Soft Matter
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Volume 5 | Number 2 | 21 January 2009 | Pages 253–480
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REVIEW
Bruno G. De Geest et al.
Polyelectrolyte microcapsules for
biomedical applications
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REVIEW
www.rsc.org/softmatter | Soft Matter
Polyelectrolyte microcapsules for biomedical applications
Bruno G. De Geest,*ab Stefaan De Koker,†b Gleb B. Sukhorukov,c Oliver Kreft,d Wolfgang J. Parak,e
Andrei G. Skirtach,d Jo Demeester,b Stefaan C. De Smedtb and Wim E. Henninka
Received 3rd June 2008, Accepted 18th August 2008
First published as an Advance Article on the web 16th October 2008
DOI: 10.1039/b808262f
In this paper we review the recent contributions of polyelectrolyte microcapsules in the biomedical field,
comprising in vitro and in vivo drug delivery as well as their applications as biosensors.
Introduction
Polyelectrolyte microcapsules,1–5 fabricated by layer-by-layer
(LbL) coating6 of a sacrificial template followed by the decomposition of this template, have gathered increased interest as
novel entities for drug delivery and diagnostic purposes.3,5,7–10
Briefly explained, the LbL technique is based on the alternating
adsorption of charged species onto an oppositely charged
substrate, using electrostatic interactions as the driving force.
The main advantage of the LbL technique is the ease of
manipulation and the unmet degree of multifunctionality,11
allowing one to tailor the surface with different kinds of functional groups,12–14 lipids,15–19 nanoparticles20–22 etc.
Polyelectrolyte capsules are made by coating a spherical
substrate with alternating polyelectrolyte layers of opposite
charge.3 Once a certain thickness of the multilayer coating is
achieved, the spherical substrate is dissolved and the obtained
capsules are thoroughly washed to remove the dissolved
decomposed products of the sacrificial template. Molecules can
be entrapped into polyelectrolyte capsules after fabrication of the
a
Department of Pharmaceutics, Utrecht University, 3584 CA, Utrecht, The
Netherlands. E-mail: br.degeest@ugent.be; Fax: +32 9 264 81 89; Tel: +32
9 264 80 74
b
Laboratory of General Biochemistry and Physical Pharmacy, Ghent
University, Belgium
c
IRC, Queen Mary University of London, London, United Kingdom
d
Max Planck Institute for Colloids and Surfaces, Golm, Germany
e
Philipps University Marburg, Department of Physics, Marburg, Germany
† Author who contributed as equally as the first author.
Bruno De Geest
Bruno De Geest graduated as
a chemical engineer in 2003
from Ghent University in Belgium, where he obtained his PhD
in 2006. Following two years of
post doctoral research at the
University of Utrecht in The
Netherlands he obtained a post
doctoral fellowship at the
Laboratory of Pharmaceutical
Technology at Ghent University.
His main interest are the overlap
between chemistry, materials
science, medicine and biology.
282 | Soft Matter, 2009, 5, 282–291
capsules by temporarily switching capsule permeability23–26 or
during the generation of the capsules by incorporating them into
the porous substrate that serves as a template for LbL
coating.27,28 When the molecular weight of the molecules is
sufficiently high or when they remain entrapped by electrostatic
interaction, they remain entrapped while the low molecular
weight degradation products (often ions as is the case for
carbonate or silica based templates) of the sacrificial template can
diffuse through the capsule’s wall.29 Fig. 1 schematically represents the fabrication of hollow polyelectrolyte microcapsules in
the case where calcium carbonate is used as a sacrificial template.
Intracellular delivery
Introduced in 1998 as a physicochemical oddity, these capsules
have evolved towards delivery vehicles for different types of
Fig. 1 A schematic representation of the synthesis of hollow polyelectrolyte microcapsules using calcium carbonate (CaCO3) as a sacrificial core template. Macromolecules are co-precipitated with CaCO3 by
mixing them with calcium chloride and sodium carbonate (A). These
macromolecule loaded particles are coated with several layers of polyelectrolytes of alternate charge (B) followed by the dissolution of calcium
carbonate core template in an EDTA solution (C). Reprinted with
permission from De Koker et al.30
Stefaan De Koker
Stefaan De Koker graduated as
a bio-engineer from Ghent
University in 2001. He started
his PhD at the VIB, at the
Department for Molecular
Biomedical
Research.
Currently, he is finishing his
PhD at the Department of
Pharmaceutics
at
Ghent
University. The main focus of
his work is evaluating biodegradable polyelectrolyte microcapsules as novel antigen
delivery tools in the field of
vaccination.
This journal is ª The Royal Society of Chemistry 2009
form a pore-like structure with an internal diameter of 10 nm.
Only molecules smaller than 40 kDa can diffuse freely through
these pores. For larger molecules, transport to the nucleus is an
active process dependent on the presence of nuclear localization
signal peptides that interact with specific transporter molecules.
Due to technical limitations the design of polyelectrolyte capsules
that are small enough to cross the nuclear membrane appears
highly doubtful. On the other hand, it could be a major challenge
to equip polyelectrolyte capsules with virus-like properties
allowing the capsules to enhance the transport of their payload
through the nuclear membrane into the nucleus of the cell.
