID 1.6 mm tubing with a length of 30 cm. The exit of the outer needle
was located in the centre of the outlet tubing. The outlet tubing entered a UV box
where it was exposed to UV light (320-390 nm wavelength) from a Dymax model
5000 Flood using a Dymax 400 W power source. Residence time within the UV box
was 30-240 seconds depending on the flow rates. Particles were collected at the end
of the outlet in a water bath.
116
1.4.3. Monomer and continuous phase preparation

All materials were used as supplied from Sigma-Aldrich with the exception of
PEGA macromonomers (Versamatrix), photoinitiator (Ciba) and Isopar M
(Multisol)
The dispersed phase consisted of an aqueous solution (10 ml ultrapure water)

containing 3 g (3.34 mmol) PEGA macromonomers, 0.15 g (2.11 mmol) acrylamide
and 0.10 ml darocure 1173 photoinitiator. 0.05 ml of red food colouring (Supercook)
was included in early experiments to give greater contrast to the monomer droplets.
The density of the solution was 1.008 g/ml. The viscosity solution as measured
using a Hydramotion Viscolite 700HP model VL700-T15HP was 4.8 cP at 20 ˚C.
The continuous phase consisted of Isopar M (viscosity of 2.1 cP) in which the
surfactant Span 20 (Sorbitan monolaurate) was dissolved, the amount of surfactant
was varied. The free radical polymerisation of the dispersed phase droplets led to the
formation of insoluble particles, these particles were collected in a water bath at
room temperature. Silicone oil (20 cSt) was used without surfactant (viscosity of 20
cP).
1.4.4. Particle size measurement

The polymerised particles were collected at the exit of the outlet tube and washed (2
x 10 ml DCM, 2 x 10 ml THF, 2 x 10 ml methanol and 4 x 10 ml water). The
diameter of the particles (fully swollen in water) was measured using an optical
microscope (a Zeiss Imager A1 microscope and Canon Powershot G6 camera).
Particles above 1500 µm in diameter were photographed without use of the
microscope over grid marker paper. ImageJ was used to determine the mean
diameter of the particles, due to the sometimes asymmetrical shape of the particles
the cross sectional area of each individual particle was measured and a mean
diameter then determined from it. The mean diameter and standard deviation were
obtained by measuring the diameter of at least 30 particles.
117
5.1. Results and discussion
1.4.5. Microfluidic system

Droplet formation in microfluidic systems is determined by the balance between the
shear forces imparted on the forming droplet by the co-flowing continuous phase
and the interfacial energy between the two phases. These hydrodynamic conditions
within the microfluidic device are usually described by two dimensionless numbers;
the Reynolds number and the capillary number.95 The Reynolds number is defined in
Equation 1 and the capillary number in Equation 2. Where ρ and µ are the density
and viscosity of the liquid, respectively, D is the diameter of the outlet tubing, V is
the velocity of the liquid and γ is the interfacial tension.95,97 It has been shown that
increasing the value of the capillary number for the dispersed phase (Cad) produces
smaller droplets.95 Therefore, by increasing the velocity or viscosity of the
continuous phase or decreasing the interfacial tension smaller droplets should be
produced.
Re ≡ ρDV/µ
Equation 1. Reynolds number
Ca ≡ µV/γ
Equation 2. Capillary number.
1.4.5.1. Variation of particle size with flow rate ratios and surfactant
concentration
Initial experiments produced droplets with diameters equal to that of the tubing
(Figure 5 -53 A & B). Increasing Qc/Qd (where Qc and Qd are the flow rates of the
continuous and dispersed phases respectively), did not lead to a significant decrease
in droplet size and the particles formed (once washed and fully swollen in water)
had diameters greater than 1800 μm (Figure 5 -53). This result suggested that the
interfacial energy between the aqueous monomer solution and organic continuous
phase (isoparaffin) was relatively high. Indeed, the interfacial tension of linear
alkanes with boiling points in the range of Isopar M (190 - 260 ˚C) have been shown
to be 53.1 - 54.5 mN/m.123 Higher interfacial tensions result in higher capillary
118
pressures, (the pressure difference between inside and outside of a droplet) as given
by the Young-Laplace (Equation 3).
Δp = γ(1/R1 + 1/R2)
Equation 3. Young-Laplace equation
Where Δp is the pressure difference across the liquid-liquid interface, γ is the surface
tension and R1 and R2 and the principal radius of curvature. 124 This means that larger
droplets have a lower capillary pressure and are therefore more thermodynamically
favourable than smaller droplets. The highest calculated Cad for the experimental
results shown in Figure 5 -53 was ~ 3 x 10-5.
This setup did not lead to control of particle size, as presumably it was not possible
to overcome the interfacial forces with shear forces.
Figure 5-53. A: Effect of Qc/Qd on mean particle diameter with no surfactant in the continuous
phase, Qc= 2 ml/min. B: Photograph of PEGA particles produced using a Q c/Qd of 10 and a Qc
of 2 ml/min (scale bar is 3500 µm).
The addition of surfactant to the continuous phase results in reduced interfacial

