Layer by Layer - 2015

Biotechnology Advances 33 (2015) 13101326
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Drug nano-reservoirs synthesized using layer-by-layer technologies

Rui R. Costa a,b,, Manuel Alatorre-Meda a,b,c, Joo F. Mano a,b,
a
3B's Research Group Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence of Tissue Engineering and Regenerative Medicine,
Avepark Parque de Cincia e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR, Portugal
b
ICVS/3B's, PT Government Associated Laboratory, Braga/Guimares, Portugal
c
Catedrtico de CONACYT en Centro de Graduados e Investigacin en Qumica del Instituto Tecnolgico de Tijuana, Blvd. Alberto Limn Padilla S/N, 22510 Tijuana, BC, Mexico
a r t i c l e
i n f o
Available online 18 April 2015

Keywords:
Layer-by-layer
Drug delivery
Tissue engineering
Biomaterials
Nanolms
Nanobers
Nanocapsules
Nanodrugs
Nanomedicine
Stimuli-responsive nanobiomaterials
a b s t r a c t
The pharmaceutical industry has been able to tackle the emergence of new microorganisms and diseases by
synthesizing new specialized drugs to counter them. Their administration must ensure that a drug is effectively
encapsulated and protected until it reaches its target, and that it is released in a controlled way. Herein, the
potential of layer-by-layer (LbL) structures to act as drug reservoirs is presented with an emphasis to nanodevices of various geometries, from planar coatings to bers and capsules. The inherent versatile nature of this
technique allows producing carriers resorting to distinct classes of materials, variable geometry and customized
release proles that t within adequate criteria required for disease treatment or for novel applications in the
tissue engineering eld. The production methods of LbL reservoirs are varied and allow for different kinds of
molecules to be incorporated, such as antibiotics, growth factors and biosensing substances, not limited to
water-soluble molecules but including hydrophobic drugs. We will also debate the future of LbL in the pharmaceutical industry. Currently, multilayered structures are yet to be covered by the regulatory guidelines that govern the fabrication of nanotechnology products. However, as they stand now, LbL nanodevices have already
shown usefulness for antifouling applications, gene therapy, nanovaccines and the formation of de novo tissues.
2015 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . .
Layer-by-layer as a drug reservoir construction tool . . . . .
Types of LbL drug nano-reservoirs . . . . . . . . . . . . .
3.1.
Thin nanometric lms . . . . . . . . . . . . . . .
3.2.
Nanocapsules . . . . . . . . . . . . . . . . . . .
3.3.
Nanotubes . . . . . . . . . . . . . . . . . . . .
3.4.
Films as boundaries of scalable devices . . . . . . . .
Loading and release mechanisms . . . . . . . . . . . . .
4.1.
Release by disruptive interactions and controlled stimuli
4.1.1.
pH . . . . . . . . . . . . . . . . . . . .
4.1.2.
Temperature . . . . . . . . . . . . . . .
4.1.3.
Ionic strength . . . . . . . . . . . . . . .
4.2.
Destruction of the carrier . . . . . . . . . . . . . .
4.2.1.
Light . . . . . . . . . . . . . . . . . . .
4.2.2.
Biochemical . . . . . . . . . . . . . . . .
4.2.3.
Electrochemistry . . . . . . . . . . . . .
Drug delivery for biomedical applications . . . . . . . . . .
5.1.
Antifouling . . . . . . . . . . . . . . . . . . . .
5.2.
Injectable drug formulations . . . . . . . . . . . .
5.3.
Gene delivery . . . . . . . . . . . . . . . . . . .
5.4.
Stem cell differentiation and tissue formation . . . . .
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Corresponding authors at: 3B's Research Group Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence of Tissue
Engineering and Regenerative Medicine, Avepark Parque de Cincia e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR, Portugal. Tel.: +351 253 510 900; fax: +351 253 510 909.
E-mail addresses: rui.costa@dep.uminho.pt (R.R. Costa), jmano@dep.uminho.pt (J.F. Mano).
http://dx.doi.org/10.1016/j.biotechadv.2015.04.005
0734-9750/ 2015 Elsevier Inc. All rights reserved.
R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326
6.
Current regulatory status of nanotechnology products
7.
Conclusions . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
The emergence of new diseases and microorganism strains demands
constant evolution and research in the eld of drug delivery and
nanomedicine. The most straightforward strategy adopted by the pharmaceutical industry has been the synthesis of new drugs that may hopefully ght against these pathologies. In turn, this triggers the demand for
appropriate drug carriers that can effectively be loaded with and protect
the drugs until they are administered and delivered. There is a multitude of carriers that have been used, including micelles, liposomes,
polymersomes or polymer particles/capsules (Blanazs et al., 2009;
Costa and Mano, 2014; Lima et al., 2012; Malam et al., 2009; Meng
et al., 2009; Szarpak et al., 2010) but there are still drugs which these
strategies fail to encapsulate efciently (e.g., hydrophobic and short
biofunctional peptides). Thus, drug delivery faces a few challenges: (i)
to elaborate advanced new functional drugs and nanodrugs, (ii) to develop delivery systems capable of encapsulating water-insoluble
drugs, and (iii) to develop drug delivery systems that provide a
sustained release of a drug within a desired therapeutic window in
order to ensure its efcacy (Antipov et al., 2001; Vergaro et al., 2011).
Some diseases, like cancer, demand extra needs, since the administration of chemotherapeutics faces problems like non-specicity, toxic effects and lack of localized administration strategies (Jain and
Stylianopoulos, 2010).
The synthesis and design of drug delivery devices have experienced
great advances towards new sustained and controlled delivery systems
for a safe and efcient administration. These include long-term stability,
high loading capacity and site selectivity. Other desired properties
include the capability of a carrier to control the delivery of multiple
biological compounds at independent time scales and to release them
within a therapeutic window (Chung and Rubner, 2002; Pavlukhina
and Sukhishvili, 2011; Radt et al., 2004).
Recently, scientists and engineers worldwide have begun to adopt
biomimetic concepts that take natural structures as inspiration to develop
new drug delivery devices, by mimicking not only their function but also
their architecture. One inspirational example is the layered organization
of nacre found in sea animal shells, which provide mechanical strength
(Luz and Mano, 2009). One can envisage the development of layered
structures in a laboratorial environment that exhibit other functionalities,
depending on their ultimate application. Scientists have developed a
strategy by which polyelectrolytes are alternately adsorbed onto solid
surfaces (Decher and Hong, 1991; Decher et al., 1992; Iler, 1966). This
technique is known as layer-by-layer (LbL), and in the last two decades
it has provided a reliable, easy, versatile and cost-effective way of modifying surfaces for tuned cell adhesion, drug delivery and improved implant
biointegration. LbL relies on the use of materials belonging to virtually
any material class, from inorganic compounds (e.g., nanoparticles and
carbon nanotubes) (Mamedov et al., 2002; Srivastava and Kotov, 2008;
Upadhyayula and Gadhamshetty, 2010) to polymers (including synthetic
and natural-based macromolecules) (Boddohi et al., 2008; He et al.,
2008) sequentially assembled by spontaneous adsorption and forming
robust coatings.
Films fabricated via LbL can be assembled on top of not only 2D planar
substrates but also on convoluted and 3D geometries. In all cases, the sole
requirement for this reaction to occur spontaneously is the exhibition of
complementary interactions between the various selected building
blocks such as electrostatic contacts, van der Waals forces, and hydrogen bonds (Borges and Mano, 2014; Boudou et al., 2010; Costa and
Mano, 2014; Decher, 1997; Hammond, 2011; McClements, 2006; Tang
et al., 2006). Table 1 summarizes the different devices that result from
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LbL being applied to different geometries (see also Section 3). The assembled lms may then provide in both cases a matrix capable of acting as
drug reservoirs. Thanks to this versatility of LbL, it is possible to construct
planar lms, three-dimensional multilayered capsules (PMCs) and nanotubes by assembling the constituents around leachable sacricial templates (Fig. 1). Additional characteristics include the lack of necessity to
resort to organic solvents and biological aggressive processing conditions
(e.g., high temperatures, extreme pH values), making LbL an attractive
strategy to be coupled with biological applications. Specialized biomaterials and ligands, such as cell adhesion enhancers, may be added in
order to render a substrate more instructive for cell adhesion, proliferation and differentiation (Costa et al., 2011; Gribova et al., 2013; Oliveira
et al., 2013). Furthermore, signicant efforts have been made to design
coatings and PMCs capable of loading and releasing small molecules,
drugs and biomolecules, in conjugation with both current and cuttingedge biomedical devices, such as implants and catheters (Karlsson et al.,
2010; Lichter et al., 2009; Wang et al., 2009b).
In this critical review, the LbL strategy will be presented as a technique able to conceive various multilayered systems with potential
use in drug delivery. We will focus in biomedical applications that
require a high level of control of drug administration, such as in biosensing, microenvironments conning chemical reactions, and microtissue
production (De Koker et al., 2011; Gribova et al., 2012; McShane and
Ritter, 2010; Tong et al., 2012). PMCs with a few micrometers in diameter are often based on microtemplates that are convenient and easy
to use, thus being considered the gold-standard of LbL strategies for
drug delivery. The topic of micrometric PMCs has been often nicely
reviewed (Becker et al., 2010; De Koker et al., 2011; Shchukina and
Shchukin, 2011; Tong et al., 2012; Vergaro et al., 2011; Wohl and
Engbersen, 2012). Herein, we will focus on drug reservoirs consisting
of planar coatings (Section 3.1), nanometric PMCs, which among other
properties offer a higher surface area per unit weight than larger
PMCs (Section 3.2), and nanotubes (Section 3.3). It is also our intention
to show the readers how such an approach is capable of rivaling with
some of the most common drug administration systems currently in
use, focusing on reports about potential applications published mostly
in the last 5 years (Section 5). Despite the tremendous efforts, only in
recent years have LbL constructs reached a level where the fabrication
parameters and drug encapsulation strategies are well-known. It is
now possible to fabricate LbL devices with properties that are desirable
for an efcient systemic delivery and that can be reproduced with great
reliability, which include low toxicity, stability in various aqueous environments, prolonged release of drugs in vitro and in vivo and targeted
delivery (Shutava et al., 2014). Due to the potential benets that LbL
devices can bring to the healthcare sector, the current regulatory status
of LbL devices and nanomaterials will be discussed in Section 6.
2. Layer-by-layer as a drug reservoir construction tool
LbL is often regarded as a surface engineering technique, grouped
together with other surface engineering approaches, such as plasma
surface modication (Chu et al., 2002), polymer grafting (Kato et al.,
2003), micro/nanofabrication (Lu and Chen, 2004) and Langmuir
Blodgett (Zasadzinski et al., 1994). All these surface engineering tools
aim to modify the properties of interfaces while retaining the properties
of the bulk materials. Although primarily appreciated as a surface engineering technique, the use of LbL to encapsulate bioactive substances
makes it identiable as a method to fabricate drug delivery reservoirs,
capable of solving many problems of conventional devices, such
as hydrogels (premature disintegration of the matrix) (Hoare and
Potential applications
Liang et al. (2003), Rejman et al. (2004), Tian et al. (2006), Yang et al. (2007),
Shimoni et al. (2013), Shutava et al. (2014), Silva et al. (2014)
Schler and Caruso (2001), Sukhishvili and Granick (2001), Serizawa et al. (2002),
Djugnat and Sukhorukov (2004), Li and Haynie (2004), Radt et al. (2004), Borden
et al. (2007), Zelikin et al. (2008), Zhao and Li (2008), Ochs et al. (2010), Poon
et al. (2011b), Morton et al. (2013), Feng et al. (2014), Liu et al. (2014)
Wound healing/surgical patches