Fig. 2 A schematic representation of a mammalian cell and the different
intracellular regions which can be aimed to deliver therapeutic molecules:
(1) the endosomal compartment, (2) the cytosol and (3) the nucleus.
These regions are each shielded by their respective membranes.
molecules that serve as therapeutic agents or allow the conducting of diagnostic assays on the capsules’ surfaces31–33 or
within their micron sized interiors.34–38 In this review we give an
overview of the recent progress that has been made in the
development of polyelectrolyte capsules for intracellular
purposes, comprising both therapeutic as well as biosensor
applications. As it is schematically shown in Fig. 2 there are
roughly three zones in a living cell that can be targeted by
microcapsules for delivering therapeutics: (1) the endosomal
compartment, (2) the cytosol and (3) the nucleus.
The endolysosomal compartment of antigen presenting cells
(such as dendritic cells, macrophages and B cells) constitutes
a highly interesting target for the delivery of antigens (i.e. for
vaccination purposes) that are subsequently cleaved into peptide
fragments and presented on the cell surface in combination with
MHCII class molecules (MHC, major histocompatibility
complex), resulting in activation of CD4 + T-helper cells. Besides
the endosomes, the cell cytosol can also be a very interesting
target for antigen delivery. Cytosolic antigens are cleaved by the
proteasome, transported to the ER and eventually presented in
combination with MHCI to CD8 + cytotoxic T cells (CTLs). To
date, only a few antigen delivery systems are able to initiate CTL
responses, which are crucial to kill virally infected cells and
tumor cells. In addition, the cytosol can be a target for drug
molecules interfering with all kinds of intracellular process, such
as e.g. siRNA which can suppress the production of specific
proteins.39–42
The nucleus is the target for drugs aiming to change the genetic
code of the cell, e.g. to introduce new genes or to repair gene
defects.42–44 To reach each of these sites a specific membrane, each
with its specific properties, has to be crossed. After being
phagocytosed, particles with diameters of up to 10 mm will end up
in endosomal/lysosomal/phagosomal compartments.45 Several
strategies have been developed to help particles escape from the
endosomal compartment to the cytosol.46–55 In theory such
strategies could also be used to functionalize the surface of
polyelectrolyte capsules. However, this has not yet been reported
in literature and the question regarding whether polyelectrolyte
capsules can escape from the endosomal compartment has not yet
been addressed thoroughly. Transport of macromolecules to the
nucleus is regulated by so-called nuclear pore complexes.44,56–58
These complexes consist of several hundreds of nucleoporins that
This journal is ª The Royal Society of Chemistry 2009
Uptake, toxicity and biodegradability
Intracellular delivery implies ubiquitously that the capsules
should be able to cross the cellular membrane and deliver their
content in the cytosol of the cells or at least reach the endosomal
compartment. The interactions between capsules and living cells
have been studied by several groups addressing different
aspects59 such as uptake kinetics and mechanisms, toxicity,
intracellular degradation as well as strategies to enhance or block
the capsule uptake. Sukhorukov et al. were the first to demonstrate cellular uptake of polyelectrolyte capsules.10 They showed
that breast cancer cells could internalize up to thirty 5 mm sized
capsules, composed of PSS–PAH polyelectrolytes, per cell, only
leading to a small capsule deformation due to mechanical stress
exerted by the cells. In subsequent studies Parak et al. addressed
the toxicity of such capsules. They demonstrated that capsules
alone do not exhibit acute cytotoxic damage on cell cultures, but
that rather nanoparticles with which capsules are functionalized
are potentially cytotoxic.60 This is in particular true for colloidal
quantum dots which have been suggested as fluorescence labels
in the wall of capsules for the purpose of visualization.61 For this
reason upon functionalization of capsules with colloidal nanoparticles the cytotoxicity of the nanoparticles always has to be
considered, as embedding the nanoparticles in the polymer walls
of the capsules does not reduce their cytotoxicity.
For therapeutic purposes there is a clear need for the capsules
to be biodegradable. During the past decade several bio-polyelectrolytes, such as polysaccharides, polypeptides or polynucleotides, which are potentially biodegradable have been used
for the fabrication of capsules. Degradable multilayers on planar
surfaces have been reported by Picart et al., both in vitro and in
vivo, using the above mentioned bio-polyelectrolytes.62 Degradation was based on enzymatic action following cellular invasion
in the multilayers. A second class of degradable multilayers on
planar substrates was introduced by Lynn and co-workers using
poly-b-aminoesters.63–70 These polymers are polycations containing biodegradable ester bonds in their backbone, leading to
the erosion of the multilayers and the release of potentially
therapeutic polyanions. Recently the same group also reported on
so-called charge-shifting polyelectrolytes which upon hydrolysis
undergo a shift from polyanionic to polycationic or visa versa.