energy due to the surfactant molecules sitting at the interface. The use of surfactants
is well established within conventional dispersion type polymerisations. 88,125
Additionally, surfactants have often been used within other publications describing
the controlled formation of droplets by microfluidics. 95 The surfactant used within
this work was Span 20 (sorbitan monolaurate), which has been shown to reduce the
interfacial tension of linear alkanes with water to between 20 – 26 mN/m (dependant
on concentration).126 The effect of including surfactant in the continuous phase was
119
dramatic (Figure 5 -54 A & B), even at the lowest Qc/Qd (10) and lowest surfactant
concentration (0.01 g/ml (three times the concentration used in the inverse
suspension polymerisation)) there was a large reduction in mean particle diameter
(625 µm compared to 2105 µm when no surfactant is present). The lowered
interfacial energy allowed the shear forces to influenced droplet size. As Q c/Qd was
increased particle size decreased reaching a plateau in mean particle diameter of
about 450 µm at flow ratio of 200. Slightly smaller particles (422 µm) were obtained
at very high values of Qc/Qd. This relationship between mean particle diameter and
Qc/Qd was seen as surfactant concentration was increased (further lowering the
interfacial forces increasing the dominance of shear forces), albeit with smaller
particles produced at higher surfactant concentrations. Upon the addition of
surfactant the value for Cad was greatly increased to ~ 9 x 10-5. Surfactant was
removed from the outside of the polymerisation particles by repeated washing in
solvents with a range of polar/non-polar characters.
120
Figure 5-54. Effect of surfactant concentration on particle size. A: Effect of Q c/Qd and
surfactant concentration (♦ (blue): 0.01 g/ml, ■ (red): 0.04 g/ml, ▲ (green): 0.08 g/ml) on mean
particle diameter, Qc= 2 ml/min. B: Effect of increasing surfactant concentration on mean
particle diameter at varying values of Qc/Qd. C: Effect of increasing surfactant concentration on
mean particle diameter at varying values of Qc/Qd excluding 0 g/ml surfactant concentration.
121
At a Qc/Qd of 10 the mean diameter of particles produced is very similar independent

of whether 0.04 g/ml or 0.08 g/ml of surfactant was used, indicating that the
minimal particle size at that flow rate had been obtained. However, as the shear
forces are increased particle diameter is also decreased. A surfactant concentration
of 0.08 g/ml and Qc/Qd of 1000 offered the smallest particles for this setup (335 µm).
Typically upon the polymerisation of a monomer droplet the resulting polymer

particle has a smaller volume. This corresponds to the increase density of the
polymer verses the monomer.91,97 With this system the dispersed phase was an
aqueous solution of the monomers. This was done to maintain a comparable method
to the inverse suspension polymerisation. It was found that monomer droplets
formed at the diameter of the tubing (≈1600 µm) initially give PEGA particles of
similar diameter (if collected in oil) which swelled after washing in water to give
much larger particles (≈2100 µm). The size of particular importance was that of the
hydrated particles as further applications of the polymer would be in aqueous
environments. Further investigation of swelling this behaviour was undertaken.
Particles were initially collected in oil then measured then washed and measured
again in water. Using this method it was determined that on average fully swollen
particles had diameter 35 % (± 3 %) larger than initial monomer droplets.
Considering this information a diameter of 335 µm which was found to be the lower
limit for particle size corresponds to droplet diameter of 248 µm, very close to the
internal diameter of the 26 gauge needle (240 µm). Earlier work using a similar
setup determined that the smallest droplets that could be produced had diameters just
above that of the needle.97 Surfactant concentrations above (0.08 g/ml) were not
assessed because above this concentration the surfactant was not completely soluble.
In summary, the introduction of surfactant to the continuous phase lowered the
interfacial tension resulting in the production of smaller particles. While increasing
Qc/Qd led to greater shear forces during droplet formation resulting in smaller
droplets and therefore particles, the lower limit in particle size was due to the
droplets formed at the needle diameter.
122
1.4.5.2. Effect of increasing total flow rate on particle size
Figure 5-55. A: Effect of total flow rate (at constant Qc/Qd) on mean particle diameter. B: Effect
of Qc/Qd and total flow rate (♦ (blue): 2 ml/min, ■ (red): 4 ml/min) on mean particle diameter,
0.08 g/ml surfactant concentration.
Very high Qc/Qd ratios did not offer efficient particle production; the rate of particles
was slow and the process was wasteful with a large amount of oil is used per particle
produced. Higher surfactant concentrations might have shown slightly reduced
diameters at lower flow ratios however, at concentrations higher than 0.08 g/ml the
surfactant did not fully dissolve in the oil. Another approach was to increase the
shear forces by increasing the total flow rate, a preliminary investigation into the
123
effect of total flow rate (at a constant Q c/Qd) on mean particle diameter showed as
flow rate was increased there was slight size decrease ( A). However, flow rates
higher than 4 ml/min did not lead to completely cured particles. Using a Q c of 4
ml/min over a range of Qc/Qd produced particles of only slightly reduced diameter
compared to 2 ml/min (Figure 5 -55 B). Hadziioannou and co-workers produced
similar findings showing that similar values of Q c/Qd led to similar diameters
independently of the total flow, Qc + Qd.97
1.4.5.3. Effect of flow ratios and surfactant concentration on polydispersity