Tuning the interface of implantable devices
Antifouling
Substrates with increased cell adhesion
Stem cell differentiation
DNA hybridization
Targeted delivery
Capsules
Gene delivery
Intracellular delivery
Adjustable drug release kinetics
Injectable formulations
Stem cell differentiation
Biosensors
Tubular constructs Assembled onto cylindrical templates or porous membranes with cylinder-shaped pores. Intracellular delivery
Caliber depends on the diameter of the template: bers (5 m1 mm) and membranes Blood vessel substitutes
Biosensors
(pore diameter around 200 nm).
Drug-doped cylindrical templates have been suggested as a preloading strategy.
Assembled onto planar substrates.
Thickness can vary between several nanometers and a few
micrometers depending on the number of layers and
building blocks used.
2D shape that can be attached to a substrate or peeled-off from it.
Drugs can be loaded by doping or using the drug itself as an ingredient during LbL.
Assembled onto spherical templates, leachable after construct.
Diameter depends on the size of the template: silica (500 nm5 m) and calcium
carbonate (35 m) particles. The high susceptibility to external stimuli may cause
signicant diameter changes.
3D shape with high surface
Drugs can be preloaded in the template or post-loaded by doping.
Key characteristics
Type of devices
Table 1
Multilayered devices with drug loading capability, their characteristics and potential applications.
Planar lms
Suggested references

Kato et al. (2009), Macdonald et al. (2010), Wong et al. (2010), Zhou et al. (2010),
Crouzier et al. (2011), Shah et al. (2011); Gilde et al. (2012), Guillot et al. (2013),
Chen et al. (2014), Zhuk et al. (2014)
1312
Kohane, 2008; Mano, 2008), micelles (the need of toxic surfactants during fabrication and loading) (Ariga et al., 2011; Kawakami et al., 2006)
and liposomes (limited stability) (Barenholz, 2001; Kogure et al.,
2008). Without intending to discredit the advances that these reservoirs
permitted to achieve in the last decades, LbL-based drug delivery devices are capable of bringing together many of their advantages:
(i) LbL devices can be loaded with both water soluble/non-soluble active agents, by taking advantage of the swelling behavior of LbL lms
or by drug/top layer interactions to link drugs to the LbL structure;
(ii) they can be designed to be robust and stable within wide ranges
of temperature, pH and ionic strength values, including in physiological
conditions; (iii) drug release can be controlled by external stimuli
by conceiving architectures containing intelligent building blocks;
(iv) the release can be controlled further by assembling a variable number of layers, which act as a controllable barrier against drug diffusion;
(v) multiple drug delivery is achievable by incorporating different
drugs along the thickness of the lms, due to the possibility of selecting
the type of materials deposited along the vertical axis; (vi) multilayer
lms can be loaded with growth factors and stored for long periods of
time (as high as one year (Crouzier et al., 2011; Gilde et al., 2012;
Guillot et al., 2013)) without extensive degradation or bioactivity loss;
(vii) nally, the versatility of LbL allows constructing systems with the
shape that is more appropriate to the desired end. As will be shown in
Section 3, they can be thin lms, tubes or spherical capsules.
However, LbL is not without disadvantages. It involves long construction times, with the assembly of a single layer taking typically a
few minutes but dependent on the nature of the constituents. From a
scale-up perspective, automated devices have been proposed for the
production of multilayer lms, namely dipping and sputtering machines
(Costa et al., 2013c; Fukao et al., 2011; Krogman et al., 2007). The latter
are much quicker than the former, with constructions times being 250
times faster than the dipping strategy (Izquierdo et al., 2005). Scalingup the production of 3D LbL devices has not received signicant attention. Nonetheless, efforts in developing PMCs in an automated way
have been reported by resorting to microuidic devices (Kantak et al.,
2011; Priest et al., 2008), so that stock rupture is prevented in future
pharmaceutical uses.
3. Types of LbL drug nano-reservoirs
3.1. Thin nanometric lms
LbL assembly is an easy, efcient, reproducible, robust, exible
and extremely versatile bottom-up strategy primarily used to modify
surfaces. When two or more building blocks are assembled onto a planar substrate in a sequential fashion, highly ordered nanostructured
thin lms are constructed, exhibiting tailored physical and chemical
properties, depending on the used ingredients. LbL lms can be perceived as very thin lms, ranging from Angstrms to nanometers.
These nanolms are layers of quasi-2D water-insoluble complexes
with compensated intermolecular interactions, including (but not limited to) electrostatic, hydrophobic, charge-transfer, hostguest, coordination chemistry, and biologically specic interactions to hydrogen
bonding, covalent bonding, stereocomplexation, and surface solgel
process (Borges and Mano, 2014). In such lms, the top layer remains
available to allow the formation of a new layer of a complementary
polymer by expressing the desired signaling cue (e.g., opposite charge),
driving the lm growth further (Sukhishvili, 2005).
LbL lms have been widely employed in drug delivery. In comparison to other conventional surface modication methods, LbL selfassembly offers several advantages regarding drug entrapment: (i) the
thickness and mechanical properties of the lms can be tailored by the
number of deposited layers; (ii) the chemical and biological composition can be tuned by selecting the proper material; (iii) the location
and sequence of the layers can be controlled on-demand; and (iv) surface labeling with targeting molecules to improve material/cell
1313
Fig. 1. Technical approaches to produce LbL-based drug delivery products. Multilayered devices can be produced by (A) dipping, (B) spraying, (C) microuidics and (D) perfusion. In all
cases, electrostatic-driven LbL assembly is represented. Representations are oversimplied.
interactions is possible (Borges and Mano, 2014; Costa and Mano, 2014;
Gribova et al., 2012; Sukhishvili, 2005). Moreover, since these assemblies can be designed in ways that permit controlled lm disassembly
under physiological conditions (see Section 4), LbL can contribute
with new methods of spatial and/or temporal control over the delivery
of therapeutics in vitro and in vivo (Jewell and Lynn, 2008).
Bioactive molecules such as drugs, proteins, peptides and even
nucleic acids can be incorporated to LbL lms following two main
routes. One involves the direct inclusion of the drug as one of the building blocks during the lm construction. Thus, the amount and the nature of the loaded bioactive agents can be regulated by changing the
number of layers. However, in multilayered structures the building
blocks may exhibit interlayer diffusion, which may be difcult to control
over their subsequent release (Wood et al., 2006). A second route to
load drugs is their immobilization by doping using concentrated drug
solutions after the construction of the carrier. Such an approach is viable
for low molecular weight drugs which diffuse easily through the pores
of the lm (Sukhishvili, 2005). Alternatively, both mechanisms can
be used simultaneously. Chen et al. (2014) developed free-standing
lms for the sequential co-delivery of antibiotics and growth factors.
The system was composed of 60 tetralayers of poly(-amino esters)
(PAE), alginate (ALG) and a recombinant human basic broblast growth
factor (bFGF), assembled using the electrostatic interactions between
PAE, ALG and bFGF. Once formed, the lms were loaded following a
post-doping process with the antibiotic drug ceftriaxone (CTX), by immersing the lms in concentrated solutions of CTX. The design of this
system ensured that CTX was released in a quick fashion by breaking
its electrostatic interaction with the drug reservoir layer, whereas
bFGF was released in a subsequent more sustained way due to the
slow degradation of PAE. This work demonstrated how LbL-assembled
polymer lms can be rationally designed to act as platforms for the
controlled release of multiple drugs for the treatment of skin injuries.
In this case, the broad-spectrum antibiotic drug was rapidly released
to kill invasive bacteria and thus avoid bacterial infection. Then, the
recombinant human bFGF was subjected to a long-term release, thereby
promoting the desired wound healing of tissue defects.
3.2. Nanocapsules
The LbL technology can be scaled to the third dimension by using
templates other than at surfaces to assemble multilayers. When
applied to three-dimensional colloidal particles, followed by particle