This approach allowed the release of both anionic and cationic
species incorporated in between hydrolysable layers.69,71,72
Similarly, polyelectrolyte capsules that can be degraded
through ester hydrolysis or enzymatic action were obtained by De
Geest et al. using poly(HPMA–DMAE) as a degradable polycation and dextran sulfate/poly-L-arginine as bio-polyelectrolytes
Soft Matter, 2009, 5, 282–291 | 283
Fig. 4 A schematic representation of the encapsulation of drug molecules (green dots) and pronase (red Pac-Man shapes) within calcium
carbonate microparticles (A–B) followed by LbL coating of the microparticles with polypeptide layers of opposite charge (B). When the
enzyme is liberated into the empty void of the capsules by dissolving
the calcium carbonate (C), it starts to hydrolyse the peptide bonds of the
multilayers, releasing the encapsulated drug molecules. Reprinted with
permission from Borodina et al.78
Fig. 3 The molecular structure of the degradable polyelectrolytes used by
De Geest et al. for the synthesis of intracellular degradable capsules.73
Confocal microscopy images of intracellularly degraded (A) [poly(HPMA–
DMAE)–poly(styrene sulfonate)]4 capsules and (B) (dextran sulfate–poly73
L-arginine)4 capsules. Reprinted with permission from De Geest et al.
(Fig. 3A)73 Poly(HPMA–DMAE) is a so-called charge-shifting
polymer,69,71,74 meaning that it shifts from a cationic charge (due
to the tertiary amine groups) to a neutral charge upon hydrolytic
cleavage of the carbonate ester which connects the cationic amine
moiety to the polymer backbone. Incubation of poly(HPMA–
DMAE) containing polyelectrolyte microcapsules in a physiological buffer at 37 C results in the hydrolysis of the ester bonds,
causing the decomposition of the microcapsules. Degradation of
dextran-sulfate–poly-L-arginine microcapsules on the other hand,
requires proteolytic cleavage, as was demonstrated by their fast
disappearance when incubated in a pronase solution (i.e.
a mixture of proteases able to cleave virtually every peptide
bond). Both these capsules were readily taken up by VERO cells
and degraded intracellularly. Sixty hours after incubation, no
intact capsules could be observed inside the cells any more
(Fig. 3A and B) while capsules based on non-degradable polyelectrolytes remained intact.73
When transferred into an in vivo situation, the poly(HPMA–
DMAE) based capsules will degrade under all physiological
conditions and will therefore differ little from traditional
degradable microparticles. However, in healthy tissue the enzymatically degradable capsules will likely remain intact in the
extracellular space and will become degraded solely after uptake
by phagocytosing cells. Thereby, these capsules can potentially
be used as a delivery system to specifically target bioactive
(macro)molecules towards the intracellular compartment of
phagocytosing cells.
Several other groups have further explored the concept of
capsule degradation. Itoch et al.76 and Wang et al.75 demonstrated that capsules based on respectively chitosan and
284 | Soft Matter, 2009, 5, 282–291
hyaluronic acid could be degraded by their specific digesting
enzymes such as chitinase and hyaluronidase.75–77 Lee et al.77
investigated the pepsin mediated degradation of capsules
constituted of alginate and chitosan. Degradation of these
capsules however requires the presence of these specific enzymes
in close proximity of the capsules. As the expression of these
enzymes is highly restricted in vivo (e.g. pepsin is only present in
the gastro-intestinal track), this could seriously limit their in vivo
degradation. An elegant approach to stimulate capsule degradation was recently reported by Borodina et al., who encapsulated shell digesting enzymes inside the confined volume of the
capsules themselves.78 Fig. 4 shows a schematic representation of
the proposed concept. The bioactive compound (being DNA)
was co-encapsulated with pronase (being the digesting enzymes)
by co-precipitation with calcium carbonate, resulting in calcium
carbonate microparticles loaded with both DNA and pronase in
their porous matrix. Subsequently these microparticles were LbL
coated with multilayers of poly-L-aspartic acid and poly-L-arginine. These polyelectrolytes are both polypeptides and should
thus be susceptible to enzymatic hydrolysis by pronase. Indeed, it
was shown that upon incubation of the microcapsules under
physiological conditions the microcapsules spontaneously
decomposed and subsequently released the encapsulated DNA.
It was further observed that the release rate of DNA was strongly
dependent on the amount of encapsulated pronase that was
initially loaded inside the microcapsules. Moreover, as pronase
activity is temperature dependent no activity was observed at
4 C, allowing the temperature controlled release of DNA.
Relatively few studies have addressed the in vivo behaviour of
polyelectrolyte multilayer assemblies. Picart et al. have shown
that polyelectrolyte multilayers can be digested by enzymatic
action when placed in the peritoneal62 and oral environments.79
The biocompatibility and in vivo fate of dextran sulfate–polyL-arginine polyelectrolyte capsules after subcutaneous injection
were recently assessed by De Koker et al.30 Injection of the
microcapsules resulted in a typical foreign body response, characterized by a fast recruitment of inflammatory cells to the
injection site (Fig. 5). The microcapsule mass behaved similarly
to a porous implant, with cellular infiltration starting at the
periphery and gradually proceeding towards the centre. Within
one week, 5–10 layers of fibroblasts surrounded the injected
volume. As time progressed, mononuclear phagocytes internalizing particles increasingly replaced polymorphonuclear cells.