The polydispersity5 of the particles increases when there is variation between droplet
to droplet formation or when coalescence occurs prior to polymerisation. In order to
determine whether the variables that were changed within the microfluidic setup
produced measureable trends in the polydispersity of the particles the data shown in
Figure 5 -56 was obtained. At the highest and lowest surfactant concentrations (0.01
g/ml and 0.08 g/ml) used there was a tendency for the polydispersity to increase at
higher values of Qc/Qd. While using 0.04 g/ml produced particles with
polydispersities in the range of 2-3 % over the range of Q c/Qd values tested. The
overall variation of polydispersity was found to be small (between 1.4 - 4.3 %).
1.4.5.4. Variation of particle production rates with the conditions assessed

In determining the optimum conditions with which to produce PEGA particles the
production rate was assessed (Figure 5 -57). Lower values of Qc/Qd led to higher
production rates (due to higher flow rate of the continuous phase) while higher
surfactant concentrations offered higher production rates as a greater number of
smaller particles were produced from the same volume of monomer solution
(dispersed phase).
5
Defined as the standard deviation in the particle diameter divided by the mean particle diameter. 91
124
Figure 5-56. Effect of Qc/Qd and surfactant concentration (♦: 0.01 g/ml ■: 0.04 g/ml ▲: 0.08
g/ml) on polydispersity (Qc=2 ml/min).
Figure 5-57. Effect of Qc/Qd and surfactant concentration on particle production rate (♦ (blue):
0.01 g/ml ■ (red): 0.04 g/ml ▲ (green): 0.08 g/ml) (Qc=2 ml/min).
125
1.4.6. Using a simplified microfluidic flow-focussing device to produce

polymer particles
The minimum size of particles of particles obtained using the microfluidic setup
described up to this point was limited to the diameter of the needle from which the
dispersed phase was introduced into the continuous phase. To overcome this
minimum size limitation an adjustment to the microfluidic setup was made (Figure
5 -58). Flow-focussing (FF) is a technique that allows for the formation of droplets
smaller than the orifice of the dispersed phase of the device by focussing the two
phases through a second orifice, this leads to greater shear forces. This technique is
well described within the literature for planar type microfluidic devices and also for
glass capillaries93,94,122,127, but to our knowledge this is the first time that this process
has been shown simply using needles. Therefore a FF type design was developed for
use at the mixing point of our microfluidic system as shown schematically in Figure
5 -58.
Figure 5-58. Schematic representation of the ‘flow-focussing’ device. The internal needle fits
within the external needle.
1.4.6.1. Orientation of device