dissolution, the LbL technology permits the production of hollow
nanocapsules holding the design features of the template, including
size and shape. Common templates include silica (with 500 nm to
5 m in diameter, dissolved in hydrogen uoride) and porous calcium
carbonate particles (with 3 to 5 m, dissolved in ethylenediamine
tetraacetic acid, EDTA) (Becker et al., 2010; Mauser et al., 2006; Tong
et al., 2012; Wang and Caruso, 2006). PMCs with diameters in the
nanometer scale have attracted great interest for biomedical applications, especially for targeted therapies. It has been demonstrated that
LbL PMCs can be phagocytized by cancer and immune cells, especially
PMCs expressing surface markers that can be recognized by specic
cells (Cortez et al., 2007; De Koker et al., 2009; Muoz Javier et al.,
2008). This possibility is further supported by the fact that micelles,
liposomes and macromolecules can accumulate at solid tumor sites,
where a hyper-developed vasculature is exhibited (Ishida et al., 1999;
Matsumura and Maeda, 1986; Matsumura et al., 2004). Thus, drug
carriers with a nanoscale dimension can be foreseen as carriers that
may accumulate at the site of pathological tissues.
The investigation of LbL PMCs comes from the late 1990s when
Caruso, Mhwald and coworkers started to explore the assembly of
polyelectrolytes onto colloidal particles of varying sources such as
silica, organic polymers and their different combinations (Caruso
et al., 1998; Donath et al., 1998; Sukhorukov et al., 1998, 1999; Qiu
et al., 2001a, 2001b). These pioneering publications paved the way
for the development of systems with unparalleled characteristics in
terms of versatility and functionality. However, the production of
hollow polymeric capsules using the LbL technique remained for
some time limited to the synthesis of constructs no smaller than
1 m in diameter, which are not safe for intravenous injections
(refer to Section 5.2) (De Geest et al., 2009). Among others, this size
limit was related to the difculty in coating increasingly smaller particles. This limitation is derived from technical difculties in wrapping
polymer chains around particles with higher curvatures and separation of coated particles from polymer solutions, leading to particle
aggregation (Gittins and Caruso, 2000). A milestone was achieved
when gold nanoparticles were used as colloidal templates. Gold
nanoparticles possess a plasmon band that is very sensitive to small
changes of the dielectric constant of the surrounding medium
(Schneider and Decher, 2004). The spectroscopic analysis of the
plasmon absorption band of gold nanoparticles facilitated the LbL
1314
fabrication of nanocapsules since it allowed analyzing the sequential

layer assembly of the coated particles in a quick and simple way,
which was helpful in avoiding aggregation during the LbL deposition.
The production of hollow PMCs using gold nanoparticlesacricial templates was rst reported by Caruso and coworkers
(Gittins and Caruso, 2000, 2001; Mayya et al., 2003). They synthesized nanometric PMCs with well-dened diameters (1550 nm)
coated with sequential layers of poly(styrene sulfonate) (PSS) and
poly(diallyldimethylammonium chloride) (PDADMAC) onto thiolcapped gold nanoparticles (Gittins and Caruso, 2000). Thanks to the
thiol cap, the bare nanoparticles were protected from aggregation prior
to LbL deposition and allowed the adsorption of the rst PDADMAC
layer. The core could be dissolved by exposure to cyanide solutions,
thereby leaving behind just the PMC shells. The authors also found that
high salt concentrations increased the exibility of the polymer chains,
but they also induced the occulation of the nanoparticles. In opposition,
lower salt concentrations stabilized the particles, but caused selfrepulsion of the unbonded ionized groups of the polymer chain.
Remarkably, it was not only simple nanoparticles but also other
nanoparticulate objects (such as quantum dots, nanorods, and siRNA
precomplexed onto gold nanoparticle surfaces) that could be coated.
Up until that point, synthetic routes towards the production of sub100 nm capsules had utilized in-situ free-radical polymerization
(Marinakos et al., 1999; Quaroni and Chumanov, 1999), crosslinking
of polymer micelle surfaces (Huang et al., 1999), and metathesis polymerization of covalently bound monomer monolayers (Watson et al.,
1998). More emergent examples include the formation of coreshell
nanoparticulate systems where the cores are composed of drugloaded liposomes (Fukui and Fujimoto, 2009; Pereira da Silva Gomes
et al., 2009; Haidar et al., 2010). Since the concept of PMCs was rst
introduced, many milestones have been reached with respect to the
type of template, of drug and administration routes. Huang et al.
(2015) have recently shown that the hydrophobic chemotherapeutic
doxorubicin (DOX) could be loaded into LbL casein-coated iron oxide
nanoparticles for oral administration, an administration route for the
treatment of cancer that is more comfortable to the patient.
Comparing to micro-sized PMCs, nanocapsules have higher surface
area which can be exploited for cellular uptake of drug-loaded PMCs,
thus delivering higher drug quantities to the target cells or tissues.
Functionalizing the surface of PMCs with bioactive ligands further
increases the interest for targeted therapies. Namely, antibodies have
been shown to signicantly increase cellular uptake by specic cell
types (Cortez et al., 2006; Mintern et al., 2013). Thus, it is expected
that drug-loaded nano PMCs will be scaled further in the near future
to meet the needs of the pharmaceutical industry.
3.3. Nanotubes
Depending on the shape of the substrate, LbL can reproduce
architectures with different levels of complexity and functionalization.
Like PMCs, the most straightforward way of constructing LbL tubular
structures is using cylindrical templates, such as glass nanobers. Although glass bers are convenient to use due to their availability and
ease of handling, so far these templates only allowed to produce hollow
tubes with micrometers in diameter (from 5 m to 1 mm) (Mueller
et al., 2007; Silva et al., 2014). Because of their diameter, their potential
use in the future could be that of large-caliber blood vessel substitutes
(i.e., tubular structures possessing diameters higher than 5 m (Bos
et al., 1998)). In comparison, tubular structures of a lower caliber
nanotubes could be better candidates as drug delivery carriers, since
they have higher surface area and aspect ratio, but could also be useful
as structural components in applications such as in catalysis, in biosensors and in scaffolds for cell growth (Murugan and Ramakrishna, 2006;
Sanchez-Castillo et al., 2004; Wang et al., 2004).
In order to develop nanotubes, porous polycarbonate membranes
have been used. The interior of their pores can be coated by perfusion
of the various polyelectrolytes across the length of the membrane.

The membrane can be then dissolved by leaching solvents, such as dichloromethane. With this strategy, Liang et al. (2003) were able to conceive hybrid polyelectrolyte/nanoparticle with around 400 nm in
diameter and length of 10 m (with the length dimension corresponding to the thickness of the membrane). Tian et al. (2006) followed the
same strategy to construct nanotubes of poly(acrylic acid) and
3,4,9,10-perylenetetracarboxyldianhydride via hydrogen bonding,
using membranes with 200 nm and pore length of 13 m. Yang et al.
(2007) focused on studying the biological properties of this type of constructs. Chitosan/alginate (CHI/ALG) tubes with 400 nm in diameter
were rst subjected to a degradation procedure using pancreatin,
which degrades chitosan. After a few hours of incubation overnight,
the diameter was reduced to 160 nm. Biological studies in vitro showed
that these nanotubes were non-cytotoxic towards human cervical cancer cells and breast cancer cells, and that they were also internalized by
the cells upon 24 h of co-incubation. This is an important result because
small-sized structures may facilitate cellular uptake (Rejman et al.,
2004), making nanotubes potential intracellular delivery carriers of active agents. However, despite their potential for intracellular drug delivery, Caruso and coworkers (Shimoni et al., 2013) reported that
cylindrical structures are less likely to be internalized than spherical
ones.
Loading nanotubes with bioactive substances has been seldom
performed. In a recent review, Shutava et al. (2014) contemplated
halloysite clay cylinders as potential substrates for LbL coating.
Halloysite can be mixed with highly concentrated solutions of a desired
drug, which can then be submitted to alternate polyelectrolyte deposition. Although the example of halloysite was given, one can envisage
other types of drug-doped cylindrical shapes to be used, such as
polymer-based bers and crystallized biomolecules with a brillar orientation. However, due to the higher surface area exhibited by PMCs,
alongside the lack of extensive reports about drug-loaded LbL tubes, it
is likely that drug delivery including intracellular delivery will
keep relying on nanometric PMCs instead of tubular structures in the
next years.
3.4. Films as boundaries of scalable devices

Versatility has been shown to be the key characteristic of LbL numerous times. With the use of diverse building block and template classes,
this technique has started to be perceived not just as a technique that
generates lms and 3D architectures but also as a means to build boundaries for compartments. Compartmental systems are useful to spatially
conne various functional components, such as particles, capsules,
bioactive molecules and even living structures like cells. LbL lms can
be assembled at the frontier of a compartment in order to conne
such elements inside a controlled environment and to modulate matter
exchange between the inner and outer media. This strategy could circumvent the fact that multilayered structures have a limited capacity
by themselves to accommodate large quantities of bioactive molecules
due to the low thickness of the lms.
One can nd parallelism of compartmental architectures with the
example of natural cells, which show hierarchical and compartmentalized organization. Cellular organelles exhibit specialized roles, in the
intracellular space, such as the transport of numerous compounds, synthesis of proteins and regulation of enzymatic reactions, contributing
to the overall maintenance of the cell as a whole (Gray et al., 1999;
Demaurex, 2002; Yates et al., 2005; Satori et al., 2013). PMCs and tubular structures mentioned in the previous sections are examples of
single LbL-delimited compartments with potential interest as drug
delivery devices. In comparison, a multi-compartmental device
where all compartments are delimited by coatings with nanometric thickness may be useful to construct systems where diverse
bioactive ingredients are entrapped, including cells and multiple
therapeutic drugs (Chandrawati et al., 2011; Costa et al., 2013a;