This journal is ª The Royal Society of Chemistry 2009
Fig. 5 Hematoxylin and eosin stainings of skin tissue sections at different
time intervals after subcutaneous injection of (dextran-sulfate–polyL-arginine)4 polyelectrolyte capsules. The insets show an enlarged picture of
a selected area (R1). Microcapsules appear as discs. One day after injection
the microcapsules have retained their shape and are infiltrated by predominantely polymorphonuclear cells. One week later the injection mass is
surrounded by fibroblasts while cellular infiltration gradually proceeds to
the centre. After one month microcapsule remnants are visible inside
mononuclear phagocytes. Reprinted with permission from De Koker et al.30
Importantly, although injection resulted in a mild to moderate
inflammatory response, no severe side effects such as tissue
necrosis were observed at any time, establishing the feasibility of
using polyelectrolyte capsules for in vivo applications.
To assess the in vivo uptake and degradation of the dextransulfate–poly-L-arginine polyelectrolyte capsules, RITC-labeled
(RITC, rhodamine isothiocyanate) poly-L-arginine was incorporated into the capsules, tissue sections were prepared and
analyzed by confocal microscopy (Fig. 6). One day after injection
and eight days after injection few cells had infiltrated the
microsphere mass. The microcapsules clearly had retained their
spherical shape and appeared scattered between the cells. No
deformed capsules could be seen outside the cells. Sixteen days
after injection many capsules had been phagocytosed and lost
their spherical shape. One month after injection microcapsules
were visible as debris inside the cells. At none of the time intervals
assessed deformed particles or particle debris could be observed
outside the cells, indicating that particle degradation exclusively
occurred after particle uptake (Fig. 6).
Different advantages can be envisaged for delivery systems
that release their content after cellular uptake. First, if they
contain a drug or toxic compound, they can be used to selectively
treat or kill cells that phagocytose the particles, while leaving
other cells unharmed. Possible strategies for targeting the
capsules towards specific cell types like cancer cells will be discussed later. Second, encapsulation of antigen into microparticles has been shown to enhance immune responses by basically
two mechanisms: (1) protecting antigens from fast degradation
and clearance (2) enhancing the uptake and presentation of the
antigen by professional antigen presenting cells (APCs) both via
the MHCI and MHCII routes. As biodegradable polyelectrolyte
capsules are readily taken up by dendritic cells in vitro30 and
appear quite resistant to extracellular degradation, they might be
excellent tools for the delivery of antigens to APCs, creating an
intracellular depot of the antigen. The real potential of these
microcapsules as vaccine adjuvants should be further evaluated
using polyelectrolyte microcapsule encapsualted antigens.
As an alternative to enzyme- or hydrolysis-sensitive capsules,
one could also be interested in using the change in physiological
environment when crossing the cellular membrane to trigger
capsule disassembly. The two most outspoken changes a particle
encounters upon cellular uptake are (1) a decrease in pH from 7.4
in the extracellular space to approximately 5.2–5.4 in the endosomal compartment and (2) the transition from an oxidative to
a strong reductive environment. Theoretically, it should be
possible to synthesize pH-sensitive polyelectrolyte capsules that
decompose upon the decrease in pH which takes place in the
endosomal compartment. To obtain this goal, weak polyelectrolytes with a pKa between 5.2 and 7.4 are needed. Upon
lysosomal acidification such polyelectrolytes would lose their
negative charges resulting in capsule disassembly due to a loss of
electrostatic interactions. However, upon complexation with an
oppositely charged polyelectrolyte a substantial shift in apparent
pKa occurs, rendering the capsules more stable over a wider pH
range than predicted by the pKa values of the individual polyelectrolytes components.80 The fundamental basics of this
phenomenon were investigated by Petrov et al.81 and nicely
illustrated by Mauser et al.82 showing that polyelectrolyte
capsules based on poly(allylamine hydrochloride) (PAH, pKa ¼
8.5) and poly(methacrylic acid) (PMA, pKa ¼ 4.5) are stable in
the pH range 2 to 11. In order to decompose or at least swell and
release their content upon lysosomal acidification polyanions
and polycations should be designed so that their apparent pKa in
a complexed state is situated between 5.2 and 7.4.
