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REVIEWBrunoG.DeGeest et al.Polyelectrolytemicrocapsulesforbiomedicalapplications

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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 decom-

position 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 func-

tional 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

Bruno De Geest

Bruno De Geest graduated as

a chemical engineer in 2003

from Ghent University in Bel-

gium, 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.

aDepartment of Pharmaceutics, Utrecht University, 3584 CA, Utrecht, TheNetherlands. E-mail: br.degeest@ugent.be; Fax: +32 9 264 81 89; Tel: +329 264 80 74bLaboratory of General Biochemistry and Physical Pharmacy, GhentUniversity, BelgiumcIRC, Queen Mary University of London, London, United KingdomdMax Planck Institute for Colloids and Surfaces, Golm, GermanyePhilipps University Marburg, Department of Physics, Marburg, Germany

† Author who contributed as equally as the first author.

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 repre-

sents 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

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 biode-

gradable polyelectrolyte micro-

capsules as novel antigen

delivery tools in the field of

vaccination.

Fig. 1 A schematic representation of the synthesis of hollow poly-

electrolyte microcapsules using calcium carbonate (CaCO3) as a sacrifi-

cial 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 poly-

electrolytes 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

This journal is ª The Royal Society of Chemistry 2009

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 con-

ducting 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

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.

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 demon-

strate 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 nano-

particles 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-poly-

electrolytes, such as polysaccharides, polypeptides or poly-

nucleotides, 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 Degra-

dation 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 con-

taining 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 poly-

cation and dextran sulfate/poly-L-arginine as bio-polyelectrolytes

Soft Matter, 2009, 5, 282–291 | 283

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–poly-

L-arginine)4 capsules. Reprinted with permission from De Geest et al.73

Fig. 4 A schematic representation of the encapsulation of drug mole-

cules (green dots) and pronase (red Pac-Man shapes) within calcium

carbonate microparticles (A–B) followed by LbL coating of the micro-

particles 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. 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 physio-

logical 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 poly-

electrolytes 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 enzy-

matically 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 demon-

strated 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 degra-

dation was recently reported by Borodina et al., who encapsu-

lated 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-argi-

nine. 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–poly-

L-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, char-

acterized 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 internal-

izing 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–poly-

L-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 pre-

dominantely 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 dextran-

sulfate–poly-L-arginine polyelectrolyte capsules, RITC-labeled

(RITC, rhodamine isothiocyanate) poly-L-arginine was incor-

porated 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 dis-

cussed later. Second, encapsulation of antigen into microparti-

cles 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

Fig. 6 Confocal microscopy images of tissue sections taken at several time

capsules. The capsule’s wall was stained with rhodamine (red fluorescence) an

the top right corners show the cellular uptake and degradation at a higher m

This journal is ª The Royal Society of Chemistry 2009

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 endo-

somal 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 poly-

electrolytes 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 poly-

electrolytes 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 ¼8.5) 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 exploi-

ted 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

points after subcutaneous injection of (dextran sulfate–poly-L-arginine)4

d the cell nuclei were stained with DAPI (blue fluorescence). The insets in

agnification. Reprinted with permission from De Koker et al.30

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 intracel-

lular gluthathione concentrations indeed cause capsule disas-

sembly. 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 streptavi-

din 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 func-

tionalized 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 func-

tionalizing 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 poly-

electrolyte microcapsule uptake.88

Fig. 7 Schematic structure of the ligands used to block/promote cellular uptak

magnetic nanoparticles. Confocal microscopy images of (A) capsules being in

cellular internalization and huA33 mAb functionalized capsules being internali

with a green fluorescent dye, in (C) the cellular membrane was stained with a re

(D) the capsules were stained with red fluorescent quantum dots. Reprinted w

286 | Soft Matter, 2009, 5, 282–291

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 internali-

zation 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 tar-

geting 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

e of polyelectrolyte capsules, being PLL–PEG, PGA–PEG, antibodies and

ternalized by cells, (B) PLL–PEG coated capsules being prevented from

zed by LIM1215 colorectal cells. In images (A–C) the capsules were stained

d fluorescent mouse mAb to show the EGF receptor (mAb 528). In image

ith permission from Wattendorf et al.,88 Cortez et al.91 and Zebli et al.89

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 microcap-

sules 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.

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

Triggered release from polyelectrolyte capsules

After reaching their target site, microcapsules need to release

their encapsulated contents.8 Among a variety of release mech-

anisms, 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 poly-

electrolyte 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 particu-

larly 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

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 poly-

electrolyte 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 interac-

tion. 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 elec-

trostatic 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 doxo-

rubicin 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 inter-

action with the anionic CMC doxorubicin and daunorubicin

Soft Matter, 2009, 5, 282–291 | 287

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

kg�1 (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

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

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 poly-

electrolytes. 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 micro-

capsules.37 SNARF is a pH-sensitive dye that changes its exci-

tation 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 compart-

ments (Fig. 11). Such pH-sensitive capsules can be used for high-

throughput-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 ion-

sensitive 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, espe-

cially 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) SNARF-

fluorescence after adding the capsules to the cell culture and 30 min equilibration. Most of the capsules are outside of the cells and exhibit red fluo-

rescence 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 cata-

lytic 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 TRITC-

apo-glucose oxidase and the FITC-dextran resulting in

a decrease in FRET efficiency from which the glucose concen-

tration 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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