Parakhonskiy et al., 2014; Sher et al., 2015a, 2015b).
Independent of the size of a compartment, which can range from
nanometers to millimeters, the LbL coating itself is nanometric and
must hold all compartmental elements together. In the pioneer work
of Kreft et al. (2007), concentric structures were used to conne chemical or enzymatic reactions within LbL-delimited capsules. PMCs of poly
(styrene sulfonate)/poly(allylamine hydrochloride) (PSS/PAH) multilayers were loaded with human serum albumin tagged with various
uorophores, individually loaded in each compartment. The authors
also encapsulated other compounds, namely glucose oxidase (GOX) in
the outer compartment and peroxidase in the inner compartment.
When adding glucose and Amplex Red to the surrounding medium, an
enzymatic reaction was detected: GOX oxidized glucose, which generated hydrogen peroxide. In the presence of the latter, Amplex Red was
converted by peroxidase into resorun. This product is a uorescent
compound, which makes it a useful and convenient optical indicator
of glucose degradation. More recently, Chandrawati et al. (2010) introduced liposomal compartments around silica leachable cores. Each liposome layer was separated by polymer separation layers and the whole
structure was held together by a capping coating of ve poly(N-vinyl
pyrrolidone)/poly(methacrylic acid) (PVP/PMA) multilayers. The liposomes were loaded with -lactamase, which was shown to be effectively encapsulated and protected using a liposome-disintegration
colorimetric reaction with nitrocen. Hosta-Rigau et al. (2010) used
the same compartmentalized structure to demonstrate further its
potential to load hydrophobic cargo. The proof-of-concept was made
using thiocoraline, an anticancer drug. This highlights the interest of
these structures for the treatment of cancer, since most chemotherapeutics have low molecular weight and are water insoluble.
Costa et al. (2013a) used a hybrid LbL-assisted ionic gelation
strategy to fabricate multi-responsive compartmentalized PMCs with
hierarchical nano-to-macro organization (Fig. 2). Inside a liqueed alginate environment delimited by CHI and ALG multilayers, micrometric
PMCs of CHI and elastin-like recombinamers (ELRs) were encapsulated.
Different uorescent molecules were loaded in each compartment,
demonstrating that this class of customizable hierarchical systems can
accommodate several biomolecules simultaneously. Thanks to the
temperature-responsive behavior of ELRs, the release of uorophores
from the innermost compartments could be further tuned by varying
the temperature of the incubation media.
Being only a few nanometers thick, LbL lms were suitable external
shells for compartments with hierarchical organization harboring bioactive molecules. Usually, aggressive solvents and processing conditions
are commonly used when constructing multi-shelled organic and
inorganic capsules (Xu and Wang, 2007; Li et al., 2010; Huang et al.,
2011). Therefore, LbL-delimited compartmentalized units may have a
reduced toxicity in comparison.
One could envisage that this strategy is not limited to loading multiple therapeutic molecules. Cells that secrete bioactive substances, such
as pancreatic cells, could be encapsulated in compartments and thus
would require more control over the substances that traverse the LbL
coatings (e.g., nutrients and gasses could go in and metabolic waste
products could go out). In the future, it is expected that a semipermeable behavior can be attributed to LbL compartment coatings thanks
to the selection of different materials and assembly of a variable number
of layers.
4. Loading and release mechanisms
There are several factors that can interfere with the release of a drug
from a LbL device. The simplest way is by changing the degree of interaction between layers, which in turn affects the swelling of the device,
the coating permeability and drug diffusion. These modications are
usually reversible and do not destroy the carrier. In some cases they
can be achieved by using stimuli-responsive nanobiomaterials as
1315
building blocks. Another option is to initiate the destruction of the multilayers, thus triggering the immediate release of a loaded drug. In this
section, we will revise the mechanisms that lead to both fates.
4.1. Release by disruptive interactions and controlled stimuli

4.1.1. pH
pH variations comprise one of the disruptive mechanisms on LbLbased devices. PMCs comprised of weak polyelectrolytes are inherently
pH-responsive due to protonation/deprotonation of charged groups.
Altering the pH of the medium allows controlling the charges of the
polyelectrolyte units in the shell and consequently the interaction
between layers (Delcea et al., 2011). When the pH is altered relative to
the pKa of the polyelectrolytes, protonation/deprotonation of ionizable
groups takes place. An increase in the number of charged groups leads
to an excess of charges, not bonded to oppositely charged groups. A
stronger repulsion leads to PMC shell swelling and thus to increased
permeability. This behavior is reversible, meaning that upon reestablishing the pH to its initial value, PMCs shrink and permeability
decreases. These phenomena also occur with planar LbL lms (Sato
et al., 2011), though most of the in-depth studies have been focused
on 3D PMCs.
The reversible pH-dependent swelling of PMCs has been often used
to encapsulate high molecular weight compounds. In the work of
Djugnat and Sukhorukov (2004) the permeability of PSS/PAH PMCs
could be switched between an open and a closed state with pH variations up to 11, to trigger the loading of large molecules, such as PSS
and dextran.
Although these variations may be at rst assumed to occur around
the polyelectrolyte pKa, it is necessary to understand that after
polyacid/polybase complexation, the pKa shifts. For all purposes, the
polyelectrolytes no longer act as two individual components but as a
composite polyelectrolyte complex with distinct acidity constants.
Petrov et al. (2003) showed that the apparent pKa of PAH (pKa of 8.7),
one of the most popular weak polyelectrolytes used in LbL formulations,
shifts to 10.7 when complexed to PSS (a strong polyelectrolyte). Thus, it
is crucial to characterize the pH-induced permeability of PMC shells to
properly tune the release prole of a LbL-based drug delivery carrier.
Loading and releasing bioactive molecules via pH variations are the
result of two phenomena: (i) the disruption of electrostatic interactions,
and (ii) the induction of physical defects. The consequences of (i) in the
permeability have already been alluded to in the given examples, but
charge variations can be used as a means to post-encapsulate drugs in
PMCs, in opposition to preloading strategies that rely on the use of
drug-functionalized sacricial templates (Petrov et al., 2005). Breaking
molecular interactions creates binding points for drugs, which is a
straightforward way of loading molecules with an opposite charge
with respect to overcharged layers. For example, Zhao and Li (2008)
have shown that loading bovine serum albumin (BSA) into poly(L-lysine) (PLL)/chondroitin sulfate capsules is dependent on pH. The interactions between BSA and chondroitin sulfate are stronger at pH values
below the isoelectric point of BSA, where BSA is positively charged,
whereas chondroitin sulfate is negatively charged. Likewise, BSA release
was higher as pH was increased, due to the disruption of protein/LbL interactions. In the work of Feng et al. (2014) mesoporous silica nanoparticles (MSNs) were coated with CHI/ALG multilayers and loaded with
DOX, a polycationic drug, at pH = 3. The release of DOX could be modulated by varying pH. Decreasing pH increased the release rate, due to
the electrostatic repulsion between DOX and CHI at low pH values. Simultaneously, the carboxylate groups on alginate were less negatively
charged at low pH values, thus reducing electrostatic attractions between alginate and DOX. The authors proposed the use of pHresponsive multilayer carriers for the treatment of cancer, since the
acidic intracellular environments would be a suitable trigger to release
polycationic chemotherapeutics (Fig. 3).
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Fig. 2. Hierarchical organization of multicompartmental capsules, highlighting the multilayer shells as compartment boundaries containing smaller elements inside.
Adapted with permission from Costa et al. (2013a), Copyright 2013, American Chemical Society.
For (ii), it has to be considered that pH variations may induce defects