The second major physicochemical change encountered when
crossing the cellular membrane is a transition from an oxidative
to a reductive environment, both in the endosome and the
cytosol. Disulfide bonds have the interesting property of being
cleavable under reductive conditions. Caruso et al. have exploited this property to stabilize hydrogen bonded capsules based on
poly(vinyl pyrolidone) and poly(methacrylic acid) (PMA) under
oxidative conditions by modifying the PMA with cysteamine
moieties.83–86 When the capsules were transferred to a reductive
environment, the hydrogen bonded multilayers were no longer
stable as the disulfide bonds were cleaved, resulting in the
Fig. 6 Confocal microscopy images of tissue sections taken at several time points after subcutaneous injection of (dextran sulfate–poly-L-arginine)4
capsules. The capsule’s wall was stained with rhodamine (red fluorescence) and the cell nuclei were stained with DAPI (blue fluorescence). The insets in
the top right corners show the cellular uptake and degradation at a higher magnification. Reprinted with permission from De Koker et al.30
This journal is ª The Royal Society of Chemistry 2009
Soft Matter, 2009, 5, 282–291 | 285
disassembly of the capsules. This approach offers an appealing
opportunity to trigger capsule disassembly by a physiologically
relevant stimulus, as the authors further showed that intracellular gluthathione concentrations indeed cause capsule disassembly. However, since these experiments were performed in
a test tube setting it still remains to be demonstrated that capsules
also disintegrate after cellular uptake.
Enhancing/blocking cellular uptake
One of the major advantages of the LbL technology is without
doubt its multifunctionality, allowing one to tailor the capsules’
surfaces with a virtually unlimited range of components. This
unique feature also introduces the possibility of modulating
capsule uptake or targeting the capsules towards certain cell
populations. In this regard, several groups tried to impede
cellular uptake of the capsules by functionalizing their surface
with an outer layer of poly(ethylene glycol) (PEG). PEG is well
known for its so-called stealth properties, blocking protein
adsorption to surfaces, a feature that has been successfully
applied to reduce recognition by macrophages. Using streptavidin as model protein Heuberger et al. showed that there was
almost no adhesion to the capsule surface through non-specific
protein adsorption in the case where the capsules were functionalized with an outermost layer of poly-L-lysine-PEG.87
However, in the case where the PEG was end-functionalized with
biotin a strong binding affinity of streptavidin to the capsules was
observed. These findings demonstrate the feasibility of functionalizing the capsules’ surfaces with ligands that could allow
a more specific cellular targeting of the microcapsules. Although
PEGylation of the polyelectrolyte microcapsules largely blocks
protein adsorption to the capsules’ surfaces,87 the effect of
PEGylation on cellular uptake was rather moderate (Fig. 7A–B),
indicating that other factors also significantly affect polyelectrolyte microcapsule uptake.88
Targeting the microcapsules towards selected tissues/cells,
would enable the selected delivery of their content to these
tissues. Clearly, achieving this can offer tremendous benefits, the
most obvious presumably in the field of cancer therapy. Selective
delivery of cytostatic agents/drugs to tumor cells not only may
drastically enhance therapy efficiency, but also significantly
decrease deleterious side effects. Several groups have tried to
accomplish this goal, using totally different approaches. The
Sukhorukov group incorporated magnetic nanoparticles in the
capsules’ shells (Fig. 7D). By applying a magnetic field gradient,
it was feasible to direct capsules to a region of interest. Due to the
local accumulation of capsules, cells in this area were found to
have taken up significantly more capsules than distant cells.89 An
alternative strategy was explored by the Caruso group,90,91 who
functionalized the capsules’ surfaces with a humanized A33
monoclonal antibody (huA33; Fig. 7C). This antibody binds the
human A33 antigen, a transmembrane glycoprotein that is
expressed by 95% of all human colorectal tumor cells as well as
on the basolateral surfaces of intestinal epithelial cells. Upon
binding of the huA33 to the A33 antigen, the cellular internalization mechanism is activated providing a mechanism for
particles to be taken up. As shown in Fig. 7C, polyelectrolyte
capsules coated with huA33 are readily internalized by colorectal
cells expressing the A33 antigen, while colorectal tumor cells that
do not express the A33 antigen fail to take up the particles.
Both of the above mentioned strategies open the way for targeting polyelectrolyte capsules towards specific tissues in the
body. However, to be applicable in clinical practice several
additional hurdles have to be overcome. First of all, to reach
a specific tissue intraveneous administration is often required.
Therefore, the size of the capsules should be limited to around
200–500 nm as they would be prone to clogging in the smallest
blood capillaries. Secondly, due to their polyionic nature, they
are very susceptible to protein adsorption, leading to clogging in
the blood capillaries as well as opsonisation and scavenging by
Fig. 7 Schematic structure of the ligands used to block/promote cellular uptake of polyelectrolyte capsules, being PLL–PEG, PGA–PEG, antibodies and
magnetic nanoparticles. Confocal microscopy images of (A) capsules being internalized by cells, (B) PLL–PEG coated capsules being prevented from
cellular internalization and huA33 mAb functionalized capsules being internalized by LIM1215 colorectal cells. In images (A–C) the capsules were stained
with a green fluorescent dye, in (C) the cellular membrane was stained with a red fluorescent mouse mAb to show the EGF receptor (mAb 528). In image
(D) the capsules were stained with red fluorescent quantum dots. Reprinted with permission from Wattendorf et al.,88 Cortez et al.91 and Zebli et al.89
286 | Soft Matter, 2009, 5, 282–291
This journal is ª The Royal Society of Chemistry 2009
macrophages. Hence, improved stealth strategies need to be
developed in order to allow the specific targeting of microcapsules either via magnetic guidance or by antibody mediated
recognition. Once this has been addressed, the challenge will be
to demonstrate that polyelectrolyte capsules have substantial
benefits compared to liposomes, and other conventional particles
from the drug delivery scene.
