320 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

Cite this: Chem. Soc. Rev., 2011, 40, 320–339

Designing polymeric particles for antigen delivery

Stefaan De Koker,ab

Bart N. Lambrecht,cMonique A. Willart,

cYvette van Kooyk,

d

Johan Grooten,bChris Vervaet,

aJean Paul Remon

aand Bruno G. De Geest*

a

Received 10th March 2010

DOI: 10.1039/b914943k

By targeting dendritic cells, polymeric carriers in the nano to lower micron range constitute very

interesting tools for antigen delivery. In this critical review, we review how new immunological

insights can be exploited to design new carriers allowing one to tune immune responses and to

further increase vaccine potency (137 references).

1. Vaccine design: challenges of the 21st century

In an environment dominated by micro-organisms, vertebrates

have developed multiple defence mechanisms to protect

themselves against microbial attack. When the epithelial

barrier gets breached by a micro-organism, the innate immune

defence gets activated and induces a relatively non-specific

antimicrobial and inflammatory defence. Besides this ancient

and conserved innate immune defence, vertebrates have

evolved a complex system of clonally expanding B-cell and

T-cells that form the adaptive immune system, adding antigen

specificity and immunological memory to the immune defence.

The principal function of B cells is to produce antibodies upon

antigen recognition via their B cell receptor. T cells recognize

antigen-derived peptides presented by antigen-presenting cells

via their T cell receptor. When activated by the appropriate

signals, naı̈ve T cells differentiate into effector T cells that

subsequently exert their immunological role. CD4 T cells

differentiate into T-helper (Th) cells which assist other white

blood cells in their immune functions (e.g. activation of

macrophages and cytotoxic T cells, promoting B cells to

secrete antibodies, etc.) mainly by secreting cytokines. CD8

T cells in contrast differentiate into cytotoxic T cells (CTLs),

which have the capacity to recognize and kill virally infected

cells. Importantly, B-cells and T-cells also have the capacity to

remember an encounter with an antigen, allowing them to

react faster and more vigorous to re-exposure to the same

antigen. This fundamental property of the adaptive immune

system is called immunological memory and underlies the

success of vaccination. By pre-exposing the immune system

to either complete but weakened or killed pathogens, or

(partially) purified immunogenic components of the pathogen,

the immune system mounts a fast and strong response upon

exposure to the native pathogen, ideally preventing illness.

a Laboratory of Pharmaceutical Technology,Department of Pharmaceutics, Ghent University, Ghent, Belgium

bDepartment of Molecular Biomedical Research, Ghent University,Ghent, Belgium

c Laboratory of Immunoregulation and Mucosal Immunology,Department of Pulmonary Medicine, Ghent University, Ghent,Belgium

dDepartment of Molecular Cell Biology and Immunology,Medical Centre, Vrije Universiteit Amsterdam, The Netherlands

Stefaan De Koker

Stefaan De Koker graduatedas a bio-engineer from GhentUniversity in 2001. He startedhis PhD at the VIB, at theDepartment for MolecularBiomedical Research, whichhe obtained in 2009. Currentlyhe is working as a post-doctoral associate affiliated tothe Laboratory of Pharma-ceutical Technology aswell as the Laboratory ofMolecular Immunology, bothat Ghent University. The mainfocus of his work is to evaluatenovel microparticulate systemsfor vaccine delivery.

Bruno G. De Geest

Bruno De Geest graduated asa chemical engineer in 2003from Ghent University inBelgium, where he obtainedhis PhD in 2006. Followingtwo years of post doctoralresearch at the University ofUtrecht in The Netherlands heobtained a post doctoralfellowship at the Laboratoryof Pharmaceutical Technologyat Ghent University. His maininterests are situated in thefield of materials chemistryand immunology.