in the multilayers. Antipov et al. (2002) were pioneers in showing that
PAH/PSS PMCs can be switched between open/closed states by varying
the pH values, in respect to the assembly pH (pH = 7). Decreasing the
pH led to an increase of the PAH positive charge, which resulted in the
formation of pores in the PMC shells and increased permeability to
FITC-conjugated dextrans. Thus, altering the pH disrupts intermolecular
interactions and increases electrostatic repulsions, by which porosity or
even shell disintegration may be induced. Liu et al. (2014) showed that
DOX preloaded PMCs of tannic acid and poly(N-vinylpyrrolidone) released the cargo only for high pH values (around 10), due to tannic
acid neutralization and complete PMC disintegration, but DOX was
protected for values close to neutral pH, at which the capsule was still
intact.
4.1.2. Temperature
Similar to pH, PMCs are inherently responsive to temperature. When
PMCs experience a temperature increase, a structural rearrangement
takes place in order to optimize the shell/water interface. This phenomenon is dependent on the number of layers. By default, when one layer is
adsorbed, the system acquires an excess of electrostatic charges. When
a second layer is adsorbed, such excess is balanced and the system
reaches a zero-net charge (Khler et al., 2005, 2006). Due to the
repeating character of the LbL assembly, for every LbL-based lm there
is either a charge excess or a balance when the number of layers is odd
(in the form of a (P1 / P2)n / P1 organization, where P1 and P2 are two
complementary polyelectrolytes) or even (in (P1 / P2)n form), respectively. In a 1:1 electrostatic ratio (i.e., even number of layers), hydrophobic interactions are prevalent and heating leads to shrinking. When the ratio is
not met (i.e., odd number of layers) electrostatic repulsions prevail, leading to swelling. These tendencies are amplied by the increase of temperature since they promote the optimization of the shell molecular
conformation.
Insight on this mechanism was given in a series of reports by Khler
et al. (2005, 2006). Using PMCs made of strong polyelectrolytes
namely PDADMAC and PSS they showed that shells with an even number of layers shrunk upon heating, accompanied by an increase of their
thickness, and PMCs with an odd number of layers had their diameter increased 5-fold of their initial size. Increasing the temperature above 55 C
led to their disassembly. The thickening of the capsule wall that accompanies PMC shrinking had been demonstrated previously by Leporatti
et al., who explained that there is a rearrangement of the macromolecular
layer constituents, assuming thus a conformation that is entropically
more favorable (Leporatti et al., 2001). Temperature-induced permeation
is a very popular mechanism used in drug post-loading procedures.
Incubating PMCs in a drug solution rst leads to its penetration to their interior. At high temperatures, the mixture is heated until the capsules
shrink and the shells become impermeable, entrapping the drug inside
(Khler and Sukhorukov, 2007). Large molecules may have higher difculty to traverse the LbL shell and may require an additional increase in
permeability. As will be discussed in the next section, there are situations
when this can be achieved by high ionic strength values.
The shrinking/swelling behavior of PMCs has been reported to occur
typically in temperature ranges between 50 and 70 C (Ibarz et al., 2002;
Glinel et al., 2003; Khler et al., 2006; Bedard et al., 2009; Huang and
Chang, 2009). Temperature-responsive macromolecules can be incorporated in the multilayers. Recently, it has been reported that incorporating temperature-responsive polypeptides in the PMC shell reduces
the temperature necessary for shrinking to occur. ELR-containing
PMCs experienced a reduction of 42% in size when varying the temperature from 25 to 37 C, which are more biological-friendly values (Costa
et al., 2013b). Introducing temperature-responsive materials into LbLbased drug delivery devices, together with an odd/even number of
layers, may constitute an additional means for controlling the release
rate of this class of drug carriers.
4.1.3. Ionic strength
Ionic strength-induced variations are caused by the presence of salt
ions within the multilayer lms. The inuence of ionic strength on LbL
lms is similar to that of pH, since both affect the electrostatic interactions between layers. The difference is that, unlike pH, strong polyelectrolytes can be also affected. Ions trigger a screening mechanism
characterized by the interaction between ions and polyelectrolyte
ionized groups (Chen and McCarthy, 1997; Schlenoff et al., 1998). An
increase in the concentration of salt results in the disruption of electrostatic bonds between polyelectrolyte layers, due to competing polyelectrolyte/polyelectrolyte and polyelectrolyte/counterion interactions. As a
result, the LbL structure becomes a looser mesh and swells. The higher
ionic content within the LbL structure also promotes swelling due to osmotic water storage (Schlenoff et al., 2008). Under high ionic strength
conditions, the permeability of a multilayer shell increases and high
molecular weight molecules can traverse through it. In PMCs, charged
groups are shielded and pores form, allowing the permeation of molecules to the interior (Peyratout and Dhne, 2004; De Cock et al., 2010).
If salt is removed from the system, PMCs become impermeable again,
accounting for the reversible nature of this stimulus.
Salt-induced permeability and changes in morphology of multilayer
lms and capsules are well-established protocols and a common strategy to load bioactive substances with high molecular weights (Fery et al.,
2001; McAloney et al., 2001; Delcea et al., 2011). Ibarz et al. (2001)
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Fig. 3. Construction schematics of CHI/ALG-coated MSNs loaded with DOX for the intracellular delivery to cancer cells.
Reprinted with permission from Feng et al. (2014), Copyright 2014 American Chemical Society.
showed that PMCs exhibit a semipermeable behavior with a molecular

weight cut-off. PAH/PSS capsules were impermeable to PAH with a
molecular weight above 15,000 Da, whereas low molecular weight
dyes could easily traverse the LbL shell. However, upon increasing the
salt concentration (above 0.02 M in NaCl), PAH with a molecular weight
up to 70,000 Da was loaded into the PMCs. Triggering permeability
changes via ionic strength variations does not seem to affect low
molecular weight substances, even if they are hydrophobic. This hypothesis is supported, for example, by the work of Qiu et al. (2001a,
2001b). These authors encapsulated ibuprofen, an acidic nonsteroidal
anti-inammatory drug, in various types of PMCs, including PAH/PSS,
poly(ethyleneimine) (PEI)/PSS and PAH/dextran sulfate. The fractional
release of ibuprofen in pure water and 0.5 M NaCl solutions was not
affected signicantly. The scheme in Fig. 4 depicts the effect of ionic
strength and other disruptive mechanisms in PMCs.
4.2. Destruction of the carrier
4.2.1. Light
Light constitutes an elegant way to control how a drug is encapsulated and released. Light-responsiveness is receiving increasing attention
for drug delivery owing to the possibility of developing intelligent systems that release active molecules in a precise, temporal and spatially
controlled fashion (Alatorre-Meda et al., 2013; Alvarez-Lorenzo et al.,
2009; Borges et al., 2014). In general, the encapsulation and controlled
release of drugs mediated by electromagnetic stimuli can be driven by
reversible and sometimes non-reversible mechanisms governing
the integrity and evolution of the physical properties of drug carriers.
Reversible processes, such as gradients in the hydrophilic/hydrophobic
character of polymeric walls, are commonly brought by the photoisomerization of light-sensitive groups. Meanwhile, non-reversible
mechanisms, such as the cleavage and degradation of polymeric chains,

can be promoted by photopolymerization, photocrosslinking/
decrosslinking,
photosensitization-induced
oxidation
and
photoexcitation.
Briey, photoisomerization is related to conformational changes
around bonds (usually double bonds) restricted in rotation. This phenomenon involves predominantly a trans-to-cis isomerization of azobenzene
groups (which have a N = N bond with phenyl rings on either
side) upon electromagnetic irradiation, and/or the generation of other
charged species (e.g., the conversion of spiropyrane to merocyanine).
Photoisomerization is usually accompanied by a change in the hydrophilic/hydrophobic balance of the photoexcitable molecules. Thus, if
these molecules are assembled in a drug delivery system, light can
serve as a remote trigger for particle disassembly and drug release
(Kano et al., 1980).
Photodegradation involves the ablation of polymeric chains all along
the drug carrier, which causes the disassembly of the drug delivery
system. Degradation of polymers in small fragments may facilitate the
clearance from the body.
Photocrosslinking and decrosslinking entail the formation and
rupture of light-sensitive junctions holding together polymer chains
within polymeric matrices, respectively (He et al., 2009). The rupture of junctions leads rst to a gradual formation of bigger and bigger pores along the polymeric matrix and nally to the disintegration
of the carrier, which is useful to increase the release kinetics of a
drug.
Photosensitization-induced oxidation implicates the generation of a
strong oxidizing agent, singlet oxygen, upon illumination of a sensitizer
molecule loaded within the drug carrier or part of the structure itself.
Irradiation at an appropriate wavelength leads to the disruption of the
drug carrier (Anderson and Thompson, 1992).
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Fig. 4. Disruptive effects of pH, temperature and ionic strength on LbL shells leading to permeability variations. The example of PMCs is represented.
Finally, photoexcitation exploits the ability of metal nanoparticles to

absorb light efciently due to coherent oscillation of conduction band
electrons in strong resonance with certain frequencies of light. This phenomenon depends on nanoparticle size, shape, level of aggregation and
composition (Prevo et al., 2008). Photoexcitation of metal nanostructures results in the formation of a heated electron gas that rapidly
cools down by exchanging energy with the nanoparticle lattice (Link
and El-Sayed, 2000). Metal nanoparticles can reach temperatures well
above 600800 C (Wu et al., 2008), which induce signicant thermal
and mechanical stresses in the structure of the drug carriers, leading
to their rupture and subsequent payload release. Alternatively, nanoparticles can transmit the energy to the surrounding medium causing
localized heating, although the temperature increase is limited to a
few degrees (Topete et al., 2014a, 2014b).
LbL systems exhibiting the above described mechanisms have been
conceived to encapsulate and release drugs from polymeric systems.
Some representative examples describing the inclusion of metal nanoparticles as a part of the LbL construct can be consulted elsewhere
(Bedard et al., 2010; Esser-Kahn et al., 2011). In other examples, Bedard
et al. (2007) made use of photoinduced structural changes in polymeric
capsules to entrap and release uorescent dextran molecules from
an azobenzene-substituted LbL construct of (PAH/PAZO)n/PVS, where
PAZO and PVS stand for poly(1-4[4-3(3carboxy-4-hydroxyphenyl-azo)
benzenesulfonamido]-1,2-ethanedyil) and poly(vinyl) sulfonate, respectively. Light irradiation caused shell shrinking along with an
increase in wall permeability, which facilitated encapsulation. The longer the irradiation time, the greater the encapsulation yield. Although
annealing effects were excluded, the permeability changes were found
to be irreversible. Park et al. (2005) used photocrosslinking as a
means to achieve rhodamine B release from LbL nanocapsules prepared
with benzophenone modied-PAH and PSS. The release rate could be
conveniently controlled by the photocrosslinking density of PAH chains.
The permeability of the PAH-BP/PSS hollow shells was reduced by ca.
50% after 3 min of UV irradiation. Finally Koo et al. (2010) employed
the LbL technique to prepare PMCs with walls containing photoacid
generators (PAGs). Upon exposure to UV light, the PAGs were activated
and the subsequent pH decrease caused by the release of protons triggered the swelling of the microcapsules. Interestingly, the open/closed
state of the microcapsules could be controlled via alternate exposure
to UV light and washing with neutral water. Prolonged exposure led
to the disassembly of the capsules and caused rapid release of the