Triggered release from polyelectrolyte capsules
After reaching their target site, microcapsules need to release
their encapsulated contents.8 Among a variety of release mechanisms, those with remote functionalities, for example by
external forces such as light,92–98 ultrasound,99–101 hydrolysis102–106
or magnetism107 represent interesting strategies for controlled
drug release after administration, by opening the capsules after
they have reached their target tissue. Recently, several research
groups have reported on the light triggered activation of polyelectrolyte capsules inside living cells. Skirtach et al. showed that
polyelectrolyte capsules functionalized with gold nanoparticles
could be opened remotely inside living cells by irradiation with
laser light.108 When operating in a tissue environment, it is desirable
to minimize the side effects of the applied irradiation. Thereby, the
near-infrared ‘‘biologically transparent’’ window appears particularly attractive. Near-infrared absorption could be induced either
by aggregates of nanoparticles109,110 or nanorods.111 The latter are
particularly attractive, because they allow wavelength tunability of
remote release.112 Shell-in-shell microcapsules could be activated
for conducting bio-reactions in confined volumes if the inner shell
is functionalized with nanoparticles.22 Various nanoparticles,
including gold,92–97 and silver113,114 are suitable for the remote
release of encapsulated materials. Alternatively, organic moieties
could be used as sensors for inducing release. In this regard, remote
activation by an IR-dye was shown.94
Another concept of light activated polyelectrolyte capsules was
introduced by Wang et al. using capsules containing hypocrellin B
(HB), a photosensitizer.115 HB is used in so-called photodynamic
therapy for treatment of diseases such as cancer, viral infections
etc. In absence of light HB is not cytotoxic, however after exposure
to light irradiation singlet oxygen (1O2) is generated which is
cytotoxic and induces cell death. As HB is not water-soluble it
should be contained in a pharmaceutical formulation allowing it
to enter living cells. Therefore HB was loaded into the capsules by
non-specific interactions applying a solvent exchange step using
ethanol as the solvent for the HB. When the HB loaded capsules
were incubated with living cells they were taken up by these cells. It
was shown that neither empty capsules nor HB loaded capsules
were cytotoxic for the cells. However, upon irradiation with 488
nm light, a 70% drop in cell viability was observed in the HB
microcapsules treated cells. Although this concept surely holds
potential to be applied in an in vivo setting, it remains a challenge
to demonstrate the benefit of using polyelectrolyte capsules instead
of more conventional delivery forms for hydrophobic drugs such
as e.g. PLGA nano- or microparticles, liposomes or micelles.
Delivery of chemotherapeutic molecules
Several classes of therapeutic molecules have an intracellular
target. Amongst them are low molecular weight compounds such
This journal is ª The Royal Society of Chemistry 2009
Fig. 8 The fluorescence intensity averaged from inside the circles shown
in the inset figure as a function of incubation time. 1 and 2 refer to the
capsule interiors, 3 refers to the bulk. Rhodamine 6G was used as a low
molecular weight model drug and (PSS–PAH)5 capsules templated on 8.7
mm sized melamine formaldehyde particles were used. Reprinted with
permission from Liu et al.119
as chemotherapeutics and high molecular weight compounds
such as proteins and oligo/polynucleotides like e.g. those in ref.
42 and 116. Several groups have addressed the encapsulation of
the chemotherapeutics doxorubicin and daunorubicin in polyelectrolyte microcapsules. It has been reported that species with
a molecular weight lower than 5 kDa can freely diffuse in and out
of the polyelectrolyte microcapsules.29 Therefore, an electrostatic
loading mechanism is often applied. This technique implies that
the interior of the capsules is filled with a compound oppositely
charged to the compound one desires to encapsulate. Following
incubtion, the low molecular weight compound accumulates
inside the polyelectrolyte capsules through electrostatic interaction. This principle has been introduced by Sukhorukov et al. for
the controlled precipitation of dyes in hollow polyelectrolyte
capsules.25 Khopade and Caruso117 and Tao et al.118 used electrostatic interaction between the cationic doxorubicin and the
polyanionic alginate as the driving force for doxorubicin
encapsulation in biocompatible capsules. The process of charge
driven loading is shown in Fig. 8 taken from Liu et al., and
illustrates the accumulation of the positively charged model drug
rhodamine 6G inside (PSS–PAH)5 capsules through electrostatic
interaction with the anionic PSS–melamine complex.119 After
having accumulated inside the capsules, the low molecular
weight drug molecules can be partially released due to
a concentration gradient between the capsule interior and the
bulk solution. Moreover, the authors demonstrated that doxorubicin loaded capsules could kill in vitro cultured HL-60 human
leukemia cells, exhibiting slower pharmacokinetics compared to
the freely soluble drug. This is an interesting observation as it
might decrease the dose-limiting toxicity. It should however be
noted that the authors of the above mentioned papers have not
yet investigated whether the capsules released their content
following intracellular internalization or whether the drug was
released in the medium surrounding the cells.