The shear forces at the point of mixing between the two phases is controlled by the
relative flow rates of the two phases. By reducing the diameter of the outlet at the
end of the needle (from which the continuous phase flows) the shear forces are
increased due to the greater velocity of the liquids. This led to small droplets being
produced at a high rate however, the high production rate of droplets often lead to
126
the coalescence of the droplets. The density of oil phase was 0.79 g/cm 3 while the
density of the monomer solution was 1.00 g/cm3. This led to the monomer droplets
settling on the bottom of the PTFE tubing. Additionally, this difference in density
meant that the orientation of setup up was especially important because the speed of
the monomer droplets was determined by both the flow rate of the continuous phase
and gravity. If the flow direction opposed gravity the monomer droplets would
cluster upon leaving the external needle, this resulted in a polydisperse sample with
a larger mean diameter as some of the droplets coalesced before being polymerised.
If the flow direction and gravity were in the same direction the distance between the
monomer droplets was greater than when the device was horizontal (i.e. flow
direction was at 90˚ to gravity) (Figure 5 -59 A & B), however, because the PTFE
tubing had to pass through the UV box in a relatively horizontal position
coalescence of droplets also occurred where the tubing was bent from vertical to
horizontal. This bend led to a reduction in the speed of the monomer droplets,
greatly reducing their separation from neighbouring droplets causing coalescence.
The optimum setup was found to be a constant slightly ‘downhill’ gradient (20˚
from horizontal) from the needle to the end of the outlet tubing. The slight slope led
to a slight increase in the separation between monomer droplets at the tip of the
needle preventing coalescence (except at very high monomer flow rates). The
constant gradient removed the problem of coalescence due to the changing of
droplet speed (and therefore separation) caused by a change in the angle of the
tubing. The FF design lead to the production of smaller droplets that the earlier
microfluidic setup, however the high production rate of droplets and the difference
in density meant that the orientation of the device had to be adjusted to minimise
coalescence of droplets.
127
Gravity
B
Figure 5-59. Effect device orientation on droplet and resulting particle formation. A: Schematic
representation of effect of gravity and flow direction on droplet formation (left; the flow of
gravity opposes gravity, right; flow direction and gravity are in the same direction. B: Optical
micrographs of particles produced (left; when flow opposes gravity, right; when flow is with
gravity) scale bar is 110 µm.
1.4.6.2. Optimising conditions with the FF device

Using the optimised device orientation, a range of flow rates were tested to
determine the conditions for the production of monodisperse particles (Figure 5 -60
A). At high values of Qd polydisperse particles were polymerised as a result of
insufficient droplet separation leading to coalescence of droplets. While values of Q c
above 1.5 ml/min produced particles that were not fully polymerised. A narrow
128
range of conditions were found to give near monodisperse particles, these conditions
were investigated to determine the variation of particle size with Q c/Qd (Figure 5
-60 B) using 0.08 g/ml of surfactant in the continuous phase. As Q c/Qd was increased
particle size decreased until an apparent minimum diameter of approximately 100
µm at Qc/Qd values of 375 and above. The effect of surfactant concentration was not
tested for the FF setup as the highest concentration (0.08 g/ml) had already been
established to give the smallest particle diameters with the earlier microfluidic setup.
The optimised flow focussing setup produced particles below the limit of the earlier
microfluidic setup down to a minimum of 100 µm in diameter. However, it offered a
less robust production with near monodisperse particles only found under a narrow
range of conditions.
Figure 5-60. Development of flow-focussing setup. A: Particle morphology as a function of the

flow rate of the dispersed phase (Qd) and continuous phase (Qc) where ■; polydisperse particles
(polydispersity > 8 %), ♦; near monodisperse particles (polydispersity < 8 %) and ▲; particles
did not fully polymerise. B: Effect of Qc/Qd on mean particle diameter using flow-focussing.
129
1.4.6.3. Effect of changing oil phase

The shear forces exerted on the disperse phase are determined by the velocities and
the viscosities of the two phases. Up to this point only the velocities have been
varied, therefore, in order to achieve smaller particle sizes the continuous phase was
changed to oil with higher viscosity. Silicone oil was chosen as it was obtainable in
a range of different viscosities additionally it had a density of 0.95 g/ml which
would reduce the problems associated with the difference in density found for Isopar
M. Initial polymerisations using silicone oil (with a viscosity of 20 cP) as the
continuous phase produced either polydisperse or highly aggregated particles
(Figure 5 -61).
Figure 5-61. Optical micrographs of PEGA particles produced using FF device with silicone oil
(50 cSt viscosity) at a variety of conditions (clockwise from top right: Q c/Qd = 100 with Qc = 0.5,
Qc/Qd = 100 with Qc = 0.1, Qc/Qd = 450 with Qc = 0.25, and top left Q c/Qd = 650 with Qc = 0.1.
Scale bar is 200 µm.
Particles with a narrow size distribution were not formed at any of the conditions
investigated. At high total flow (Qc + Qd) rates polydisperse particles were formed,
while at lower flow rates aggregated particles were produced (Figure 5 -61). The
aggregated particles appear to be of a relatively narrow size distribution with a mean
diameter of approximately 60 µm. Presumably, droplets underwent coalescence
130
prior to polymerisation at higher flow rates while at lower flow rate droplets began
to polymerise before coming into contact with one another resulting a mass of
aggregated particles. An increase in surfactant concentration within the continuous
phase was investigated however it was still not possible to produce individual
particles. Coalescence occurred as a result of slight variations in the speed of
movement of the dispersed droplets causing the separation between droplets to
become insufficient. It is likely that this is an intrinsic limitation of microfluidic
devices fabricated from flexible tubing. This problem is not reported in planar ‘chip’
type designs. As a result of these findings silicone oil was not further investigated as
the continuous phase.
1.4.7. Optimum conditions for particle production