entrapped substances.
4.2.2. Biochemical
Biochemical recognition is another widespread approach to induce
the assembly and disassembly of LbL constructs in drug delivery. In a
recent review Borges and Mano (2014) presented a comprehensive
description of diverse mechanisms based on biologically specic interactions, including the avidinbiotin, antibodyantigen, and lectin
carbohydrate binding processes, as well as hybridization of DNA base
pairs. In the context of these particular interactions, stimuli-responsive
lms were developed by the LbL deposition of 2-iminobiotin-labeled
PEI (ib-PEI) and avidin at pH 812 (Inoue and Anzai, 2005; Inoue
et al., 2005). Such systems were characterized in terms of binding
strength as a function of the working pH and the presence of biotin derivatives. It was found that slightly acidic conditions (pH 56) favored
the disassembly of the studied systems due to protonation of the
iminobiotin residues in ib-PEI. In turn, the presence of biotin (and
some analogues) at alkaline pH values (pH 812) appeared to establish
a competition with ib-PEI for the molecular binding with avidin, also
resulting in system disassembly. Smart systems based on this mechanism have proved to be effective for in vivo targeting of tumor hypoxia
(Poon et al., 2011a) and biosensing (Inoue and Anzai, 2005; Inoue et al.,
2005). It is also worth analyzing LbL systems based on DNA hybridization (Johnston et al., 2005, 2006; Kato et al., 2009). Multilayered lms
consisting solely of DNA were successfully assembled by using oligonucleotides. The thickness and swelling of the lms could be controlled by
the extent of hydrogen bonding (i.e., the guanine/cytosine content of
the oligonucleotide) and orientation of the oligomers. Urea treatment
of the lms induced morphological changes, while exposure to low
ionic strength solutions led to lm disassembly. Interestingly, DNA multilayer lms were also assembled onto silica particles, and DNA hollow
capsules were obtained following dissolution of the core template
(Johnston et al., 2005, 2006). Other architectures based on hybridization of DNA base pairs include the surface modication of electrospun
bers (Mller et al., 2006) and the formation of highly ordered 2D
patterned DNA nanoarrays by using uniform oligonucleotide lms and
lithographic processes (Noh et al., 2009). The assembly of propagating
structures through DNA hybridization is likely to nd application in
delivery and sensing devices where switchable lm morphology is

required.
4.2.3. Electrochemistry
Electrochemical stimuli are another appealing way to control the
assembly and disassembly of LbL systems for chemical sensing and
drug release (Zhang et al., 2004; Rodrguez Couto and Toca Herrera,
2006; Wang et al., 2009a). The assembly process, referred to as electrochemical LbL deposition, is a potentio-dynamic method that promotes
the interlocking of oppositely charged compounds by the
enhancement of their electrostatic interactions and/or the promotion
of redox reactions, which are triggered by the selective ionization of
the involved species (Cheng and Dong, 1999; Cheng et al., 1999). Briey,
the method consists in applying an external electric eld during the
sequential deposition of the compounds onto an electrode. The process
is similar to electrostatic LbL assembly but progression in the adsorption
involves a conductive substrate and an applied voltage, which should
be opposite in charge relative to the species to be deposited
(Ngankam and Van Tassel, 2005; Shi et al., 2003; Sun et al., 2002).
Some aspects of this methodology are more advantageous than the conventional electrostatic-driven lm growth, since the lms are usually
more uniform, and it is a more suitable technique to be performed in
the presence of salt without risk of competitive adsorption from the
salt ions (Cheng and Dong, 1999; Cheng et al., 1999).
By exploiting electrochemical interactions, it is possible to trigger the
release of loaded drugs on demand. Dong and coworkers (Wang et al.,
2009a) reported the tunable release of DNA from inorganic ions/DNA
multilayer lms. At rst, they immobilized DNA on the surface of a gold
thin lm by the coordination/electrostatic interactions between inorganic zirconium ions and phosphate groups in the backbone of the DNA
chain. Then, the biopolymer was selectively released upon the exclusive
control of an external electric potential. The selective electrodissolution
of the lm was attributed to the migration of ions in the multilayer lm
and the electrodeposition of ions on the gold surface, a phenomenon
being dependent on both the applied potential and the ionic strength of
polyelectrolytes. Remarkably, the electrodissolution process could be
switched off by removing the external electric potential and restored by
reapplying the electric eld at physiological pH. As claimed by the authors, this approach can be generalized to other negatively charged biomolecules, including RNA and some proteins. Thus, it is an attractive
approach for biomedical applications where spatio-temporal control of
biological material is required (Wang et al., 2009a).
5. Drug delivery for biomedical applications
The inclusion of therapeutic molecules as building blocks of LbL lms
and PMCs has allowed addressing several biomedical applications that
require at some point the delivery of drugs. Herein, we review the
applications that have recently beneted from the development of
LbL-based devices, with special emphasis on nano-sized devices
lms, tubular structures and PMCs.
5.1. Antifouling
Antifouling is the capability of a surface to inhibit protein adhesion,
microorganism growth and biolm formation (Banerjee et al., 2011).
When any of these events occur, the most likely results are contamination, alteration of the surface properties (with respect to the original
properties of the interface) and degradation, culminating in decreased
device durability. Antifouling LbL strategies have been mostly exploited
to prevent decline of water ux and loss in the salt rejection properties
of reverse osmosis membranes (Chen et al., 2013; Kristensen et al.,
2008) and to minimize costs and losses in construction materials of maritime structures due to corrosion (Xu et al., 2014; Zhu et al., 2013). For
biomedical devices, it is crucial that their surfaces exhibit antimicrobial
properties to prevent contamination. Microbial contamination is one of
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the major causes that lead to the premature failure of biomedical devices,
such as implantable devices and catheters (Brown et al., 1997; Costerton
et al., 1999). However, when designing antifouling coatings to be exposed to living organisms, one needs to consider that these environments
can be as complex and hostile if not more than those in the naval
industry: there are bacteria and viruses with highly specic binding
mechanisms, as well as proteins that may affect the performance, durability and integration of biomedical devices in a living organism.
Since LbL allows using therapeutic molecules as multilayer building
blocks, the most straightforward strategy to avoid deleterious effects
caused by microorganisms is an assembly using drugs. For instance,
Hammond and coworkers (Wong et al., 2010) developed bactericidal
and virucidal lms with N,N-dodecyl, methyl-poly(ethyleneimine)
(DMPEI), a polycation with microbicidal activity, and various polyanions.
The surfaces proved to be effective against Gram negative/positive
airborne/waterborne bacteria (Escherichia coli and Staphylococcus
aureus) and viruses (inuenza virus, strain H1N1). Zhuk et al. (2014)
followed the same strategy to conceive lms with self-defense capability. Films made of tannic acid and one of several cationic antibiotics
were rst assembled. In bacterial cultures, the release of antibiotics
was triggered by the local acidication caused by bacteria adhesion,
growth and initiation of lm degradation. High antibiotic efcacy was
detected not only at the surface but also in a relatively large volume in
the vicinity. This is an interesting approach since it ensures that an
antibiotic is protected until it is strictly required, i.e., when bacteria
need to be eliminated. It also reduces unnecessary exposure of bacteria
to antibiotics and possible development of resistant bacterial strains.
Adding camouage to a biomedical device is useful not only to avoid
undesired microorganism adhesion but also to avoid recognition and internalization by cells. Zhou et al. (2010) coated poly(lactic-co-glycolic
acid) (PLGA) nanoparticles with CHI/ALG multilayer lms. In a preliminary stage, CHI/ALG planar lms prevented the adhesion of a model protein, BSA. This assay is an indicator of how a drug carrier interacts with
serum proteins inside the body, which may affect their circulation time
and cellular uptake. Then, the internalization of CHI/ALG-coated PLGA
nanoparticles by HepG2 liver cancer cells was shown to be about 20%,
whereas bare nanoparticles showed an uptake ratio of about 90%. This
study shows that it is possible to properly engineer a surface in order to
provide a shield that prevents non-specic cellular uptake interactions.
5.2. Injectable drug formulations
Therapeutic substances can be administered by injection (intramuscular, intradermal or subcutaneous), orally or by intranasal spray application (Mitragotri, 2005; Giudice and Campbell, 2006; Becker et al.,
2010), often available as colloidal formulations. As LbL-based devices
move towards clinical translation, coated particles and PMCs have
been suggested for injectable formulations that benet from the advantages of the LbL technique (Morton et al., 2013). Today, micrometric
PMCs have been used mostly to study the fundamentals of LbL spherical
drug delivery carriers and have been even suggested to be used in
vaccine formulations (De Geest et al., 2012). However, micrometric
components may pose a risk of obstruction in blood vessels with the
lowest caliber (ca. 5 m), thus nanometric PMCs have the highest
potential to be used in LbL nanovaccine formulations.
Drug stabilization is crucial for injectable drug formulations. When a
drug is released from its carrier in a living organism, it is rapidly cleared
and may produce signicant off-target cytotoxicity. This is particularly
important with low molecular weight drugs, which tend to be released
faster and thus it is necessary to ensure proper distribution at cellular
and tissue levels (Chen, 2010). Hammond and coworkers have recently
studied the stability and biodistribution of gold nanoparticles and
quantum dots coated with PLL and dextran, capped with a layer of HA.
Off-body imaging techniques were used to trace their retention inside
BALB/c mice. These showed low liver accumulation and a blood elimination half-life of 9 h (Poon et al., 2011b). In another work (Morton et al.,
1320
2013), PLGA nanoparticles were coated with PLL and dextran sulfate
multilayers. The PLGA cores were loaded with cardiogreen (CG), a
polymethine dye. Using a nude female mice animal model, nanoparticle
formulations were administered via the tail vein. In comparison to noncoated PLGA nanoparticles, LbL architectures signicantly reduced liver
accumulation (Fig. 5). Furthermore, drug half-life increased from 1.87 h
to more than 4 h for the coated systems.
These studies show how LbL architectures may help improve drug
delivery, efcacy and biodistribution in living organisms. It may inclusively be an interesting approach to deliver short bioactive polypeptides, which are rapidly degraded by the body and, as such, have met
limited clinical success. By encapsulating such peptides within a multilayered carrier, there is potential to increase their half-life in the body in
order to have a signicant therapeutic effect. In this regard, there are
also recombinant antigens, which hold high potential as vaccines
against lethal intracellular pathogens and cancer, but are poorly immunogenic and fail to induce potent cellular immunity (De Geest et al.,
2012). Recent works with micro PMCs, like the report of De Geest
et al. (2012), show that LbL architectures are adequate containers to improve the immune response to vaccine antigens.
5.3. Gene delivery