Chem Soc Rev Dynamic Article Links

www.rsc.org/csr CRITICAL REVIEW

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 321

Since the pioneering work of Jenner and Pasteur over

200 years ago, vaccines have dramatically improved human

health by preventing numerous infectious diseases and are now

estimated to save 3 million lives annually.1 Nevertheless, many

challenges still remain. First, there is an urgent need to develop

effective but safe preventive vaccines against insidious

pathogens such as HIV, Plasmodium (the causative agent of

malaria), Dengue and Mycobacterium tuberculosis that affect

millions of people. Second, given the high virulence of these

pathogens, vaccines composed of live attenuated strains im-

pose serious safety issues and are unlikely to become approved

by the regulatory authorities. This is now enforcing the

vaccination field to move towards entirely synthetic vaccines

composed of recombinant antigens. Although much safer,

such recombinant vaccines are far less immunogenic and will

require the development of new adjuvants that can increase or

modulate the adaptive immune response elicited to become

effective. Third, the design of therapeutic vaccines that can

induce strong cytotoxic T cell responses might lead to a

successful immunotherapeutic treatments of cancer and

chronic viral infections such as HIV and HCV.2

Despite their tremendous impact on public health, vaccines

have been developed largely on a trial and error basis. Only

recently, it has become more and more clear how pathogens are

recognized by the innate immune system, and perhaps even more

importantly, how these initial interactions between pathogen and

innate immune system shape the subsequent adaptive immune

response.3 These insights have led to the realisation that although

designing a vaccine starts with the choice of an appropriate

antigen, the selection of the immune potentiator to activate the

innate immune system and the way both antigen and immune

potentiator are delivered to the immune system are equally

important in determining the ultimate success of the vaccine.

In this review, current knowledge on how dendritic cells (DCs)

prime and modulate effector T cell responses is summarized.

Then, we explore how this knowledge can be exploited to design

better vaccines, with the focus on new materials and delivery

strategies allowing us to present the antigen to the immune

system in an optimal immunogenic way.

2. Dendritic cells: potent inducers of effector T cell

responses

To prime a naı̈ve T cell to become an effector T cell (Th or

CTL) three signals are needed (Fig. 1). First, the antigen needs

to be processed and presented as a peptide to the cognate

T-cell receptor (TCR) by major histocompatibility complex

(MHC) molecules at the cellular surface of professional

antigen presenting cells (APCs). Two generally distinct

pathways are used for presentation of antigens via MHCI

and MHCII to respectively CD8 and CD4 T cells. The MHCI

presenting pathway is present in almost all cell types, and is

responsible for the processing and presentation of cytosolic

proteins, which are cleaved by the proteasome, transported to

the endoplasmatic reticulum (ER) and subsequently loaded

onto MHCI molecules. By this, the internal proteome of the

cell is made accessible for surveillance by cytolytic CD8 T cells,

allowing them to recognize and kill virally infected and

transformed cells. The MHCII processing pathway in

contrast is restricted to professional APCs, including B cells,

macrophages, DCs and probably also basophils. Endocytosed

proteins are degraded in endo-lysosomal compartments,

loaded onto MHCII and subsequently presented at the cell

surface.4 Although B cells and macrophages can present

antigens, they are far less efficient compared to DCs, which

have the unique capacity of priming naı̈ve T cells.5 As will be

elaborated later on, DCs also have the capacity to present

endocytosed antigens in combination with MHCI instead of

MHCII, a feature called cross-presentation, which is essential

for the induction of CTL responses against viruses and

intracellular bacteria that do not infect DCs. In addition,

cross-presentation is also crucial for inducing CTL responses

against tumour cells.

Besides antigen being presented by MHC molecules,

priming of naı̈ve T cells requires a co-stimulatory signal being

delivered by the APC, which is mediated by interactions

between the co-stimulatory ligands CD80 and CD86 on the

DC and the receptor CD28 on the T cell. Expression of these

co-stimulatory ligands is typically low on immature DCs

Fig. 1 Initiation of effector T cell responses requires three signals. Stimulation of the TCR by MHC/peptide complexes delivers signal 1,

interactions between co-stimulatory ligands on the APC and CD28 on the T cell provide signal 2 and the secretion of inflammatory cytokines that

polarise T cell responses delivers signal 3.

322 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

present in peripheral tissues, making them weak APCs.