The aim of this work was to produce particles with the smallest possible diameter
with a very narrow size distribution. However, the rate of particle production was
slower at higher values Qc/Qd. With these factors in mind the optimum conditions
used in the FF design were: 0.08 g/ml of surfactant in the oil phase, a Q c/Qd of 175
and a Qc of 1.5 ml/min.
1.4.7.1. Particle size distribution

Optimum conditions (described above) were used to produce approximately one
gram of polymer. These particles were further analysed by optical microscopy
(Figure 5 -62 A) and a size distribution obtained (Figure 5 -62 B). The majority of
the polymer volume was in the size range (150-170 μm) a low volume of smaller
satellite were also observed. The mean particle diameter was 161 μm with a
polydispersity of 3.3 %. The rate of particle production was 4000 particles min-1.
131
Figure 5-62. PEGA Particles produced under the optimum conditions. A: Optical microscopy
of particle (scale bar is 160 μm). B: Particle size distribution.
1.4.8. Morphology of particles

The particles produced by this technique often had an elliptical morphology with
aspect ratios128 ranging from 1.006-1.098. While Hadziioannou and co-workers97 did
not report this; no images of the particles that were produced were shown in the
paper. Within this work the formation of these asymmetrical particles seems to be a
result of the difference in density of the water (dispersed phase) and the oil
(continuous phase). This led to the monomer droplets descending and flowing along
the bottom of PTFE tubing. This contact with the tubing wall would have lead to
asymmetrical flow around the droplets.
132
Figure 5-63. The typically slightly asymmetric shape of particles produced by microfluidics
each image is of particles produced under different conditions. Scale bar is 200 µm.
5.2. Conclusions
This work provides an overview of using simplified microfluidic devices to produce
PEGA particles. The creation of the device required no specialist fabrication
methods and could be assembled using readily available lab supplies. Surfactant was
required to obtain control of the particle size by varying Q c/Qd, upon increasing
Qc/Qd smaller particles were obtained. Additionally, increasing the surfactant
concentration led to a reduction to the particle size. A minimum particle diameter of
335 µm was determined. A flow-focussing setup was devised to overcome this
limitation and allowed for the production of smaller particles with a diameter of 99
µm and a polydispersity of 3.2 %. This low value for polydispersity was a dramatic
improvement compared to the particles prepared by inverse suspension
polymerisation (43 %).
133
6 Conclusions
This thesis describes the design, synthesis and application of enzyme responsive
hydrogel particles through the use and development of peptide actuators.
Initially, PEGA hydrogel microparticles (μPEGA) were produced as the support for
the enzyme responsive system based on peptide actuators. These particles had a
homogeneous distribution of amines and were used as the chemical ‘handle’ from
which peptides could be synthesised. Enzymatic hydrolysis on the coupled peptides
was shown to be homogeneous throughout the particles. Additionally, μPEGA was
found to give rise to faster enzyme hydrolysis than commercially available large
PEGA particles (macroparticles) due to reduced diffusion distance. μPEGA was then
functionalised with linear peptide actuators. These zwitterionic molecules were
shown to actuate changes in the accessibility of the crosslinked PEGA particles. This
occurred through a switch in the overall charge balance of the polymer network
(from neutral to cationic) upon enzymatic hydrolysis of the enzyme cleavage peptide
(ECP) within the peptide actuator. The resulting electrostatic repulsion between
neighbouring peptide actuator fragments led to an increase in the mesh size of the
polymer network. By selecting the composition of the ECP to match an enzyme’s
specificity it was possible respond to different enzymes. The pH responsiveness of
the linear actuator functionalised particles was demonstrated. This behaviour was
utilised for the loading of the particles with a fluorescently labelled macromolecule.
Enzyme specific release of this payload was then possible. However, at
physiological ionic strength no enzyme triggered swelling of the functionalised
particles was observed. This was a result of the mobile ions in solution screening out
the interactions between the peptide actuators. This limitation was addressed with
the design of branched peptide actuators.
134
Branched peptide actuators consisted of two linear actuators using the amino acid
lysine as the branch point. This structure was built up from each of the amines
within μPEGA. Upon enzyme hydrolysis there was double the charge density within
particles (compared to when using linear peptide actuators) and the distance between
charged groups was likely reduced. Therefore, particles functionalised with
branched peptide actuators capable of specifically responding to enzymes at
physiological ionic strength (by overcoming electrostatic screening). Much like the
linear peptide actuator functionalised particles this system capable for physically
entrapping a macromolecular payload, was could be released only in response to a
defined enzyme at physiological ionic strength.
The final experimental chapter provided an overview of using simplified