Gene therapy is the process by which a foreign, corrective (or missing) gene is inserted in biological tissues or cells aiming to alleviate
symptoms or prevent disorders (Anderson, 1990, 1998). Conceptually
speaking, this innovative approach of using a gene as the drug was conceived to constitute a promising alternative to conventional procedures
to treat some diseases (Crystal, 1995; Verma and Somia, 1997). However, due to therapeutic biological limitations (such as adverse immune
responses of the body) and a poor understanding of the physicochemical motifs involved in the DNA compaction and delivery processes
(i.e., transfection), gene delivery is not 100% effective yet (Mhashilkar
et al., 2001; Alatorre-Meda et al., 2011a, 2011b).
The entrance of naked exogenous DNA to the cell nucleus is problematic due to different extra and intracellular barriers. On the one
hand, systemic circulation of DNA is hindered by nuclease degradation
(Nguyen et al., 2009). On the other hand, the electrolytic nature of
DNA gives rise to electrostatic repulsions as DNA approaches to cells,
provided that both DNA and cell membranes are negatively charged
(Tros de Ilarduya et al., 2010). Also, once inside cells, steric restrictions
hinder DNA transportation to the cell nucleus (Dowty et al., 1995).
Thus, in order to properly transfer exogenous DNA into living cells, all
extra/intracellular barriers must be circumvented.
Current gene transfer protocols rely on natural and synthetic DNA
complexing agents (referred to as vectors or gene carriers) to surpass
such biological barriers. The ideal vector must protect DNA from enzymatic degradation, bind to target cells without inducing toxicity or an
immune response, cross the cell membrane, escape from the endosome,
and nally release the therapeutic DNA or RNA, resulting in an induced
gene expression. In this context, the LbL technology presents itself as an
attractive unconventional technique to produce gene delivery vectors.
It allows a precise tuning of the chemical, mechanical and biological
properties of the engineered systems depending on the choice of the
constituent materials, assembly conditions and number of layers. Moreover, LbL assembly can also be applied to a range of templates, including
at surfaces and colloidal particles, which can contain the genetic material to be released or can simply be employed as sacricial substrates to
produce thin lms and PMCs.
Thin lms constitute the simplest LbL vectors in gene delivery, using
DNA or RNA as the anionic building block in the construct. Essentially,
the DNA layers can be intercalated with layers of cationic polymers
such as PLL, poly(-amino ester)s, PEI and CHI, among others (Jewell
and Lynn, 2008; Blacklock et al., 2009; Li and Zhang, 2011). This setup
takes advantage of the attractive electrostatic forces between the oppositely charged species. The incorporation of different DNA plasmids into
different layers of the lm and the application of materials with different properties might provide multilayered lms with sophisticated
levels of temporal control over the release and expression of multiple
Fig. 5. Stability assessment of different nanoparticle architectures using in vivo imaging, up to 30 min after injection. The uorescence images depict cardiogreen (CG) as green
and poly(L-lysine) as blue, organized as shown on top. The numbers indexed to these two components represent the emission wavelength. Each line represents (i) free CG820,
(ii) PLGA50:50
CG , (iii) HA-terminated coatings, (iv) ALG-terminated coatings, and (v) dextran sulfate-terminated coatings. The liver is identied by the white dashed circle with an arrow.
Reprinted with permission from Morton et al. (2013), Copyright 2013 Elsevier.
genes in physiological conditions and low cytotoxicity (Zhang et al.,

2004; Jessel et al., 2006). Moreover, multilayered systems can also be
constructed to combine cell targeting molecules (e.g., antibodies),
membrane entry facilitating molecules and intracellular targeting
molecules (Singh et al., 2015; Costa et al., 2014; Hagemeyer et al., 2015).
PCMs represent another suitable architecture for gene release. Two
main conformations have been explored so far. In one case, the genetic
material is complexed with a selected vector to be encapsulated via
supercial adsorption of polycation/polyanion multilayered lms
(Zelikin et al., 2007; Soto and Ostroff, 2008; Lomas et al., 2011; Ng
et al., 2011; Santos et al., 2012). This particular conformation is anticipated to endow the genetic material with the highest possible protection against enzymatic attacks since it is covered by both the core and
the multilayered shell. However, this setup renders delivery systems
with minimal doses of encapsulated DNA, hindering to some extent its
tentative clinical impact. In a second architecture, the gene is bound to
preformed templates through electrostatic attraction to be subsequently coated by additional polyelectrolyte layers (Schler and Caruso,
2001). Similar to thin lms, in this case the gene is the anionic building
block, offering the possibility of adding as many gene layers as necessary, increasing the DNA loading capacity.
Finally, it is worth mentioning that independent of the chosen
architecture, the proper gene release requires an efcient defoliation,
or erosion, of the polyelectrolyte layers once in contact with cells and
tissues (Lynn, 2007; Saurer et al., 2013). Pathways that do not trigger
the erosion of layers, such as passive diffusion, are inadequate since
nucleic acids, in particular DNA, are large molecules and do not pass as
readily through multilayered polyelectrolyte assemblies as small molecules do (Jewell and Lynn, 2008). Approaches to trigger degradation
of LbL lms and capsules include light irradiation (Radt et al., 2004), ultrasound insonication (Borden et al., 2007), changes in pH (Sukhishvili
and Granick, 2001) and ionic strength (Schler and Caruso, 2001),
exposure to enzymes (Serizawa et al., 2002; Ochs et al., 2010) and
redox reactions (Li and Haynie, 2004; Zelikin et al., 2008).
5.4. Stem cell differentiation and tissue formation

LbL nanodevices can be used not only to ght well-known diseases
but also to drive the differentiation of cells and ultimately lead to the
formation and regeneration of de novo tissue. LbL can also be seen as
a technology able to deconstruct the microenvironment of the intracellular matrix (Mano, 2015). Differentiation of stem cells can be achieved
by loading LbL devices with differentiation agents, such as growth factors. Strategies to obtain osteoblasts from stem cells have been reported
using LbL-based devices. For example, Hammond and coworkers loaded
various growth factors in multilayers of polycationic poly(-aminoesters) and one of two polyanions heparin sulfate and chondroitin
sulfate. In one case, broblast growth factor 2 (FGF-2) was intercalated
with the primary polymers during lm assembly (Macdonald et al.,
2010). Assembling a higher number of layers led to higher quantities
of FGF-2 being loaded, and also released with faster kinetics. This
growth factor-loaded system also promoted the proliferation of
MC3T3 osteoblast precursor cells. In another work (Shah et al., 2011),
a cocktail of growth factors rhBMP2 and rhVEGF was studied instead
of single-encapsulated growth factors. Comparing with lms loaded
with just rhBMP2, the cocktail proved to be up to 33% more effective
in the formation of de novo bone.
Following the same concept, Picart and coworkers studied
carbodiimide-crosslinked PLL/HA lms as reservoirs of BMP. For various
substrates tricalcium phosphate/hydroxyapatite (TCP/HAP) macroporous granules (Crouzier et al., 2011) and titanium (Guillot et al.,
2013) BMP-loaded lms exhibited osteoconductive behavior in a rat
ectopic model. The substrates used in these works are appropriate for
applications dedicated for bone regeneration, since a great deal of
implantable devices in bone rely on titanium and calcium phosphate
1321
compounds (Fini et al., 2004; de Jonge et al., 2008; Alghamdi et al.,

2013).
Another interesting approach is to encapsulate differentiation factors in spherical shapes, which show a higher surface area and improved
cell accessibility when compared to planar substrates. Facca et al.
(2010) conceived PLL and PLGA PMCs with around 1 m embedded
with BMP-2 and TGF-1. They demonstrated that the formation of
new bone cells could be induced in vitro and in vivo. Embryoid bodies
and PMCs loaded with the growth factors were mixed in an alginate
gel and injected subcutaneously in mice, where the embryoid bodies
showed osteoblastic differentiation after 21 days. No brosis was detected at the implantation site. The use of submicron capsules can be advantageous since the smaller the structure, the easier the
internalization, thus delivering a differentiation agent more efciently
to stem cells (Rejman et al., 2004).
Loading multiple growth factors is a crucial strategy for tissue engineering strategies, a eld that relies on cells, scaffolds and biochemical
signals to help regenerate biological structures and create tissue analogues in a laboratorial environment (Richardson et al., 2001). Bone formation has been one of the most studied applications with LbL-based
strategies. This can be justied by the fact that much is already known
about biomineralization and bone regeneration (Alves et al., 2010)
and has become a model tissue when it comes to new differentiation
strategies. It is expected that soft tissues are addressed in the next
years, such as cartilage and neurons, as soon as the potential of LbL
devices for de novo tissue formation is better understood.
6. Current regulatory status of nanotechnology products
At present, there is no legal regulation worldwide related to the
usage and disposal of nanomaterials neither for industrial nor for
medical applications. That includes products that are derived from LbL
strategies, given that the assembly of multilayers occurs at the nanoscale. The existence of such regulations should be determinant for the
industrial and clinical translation of LbL for the fabrication of medical
nanostructured devices, including nanosized carriers for drug delivery.
Nonetheless, as a public health agency using scientic information to
make regulatory decisions about emerging products to be employed
in the food and biomedical elds, the Food and Drug Administration of
the United States of America (FDA) has been committed to gather information related to nanotechnology-derived products.
The current thinking of FDA regarding the use of nanotechnology or
nanomaterials in FDA-regulated products is reected in a series of draft
guidance documents that are published by the agency and continuously
updated. These documents provide a means for manufacturers and
consumers to consult available information on the safety, effectiveness,
or other product attributes on a case-by-case basis. FDA's guidance
documents do not establish legally enforceable responsibilities. Instead,
and unless specic regulatory or statutory requirements are cited, the
guidance documents should be viewed as recommendations. These
documents can be found in FDA's website (US Food and Drug
Administration [Internet], 2015).
In the Guidance for Industry: Considering Whether an FDARegulated Product Involves the Application of Nanotechnology, FDA
provides criteria to help Industry and others identifying when potential
implications that may arise with the application of nanotechnology
should be considered (US Food and Drug Administration [Internet],
2014a). This guidance advises Industry to consult FDA early in the
development process to facilitate a mutual understanding of the specic
scientic and regulatory issues for their nanotechnology products. Briefly, two points are asked to be considered in order to evaluate whether
the FDA-regulated product involves the application of nanotechnology.
Namely, it must be considered whether (i) a material or end product is
engineered to have at least one external dimension or an internal or
surface structure in the nanoscale range (approximately 1 to 100 nm),
and (ii) a material or end product is engineered to exhibit properties
1322
or phenomena, including physical or chemical properties or biological