Recently two Chinese groups performed in vivo studies with
such chemotherapeutic loaded capsules.120,121 Both groups used
CaCO3 microparticles doped with carboxymethyl cellulose
(CMC) as a sacrificial template for LbL coating with 5 bilayers of
the biopolymers chitosan/alginate. Through electrostatic interaction with the anionic CMC doxorubicin and daunorubicin
Soft Matter, 2009, 5, 282–291 | 287
weeks, the tumors were dissected and their size was compared to
either untreated or non-encapsulated daunorubicin treated
tumors. As shown in Fig. 10 the mice treated with encapsulated
daunorubicin showed the lowest increase in tumor size.
Polyelectrolyte capsules as biosensors
Fig. 9 Dual channel CLSM (confocal laser scanning microscopy)
images to show the apoptotic BEL-7402 cells induced by the encapsulated
DNR (daunorubicin). (a) Excitation at 488 nm, (b) excitation at 543 nm,
(c) transmission mode, and (d) is an overlapping image of (a) and (b). The
cells are stained by AO (acridine orange). Reprinted with permission
from Han et al.121
could be incorporated inside the capsules. Fig. 9 shows the effect
of daunorubicin loaded capsules upon incubation with cultured
BEL-7402 cancer cells. Acridine orange was used to stain the
chromatin, which is present only in the nucleus in the case of
healthy living cells. The dispersion of the red fluorescent signal
throughout the whole cell indicates that the nuclear membrane of
the cells had disappeared, meaning that cell apoptosis is induced
by the encapsulated daunorubicin. In a next step BEL-7402 cells
were implanted in nude BALBc mice and the daunorubicin
capsules were directly injected into the tumor tissue. After 4
Fig. 10 Overview of the BEL-7402 BALB/c/nu tumors. From top to
bottom: control (without treatment), treated by free DNR and treated by
encapsulated DNR. Encapsulated and free DNR with a dosage of 1 mg
kg1 (against the weight of mice) was injected into the tumors once a week
for 3 weeks (qw3). Reprinted with permission from Zhao et al.120
288 | Soft Matter, 2009, 5, 282–291
Since both the capsules’ interiors and their surfaces can be
rendered sensitive to specific physicochemical stimuli, the
capsules might be applied as biosensors for diverse applications.
Theoretically, polyelectrolyte capsules composed of one or two
weak polyelectrolytes could be directly used as pH sensors.
Such capsules lower their charge density when the pH of the
surrounding medium passes the pKa of one or both polyelectrolytes. This decreases the electrostatic interaction between
the polyelectrolyte layers, resulting in swelling and eventually
decomposition of the capsules. However, due to differences
between the pKa of the polymers in solution and their apparent
pKa values after complexation, polyelectrolyte capsules retain
their structural integrity over a broad pH range, even when
composed of weak polyelectrolytes, impeding the measurement
of swelling as a reliable pH sensor.80 An attractive alternative for
overcoming this problem has recently been proposed by Kreft
et al. by using SNARF-dextran loaded polyelectrolyte microcapsules.37 SNARF is a pH-sensitive dye that changes its excitation and emission spectra as a function of the pH of the
surrounding medium. At high pH (i.e. pH 9) red fluorescence is
emitted, whereas at low pH (i.e. pH 4) green fluorescence is
emitted. In this way the incorporation of capsules by cells could
be visualized. Whereas SNARF loaded capsules in the slightly
alkaline cell medium were red fluorescent, the capsules became
green fluorescent when incorporated by cells due to the acidic
environment in endosomal/lysosomal/phagosomal compartments (Fig. 11). Such pH-sensitive capsules can be used for highthroughput-analysis of capsules by flow cytometry.122 Though
this first demonstration was limited to the detection of protons
the same principle may be applied also for other ions. This might
be in particular interesting for multiplexed detection of several
ions in parallel. Capsules could be loaded with different ionsensitive fluorophores in their cavity. Each capsule can then be
labeled with a fluorescence barcode in its wall. In this way, the
capsules’ wall fluorescence indicates the type of ion being
measured, while its inner fluorescence is related to the ion’s
concentration. Although such an approach will be primarily
applicable for measuring ion concentrations in solution, it might
also be useful to measure intracellular ion concentration, especially of ions such as Ca2+ or K+, which exerts crucial functions in
cell signaling. As cells can take up several capsules this would
allow the concentration of several ions to be measured in parallel.
However, for this purpose investigation of whether the capsules
can escape the endosomes and reach the cytosol will also be
required as this is a particularly interesting region to monitor ion
fluctuations.