microfluidic devices to produce PEGA particles. Rather than requiring specific
microfabrication techniques it was possible to produce the device using a needle and
tubing. It was found that by varying the surfactant concentration and the flow rates
of the continuous and dispersed phase particles of different sizes were obtained.
Increasing the surfactant concentration or increasing the ratio of continuous to
dispersed phase flow rates led to smaller particles. The minimum particle diameter
possible was found to be determined by the diameter of the needle. Therefore, a
flow-focussing setup was devised to overcome this limitation and allowed for the
production of smaller particles.
135
7 Future work
The most obvious future work at this stage would be using PEGA particles produced
by microfluidics as the basis of the peptide actuator system. These particles with
their narrow size distribution would allow for the kinetics of both enzyme and pH
responsive swelling behaviour to be characterised in greater depth. As narrow size
distribution of these PEGA particles would allow for a relatively small number of
particles to be measured in order to obtain the change in swelling.
It would also be of interest to use microfluidics to prepare polymer particles

crosslinked by enzyme cleavable peptides. This would be possible by employing a
strategy similar to Moore and co-workers. 11 Monomers capable disulphide transfer
reactions to thiols would be synthesised (this has been carried out details are
contained in appendix section 8.2.2. These could then be conjugated to peptides
containing two cysteine residues resulting in a peptide crosslinker. A mixture of
monomer and peptide crosslinker could then be polymerised into particles using the
microfluidic device. Much like the enzyme responsive particles using peptide
actuators, these particles would also allow for enzyme responsive release of a
macromolecule (by tuning the crosslinking density). However, if required these
peptide crosslinked particles could be formulated so that they undergo complete
dissolution upon enzyme action (along with release of the payload). Additionally, it
may be possible to combine an enzyme cleavable peptide crosslinker and peptide
actuators into the same polymer. These systems could potentially have greater
amplitudes of response or by using different peptide substrates in the crosslinker and
ECP only response to a solution containing two different target enzymes.
Rapid and high throughput analysis of the response of the functionalised particles
may be possible if monodisperse PEGA particles of around 20 μm in diameter could
be produced (using a microfluidic chip or possibly by membrane
136
emulsification129,130). These particles could then be passed through an automated cell

sorter (flow cytometry)131. Using a split and mix method it would be possible to
prepare PEGA particles functionalised with a variety of different ECP compositions.
If these particles were then exposed to an enzyme and then sorted by size it would
be possible to screen for effective substrates for the enzyme. By designing the
peptide actuator and tuning the molecular weight cut-off of the functionalised
particles it may be possible to use enzyme hydrolysis of the ECP to trigger a
deswelling of the particles. This offers the potential for the specific entrapment of
the enzyme responsible for hydrolysing the ECP and additionally, might allow for
removal of a desired protease from a complex solution.
Ultimately, there are a number of steps that need to be overcome in order to use
particles functionalised with peptide actuators as drug delivery vehicles: Depending
on target site and method of introduction to the body (i.e. into circulation or injected
directly into the tissue) particles of smaller size may need to be produced.
Emulsion132 or miniemulsion133,134 may offer this possibility. Additionally, the
particles must respond only to the target enzyme (i.e. the marker for the disease to be
treated), to achieve this goal the ECP must be a substrate of that enzyme. This would
be assessed by synthesising the ECP as the substrate of a known disease specific
protease, then exposing these particles to that enzyme in an appropriate complex
mixture. Finally, the ultimate destiny of the particles within the body must be
determined. If the particles are not able to be cleared from the body then some form
possible degradation must be introduced into the polymer.
137
8 Appendices
8.1. Background
8.1.1. Polymers
Polymers have been used as structural materials within nature since life began with
substances such as DNA, polysaccharides and peptides playing crucial roles in
animal and plant life. The term polymer is defined from the Greek words poly and
meros, meaning many parts. In the strictest sense a polymer is a substance composed
of molecules consisting of one or more types of atoms linked to each other by
primarily, usually covalent bonds.87,135 Polymers are created through the linking
together of the small monomer molecules through chemical reactions known as
polymerisation reactions. There are two main types of polymerisation reaction;
addition or chain-growth polymerisation in which, typically a vinyl monomer
(CH2=CHX) is attacked by an initiator to yield an active centre that can then attack
another vinyl monomer linking them together by covalent bonds. Condensation or
step-growth is the second type of polymerisation, here, monomers have different
functional groups (e.g. A-A + B-B → A-a-b-B or A-B + A-B → A-b-a-B) that react
together in a condensation type reaction to form larger molecules consisting of
covalent bonds.
Individual polymer chains can be linked together through the introduction of a di-
functional (or multi-functional) monomer or crosslinker and high levels of
crosslinking result in the formation of a three-dimensional network that is insoluble.
8.1.1.1. Peptides and proteins