effects that are attributable to its dimension(s), even if these dimensions fall outside the nanoscale range, up to 1 m (1000 nm). An
afrmative nding to either of these points might suggest the need for
particular attention by FDA and/or Industry to the product in order to
identify and address potential implications for safety, effectiveness,
public health impact, or regulatory status of the product.
The Guidance for Industry: Safety of Nanomaterials in Cosmetic
Products describes safety issues that should be considered to ensure
that cosmetic products made with nanomaterials are safe and not
adulterated (US Food and Drug Administration [Internet], 2014d).
The guidance is intended to assist Industry and other stakeholders
in identifying the potential risks of nanomaterials in cosmetic products and developing a framework for evaluating them. In particular,
it summarizes important factors to be elucidated in order to assess
the impact of the nanomaterial in question on the end product, including (i) the physicochemical characteristics, (ii) agglomeration
and size distribution of nanomaterials under the conditions of toxicity testing and as expected in the nal product, (iii) impurities, (iv)
potential routes of exposure to the nanomaterials, (v) potential for
aggregation and agglomeration of nanoparticles in the nal product,
(vi) dosimetry for in vitro and in vivo toxicology studies, and (vii)
in vitro and in vivo toxicological data on nanomaterial ingredients
and their impurities, dermal penetration, potential inhalation, irritation (skin and eye) and sensitization studies, and mutagenicity/
genotoxicity studies. All this pertinent information, together with a
complete description of the testing methods, is requested by the
agency to the manufacturer in order to substantiate the product's
safety prior to its commercialization.
In the Guidance for Industry: Assessing the Effects of Signicant
Manufacturing Process Changes, Including Emerging Technologies, on
the Safety and Regulatory Status of Food Ingredients and Food Contact
Substances, Including Food Ingredients that are Color Additives, FDA
describes factors that should be considered by manufacturers of food
ingredients for human ingestion and food contact substances when
determining whether a signicant change in the manufacturing process
for a food substance already in the market affects its properties and
safety of use (US Food and Drug Administration [Internet], 2014c). The
manufacturing process for a food substance may evolve, for example,
to use a more efcient catalyst, to replace an expensive solvent with a
more cost-effective solvent, to introduce a treatment to reduce the
presence of contaminants such as lead, or to implement emerging
methodologies, such as nanotechnology. Some of these changes may
be considered signicant: it is these signicant changes in manufacturing process that are the subject of this guidance. The discussion of nanotechnology in this document in particular, intentional alterations of
particle size distribution on the nanometer scale addresses primarily
circumstances in which there has been a manufacturing change to a
food substance already used in food. Special attention is given to the
fact that particle size, surface area, aggregation/agglomeration and
shape may impact on the safety of nano-engineered food substances
in as much as their absorption, distribution, metabolism, and excretion
(ADME) might be considerably altered with respect to their larger scale
counterparts. Importantly, the variation in biological activity that may
result from engineering food substances in the nanometer range may
also raise questions about the applicability of traditional safety tests.
In this regard, the present guidance highlights that studies should
be thoroughly validated to establish the safety of food substances
manufactured using nanotechnology, as with any studies to support
the safety of food substances.
Finally, in the Guidance for Industry: Use of Nanomaterials in Food
for Animals, FDA provides Industry and other stakeholders with information required to identify potential issues related to the safety or
regulatory status of food for animals containing nanomaterials or otherwise involving the application of nanotechnology (US Food and Drug
Administration [Internet], 2014b).
In addition to FDA, there are other regulatory agencies studying the

pertinence of nanotechnology-derived products and synthetic processes employed in their construction. One such agency is the National Institute for Occupational Safety and Health (NIOSH), the leading federal
agency of the USA, providing guidance and conducting research on the
occupational safety and health implications and applications of a variety
of technological procedures. Regarding nanotechnology, NIOSH is active
in the following actions: (i) identifying critical issues related to possible
health hazards of nanomaterials; (ii) protecting the safety and health of
workers involved in this emerging technology; (iii) implementing
a strategic plan to develop and disseminate methods for safely advancing the technology through workplace controls and safe handling procedures; and (iv) investigating the possible applications of
nanotechnology to solve workplace safety and health issues (National
Institute for Occupational Safety and Health, 2013). To achieve these
goals, NIOSH proposes a hierarchy of controls to be used as a means to
implement safe working conditions. This hierarchy is established by
ve levels of controls: (i) elimination of risky materials/conditions; (ii)
substitution of risky materials/conditions; (iii) engineering controls;
(iv) administrative controls; and (v) personal protective equipment.
Following this hierarchy normally leads to the implementation of inherently safer conditions, where the risk of illness or injury is substantially
reduced. As for the European regulatory framework, information and
research needs can be found in another article by Rickerby (2007).
Despite the inexistence of specic regulation regarding LbL, there is a
massive amount of norms that guide the safe development of nanotechnology products. As was discussed in previous sections, LbL could be
scaled to industry if its inherent slow construction was solved, under
the risk of not being able to meet supply and demand needs. Efforts
have already been made to mass produce LbL devices using automated
devices based on dipping, sputtering and microuidic approaches.
Eventually, its industrial translation will follow the established guidelines for the production of safe multilayered products. The above cited
documents refer to various nanotechnology applications that require
the contact with the human body. They should thus be of extreme
awareness when developing LbL devices envisaging biomedical applications, including nanosized drug carriers to be administered in humans.
7. Conclusions
In this review, we debated the state-of-the-art of LbL nanodevices in
drug delivery. The adaptability of multilayers to substrates of various
shapes has allowed the development of planar (e.g., lms) and threedimensional (e.g., capsules) that can be used as reservoirs for active
agents. Distinct classes of materials have been employed in such
structures, from stimuli-sensitive and bioactive polymers to drugs
themselves. Thanks to the wide variety of reservoirs that LbL can
provide, various encapsulation strategies can be exploited, allowing
the incorporation of not only water soluble drugs but also hydrophobic
ones.
From all the constructs and geometries that LbL can produce, planar
multilayered devices are likely to show the most immediate potential in
the biomedical eld, namely drug delivery, due to their ease of fabrication and scale-up perspectives towards automated and mass production. The surface of orthopedic devices could be modied with LbL
nano-coatings loaded with antibiotics or anti-inammatory drugs to
prevent premature implant failure and rejection by the patients' body.
Growth factors could also be embedded to trigger the formation and
regeneration of de novo tissue. In the case of capsules, we witnessed
to the consolidation of micro-capsules as the gold standard of sphericalshaped LbL carriers, thanks to the convenient synthesis of their
template and drug encapsulation versatility. However, we believe that
nano-capsules will be more useful in the future. Due to their high surface area in respect to their diameter, they can be internalized by cells
more easily. Therefore nanocapsules show great potential as carriers
for the delivery of bioactive substances directly to cells, minimizing
drug losses and increasing the efcacy due to premature drug leakage.
Furthermore, tagging their surface with specic markers can be useful
in targeted therapies, like in the treatment of cancer, while not affecting
healthy cells. Due to the potential of LbL in the drug delivery eld, we
envisage that scaling opportunities will emerge towards their mass production in the pharmaceutical industry. Namely, microuidic devices
have been proven to be useful for the automated synthesis of PMCs
and may be further improved for the high-throughput fabrication of
various types of PMCs to meet different therapeutic needs. It is also
envisaged that scaling and industrial/clinical translation opportunities
will emerge. In the future, one may need to take into consideration
that they will have to follow the existing guidelines for nanotechnology
products and possibly lead to the creation of new regulatory drafts to accommodate specic criteria for LbL processing conditions and devices
thereof.
Acknowledgments
We acknowledge Fundao para a Cincia e Tecnologia (grant SFRH/
BPD/95446/2013), Fundo Social Europeu (FSE), and Programa
Operacional de Potencial Humano (POPH).
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Biotechnology Advances 33 (2015) 13101326

Contents lists available at ScienceDirect

Research review paper

Drug nano-reservoirs synthesized using layer-by-layer technologies

Available online 18 April 2015

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

Wound healing/surgical patches

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

applied to three-dimensional colloidal particles, followed by particle

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

fabrication of nanocapsules since it allowed analyzing the sequential

of the various polyelectrolytes across the length of the membrane.

3.4. Films as boundaries of scalable devices

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

therapeutic drugs (Chandrawati et al., 2011; Costa et al., 2013a;

4.1. Release by disruptive interactions and controlled stimuli

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

For (ii), it has to be considered that pH variations may induce defects

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

showed that PMCs exhibit a semipermeable behavior with a molecular

mechanisms, such as the cleavage and degradation of polymeric chains,

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

Finally, photoexcitation exploits the ability of metal nanoparticles to

to the disassembly of the capsules and caused rapid release of the

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

delivery and sensing devices where switchable lm morphology is

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

5.3. Gene delivery

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

genes in physiological conditions and low cytotoxicity (Zhang et al.,

5.4. Stem cell differentiation and tissue formation

compounds (Fini et al., 2004; de Jonge et al., 2008; Alghamdi et al.,

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

or phenomena, including physical or chemical properties or biological

In addition to FDA, there are other regulatory agencies studying the

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

Boddohi S, Killingsworth CE, Kipper MJ. Polyelectrolyte multilayer assembly as a function

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

Pavlukhina S, Sukhishvili S. Polymer assemblies for controlled delivery of bioactive

R.R. Costa et al. / Biotechnology Advances 33 (2015) 13101326

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