Another promising diagnostic application of polyelectrolyte
capsules is being developed by the McShane group for the
detection of glucose. Their ultimate goal is to developed
a so-called ‘‘smart tattoo’’ implanted in the skin which allows the
monitoring of the glucose level by remote interrogation using
visible or near-infrared excitation light. For this purpose,
This journal is ª The Royal Society of Chemistry 2009
Fig. 11 SNARF loaded capsules change from red to green fluorescence upon internalization by MDA-MB435S breast cancer cells. (A) SNARFfluorescence after adding the capsules to the cell culture and 30 min equilibration. Most of the capsules are outside of the cells and exhibit red fluorescence due to the alkaline pH of the medium. (B) The same cells after another 30 min of incubation. Capsules remaining in the cell medium retain their
red fluorescence (red arrows). Capsules that were already incorporated in the acidic endosome in the first image retain their green fluorescence (green
arrows). Some capsules were incorporated in endosomal/lysosomal compartments inside cells within a period of 30 min, which is indicated by their
change in fluorescence from red to green (red to green arrows). Both images comprise an overlay of microscopy images obtained with phase contrast,
a red and a green filter set. (C) Schematic presentation of the endocytotic capsule uptake. Reprinted with permission from Kreft et al.37
they have incorporated a competitive fluorescence resonance
energy transfer (FRET) assay in the microcapsules’ cavity.
This technique is based on the competitive replacement of one
partner of the FRET couple by the analyte, resulting in
a decrease in the amount of fluorescence measured that correlates
with the amount of analyte present. In a first attempt, these
authors incorporated TRITC-labeled con A, a sugar binding
lectin, and FITC-dextran (FITC, fluorescein isothiocyanate) as
a FRET couple in the microcapsules’ shell. In the presence of
glucose, FITC-dextran became displaced from Con A, resulting
in a decrease of FRET efficiency that could be correlated to
glucose concentration.33 As this system lacked robustness the
authors have optimized their concept36 using apo-glucose
oxidase instead of Concanavalin A.36 Apo-glucose oxidase is the
inactive form of the glucose oxidase enzyme, which lacks catalytic activity but retains a high binding affinity for b-D-glucose.
TRITC-labeled apo-glucose oxidase and FITC-dextran were
loaded simultaneously in polyelectrolyte capsules and formed
complexes in the capsules’ interiors. Addition of glucose, which
freely diffuses through the capsules’ membrane due to its low
molecular weight, induced decomplexation between the TRITCapo-glucose oxidase and the FITC-dextran resulting in
a decrease in FRET efficiency from which the glucose concentration can be estimated.
Conclusions
In this paper we have reviewed several contributions made in the
field of polyelectrolyte microcapsules for the purpose of
This journal is ª The Royal Society of Chemistry 2009
biomedical applications ranging from drug delivery to sensing
purposes. Whereas the advent of polyelectrolyte capsules in 1998
was followed by the thorough characterization of capsules’
physicochemical applications there are now more and more
systems coming to a point where they could start to play a role in
a biomedically relevant context. Both low molecular weight (such
as the chemotherapeutics doxorubicin and daunorubicin) as well
as high molecular weight drugs (such as e.g. protein antigens) can
be encapsulated inside the capsules and delivered to living cells in
vitro. Although it is clearly possible to use polyelectrolyte
microcapsules for intracellular delivery of different compounds,
their exact cellular localization and possible endosomal escape to
the cytosol have not yet been thoroughly addressed. Similarly, it
remains unknown if these capsules can be applied to transfect
cells. Moreover, little is known about their in vivo behaviour. As
was demonstrated by De Koker et al.,30 microcapsules composed
of the biodegradable polyelectrolytes poly-L-arginine and
dextran sulfate induce a moderate inflammatory reaction after
subcutaneous injection. Although this may well be an interesting
feature for vaccination purposes, it is an unwanted side effect for
most other applications including drug delivery. The same study
also demonstrated that these capsules are readily taken up by
mononuclear phagocytes, similar to other particles in this size
range. In many cases, it may be attractive to target microcapsules
and their contents towards other cells, e.g. tumor cells. In vitro
uptake of polyelectolyte capsules by tumor cells has been
described by several authors, either with or without targeting
antibodies. However, their size and polyionic nature raise
important issues for in vivo intraveneous administration.
Soft Matter, 2009, 5, 282–291 | 289
However the deformability of polyelectrolyte microcapsules
upon shear stress and flow through constricted pores has been
demonstrated,123,124 to the best of our knowledge, circulation of
polyelectrolyte capsules in the bloodstream after intraveneous
injection has not yet been reported in the literature. In case the
capsules would be small enough, i.e. below 200 nm, and shielded
from protein adsorption and uptake by macrophages, they could
be applied for the delivery of therapeutic agents to tumor cells
exploiting the EPR (enhanced permeability and retention) effect
(i.e. leaky vasculature in tumor tissue). Such small capsules with
diameters down to 50 nm have been reported by Schneider and
Decher125 which should, at least theoretically, make it possible to
fabricate polyelectrolyte microcapsules which could freely
circulate in the blood stream and make use of the EPR effect.
For the scientists active in the field of polyelectrolyte capsules
this offers an exciting challenge to take advantage of the unique
properties of these capsules to develop highly sophisticated drug
delivery or biosensor systems which are unmet by any other
fabrication technique. Moreover this would also elucidate for
which specific applications polyelectrolyte capsules would be
really advantageous compared to more established drug delivery
systems such as liposomes, micelles and polymeric particles.
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