Peptides are polymers of amino acids. These substances exhibit a wide range of
differing biological properties allowing them to act as; antibiotics, hormones, food
additives, poisons or pain-killers. Peptides are formed by a condensation reaction
between a carboxylic acid and an (primary) amine forming a peptide bond.
138
Essentially peptide and proteins are the same at the molecular level. The term
protein is used to refer to large molecules typically containing at least fifty amino
acids with a well defined three-dimensional structure. There are twenty amino acids
that are encoded by DNA, each amino acid has the same generalised structure but
with a different side group. These twenty amino acids offer a wide range of
functionalities that can be incorporated into peptides (Figure 8 -64). This gives as
the possibly of preparing a huge number of different peptides, for example, for a
pentapeptide there are 3,200,000 possible combinations. 136 Within this thesis amino
acids will be referred to either by their full name or the 1-letter abbreviation shown
in Figure 8 -64.
139
Figure 8-64. The structures of the twenty DNA encoded amino acids. The single letter
abbreviation is indicated with the brackets.

Two-photon microscopy (TPM)78 is a fluorescent microscopy technique traditionally
used in the fluorescent imaging of biological cells. In this technique the sample is
irradiated with a laser with a wavelength approximately twice that of the excitation
140
length wavelength of the fluorophore. This means that excitation can only occur
when two photons are absorbed simultaneously. This is extremely unlikely and
therefore only occurs at very high photon density; the focal point of the laser beam.
TPM has a number of advantages over the similar technique, confocal microscopy
(in which the sample is irradiated with ultraviolet light with a wavelength equal to
that of the excitation wavelength of the fluorophore) seen in Figure 8 -65. The use
of a longer wavelength from the laser for excitation allows for deeper penetration
into the sample. Additionally, because excitation of the fluorophore only occurs at
the focal point the problem of photobleaching is greatly reduced.131
Figure 8-65. Jablonski energy diagram showing a comparison of the excitation of a fluorophore
with a single photon (confocal microscopy) and two photons (two-photon microscopy).
8.1.3. Fluorescence resonance energy transfer

Fluorescence (or Förster) resonance energy transfer (FRET) microscopy is a
technique used for quantifying the distance between two different fluorophores. 137,138
FRET involves the transfer of energy from a fluorescent donor in its excited state to
another excitable moiety, the acceptor, by a non-radiative dipole-dipole interaction
In order for FRET to occur the distance between the donor and the acceptor must be
small (1-10 nm) and results in a decrease in donor emission and an increase in
acceptor emission.
FRET allows the determination of whether there is a close association between the
donor and the acceptor. This technique has been used to assess (amongst others)
141
calcium ion concentration,139 protein-protein colocalisation140 and enzyme

hydrolysis.141
8.2. Supplementary data
8.2.1. HPLC solvent gradient
Figure 8-66. HPLC solvent gradient used in analytical runs. Concentration of buffer B over the
length of a HPLC run.
8.2.2. Synthesis of activated disulfide-methacrylamide monomer

An activated disulfide-methacrylamide monomer was synthesised with the aim of
preparing enzyme responsive particles using on peptide crosslinkers based on the
work of Moore and co-workers.11 This monomer, N-[2-(2-pyridyldithio)]ethyl
methacrylamide (PDTEMA) (Figure 8 -67 was initially described by Ruffner and
co-workers for conjugation to oligonucleotides and oligopeptides.142 The procedure
described in this paper was followed, resulting in the successful synthesis of the
monomer (as determined by NMR, Figure 8 -68).
142
Figure 8-67. Synthesis of PDTEMA.142
Figure 8-68. Characterisation of PDTEMA by NMR. 1H NMR (CDCl3, 200 MHz): δ (ppm) 7.0-8.6
(m, 5H, Ar-H and -NH), 5.809 (s, 1H, one of d, CH 2), 5.361 (s, 1H, one of dCH2), 3.603 (m, 2H, -CH2-
NR), 2.960 (t, 2H, -S-CH 2-), 2.002 (d, 3H, -CH3, the split of this peak is due to the tautomerization of
the adjacent double bond).
143
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Design, Synthesis and

A thesis submitted to the University of Manchester for the degree of

1.1. MOTIVATION OF THE PROJECT...................................................................19

3 PEGA POLYMERISATION, CHARACTERISATION AND ENZYME

3.4.4.4. Enzyme responsive increase in swelling; effect of ionic strength. 97

4 DESIGNING NEW PEPTIDE ACTUATORS FOR IMPROVED

5 MICROFLUIDIC PREPARATION OF LOW POLYDISPERSITY PEGA

5.4. RESULTS AND DISCUSSION.......................................................................121

Figure 2-16. Biomimetic synthesis and optimization of cyclic peptide antibiotics.......

Figure 5-12. The typically slightly asymmetric shape of particles produced by

HPMC Hydroxypropyl methyl cellulose ether

Span 20 Sorbitan monolaurate

The first experimental chapter describes the synthesis of poly(ethylene glycol)-co-

Biomaterials science is an interdisciplinary field that encompasses medicine,

A common strategy to incorporate responsiveness into a biomaterial is based on

networks can be altered by changing the polymer’s interactions with neighbouring

1.1. Motivation of the project

1.2. Layout of the thesis

The first experimental chapter, Chapter 3 investigates the synthesis and

Figure 1-1. The inverse suspension polymerisation of µPEGAs.

The final experimental chapter explores the application of microfluidic devices in

Figure 1-4. Production of PEGA particles by microfluidic polymerisation. Schematic

2.2. Stimuli responsive materials

2.2.1. Chemical hydrogels

applications. Currently the main application of these biomaterials in medicine is as

Stimuli responsive hydrogels have been shown to respond to numerous stimuli

2.3. Bioresponsive Hydrogels1

Figure 2-5. Schematic representation of the different categories of bioresponsive hydrogels.

2.3.2. Actuation based on changes in crosslinking density

2.3.2.1. Systems incorporating peptide crosslinkers

Table 2-1. Selected studies of bioresponsive hydrogels based on peptide crosslinkers.

2.3.2.1.1 Enzyme catalysed changes in peptide crosslinker density

Addition of matrix metalloproteinase (MMP)-1 solution to the gel induced a volume

Furthermore, Healy and co-workers demonstrated a similar system incorporating

In another study a system that responded to enzymes by hydrogel formation rather

In 2004 Moore and co-workers published an enzyme responsive hydrogel utilising a

Messersmith and co-workers again made use of a transglutaminase (TG) enzyme

2.3.2.1.2 Enzyme hydrolysis of hydrogel network

2.3.2.2. Non-covalent crosslinking interactions

Table 2-2. Selected studies of bioresponsive hydrogels based on non-covalent crosslinking

Figure 2-7. Antigen responsive hydrogel. A: Synthesis of the antigen-antibody semi-IPN

exposed to a solution of anti-biotin (antibody), leading to antigen-antibody binding

Figure 2-8. Displacement-Induced Switching Rates of Bioresponsive Hydrogel Microlenses. A:

2.3.3. Actuation based on electrostatic interactions

Table 2-3. Selected studies of bioresponsive hydrogels based on electrostatic interactions

2.3.3.1. Electrostatic interactions between charges on the polymer network

Figure 2-11. Characterization of glucose-sensitive insulin release systems in simulated in vivo

2.3.3.2. Electrostatic interactions between charges present in pendant actuators

approaches utilise enzyme cleavable peptides as the sensing element. Thornton et al

2.3.4. Actuation based on conformational changes

The use of proteins as actuators is a new development in bioresponsive hydrogels. 64

2.4.1. The history of PEGA

Figure 2-15. Chemical structure of PEGA.

placed in an aqueous environment. The open structure of the polymer allowed

Figure 2-16. Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor

investigate enzyme accessibility. Three different supports for SPPS; TentaGel,

An alternative approach to that used by Meldal and co-workers to increase the

In 2003 a new development in the use of combinatorial libraries on PEGA was

In 2006 Ulijn and co-workers described a different morphology of PEGA in the

Figure 2-19. Real-time imaging of protease action on substrates covalently immobilised to

2.4.2. Enzyme catalysed synthesis on PEGA

prepared using solid phase methodology, enzymatically catalysed amide bond