Nevertheless, even in the absence of inflammation or infection,

peripheral tissue DCs appear to undergo a functional maturation

program, causing them to migrate to the lymph nodes and to

express co-stimulatory ligands. Antigen presentation by these

matured DCs to naı̈ve T cells however leads to tolerance rather

than immunity, by causing T cell anergy, clonal deletion or T cell

differentiation towards immunosuppressive regulatory T cells,

and constitutes an important mechanism for the maintenance of

tolerance to self-antigens.6 Induction of effector T cell responses

indeed requires the secretion of inflammatory and polarising

cytokines.7 In case of microbial encounter, DCs become rapidly

activated to upregulate co-stimulatory ligands and to secrete

inflammatory cytokines via triggering of a group of germ-line

encoded pattern recognition receptors (PRRs). These PRRs

typically recognize conserved microbial associated molecular

patterns (MAMPs) that are essential for microbial survival and

thus difficult to alter.8–11 An overview of the most important

PRRs and their microbial triggers is given in Fig. 2.

Depending on the set of PRRs triggered, DCs secrete

different cytokine profiles which will in turn largely determine

the nature of the induced immune response. As a result, DCs

link the recognition of a certain pathogen with the induction

of the appropriate adaptive response by integrating the signals

received from PRR triggering.12,13

The capacity of DCs to initiate different types of immune

responses depending on the set of PRRs triggered is of crucial

importance, as totally different types of immune defence are

needed to combat distinct pathogen spectra. These different

types of immunity are mainly regulated by different subsets of

CD4 T helper cells. Th1 cells secrete IFN-g and provide help

to macrophages and cytotoxic T cells to kill intracellular

pathogens. Th2 cells, in contrast, secrete IL-4 and IL-5 and

combat helminth infections by recruiting mast cells and

eosinophils.14 More recently, IL-17 secreting Th17 cells have

been identified as a new subset of T-helper cells that mediate

protection against extracellular bacteria and potentially fungi

by recruiting neutrophils and stimulating the release of

antimicrobial peptides.15–17 In response to a DC presenting

the antigen as a MHCII/peptide complex, a naı̈ve CD4 T cell

can differentiate in either of the aforementioned T helper

subsets depending on the cytokine microenvironment present.

An overview on how different Th responses are elicited, and

their roles in the immune defence are given in Fig. 3.

Fig. 2 Overview of the most important classes of PRRs, their cellular localisation and microbial ligands. TLRs can be localized at the plasma

membrane or the endosomal membrane, while NLRs and RLHs are located in the cytosol. Triggering of TLRs, NLRs and RLHs results in the

initiation of potent pro-inflammatory and antimicrobial responses. CLRs and scavenger receptors are mainly involved in host–host interactions,

but also recognize a wide variety of micro-organisms and have important roles in phagocytosis and antigen presentation. DCs have the capacity to

integrate the signals received from triggering of different sets of PRRs and to translate them to induce the right type of response.

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 323

3. Why vaccines need adjuvants

Adjuvants are generally defined as components that can

enhance or modulate the intrinsic immunogenicity of an

antigen in vivo. Most adjuvants have been derived empirically,

based on their capacity to increase adaptive immune responses

to co-delivered antigens but their mode of action has remained

Fig. 3 Overview on how APCs translate pathogen recognition into the induction of the appropriate Thelper (Th) response and of the role of

different Th subsets in the immune defence against different pathogen spectra. Recognition of viruses and intracellular bacteria by DCs results in

the secretion of IL-12 and type I interferons, which stimulate Th1 differentiation. By secreting IFN-g, Th1 activate macrophages, provide help to

CTLs and promote the secretion of neutralizing antibodies by B cells, which are all important to combat viruses and intracellular bacteria. Fungi

and extracellular bacteria in contrast activate DCs to secrete IL-23, thereby promoting Th17 differentiation. Th17 cells have been demonstrated to

enhance epithelial barrier function, and to recruit and activate neutrophils and monocytes. Helminth infections stimulate the generation of Th2

responses, which activate mast cells, basophils, eosinophils and provoke smooth muscle cell contraction in order to expel the helminth. Th2

differentiation is thought to require the cytokines IL-4 and TSLP, which might be basophile derived.

324 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

for a long time largely elusive.18 This empirical approach has

given us only one FDA approved adjuvant for human use,

aluminium hydroxide,19 and a second adjuvant approved by

the EU, the oil-in-water emulsion MF59. Both these adjuvants

are very useful for eliciting antibody responses, but largely fail

to activate the cellular arm of the immune response,

making them ineffective against many intracellular pathogens

including Mycobacterium tuberculosis, HIV and malaria.

Consequently, there is an urgent need to develop new

adjuvants that also allow the induction of Th1, Th17 and

CTL responses.

Only during the last decade, due to our increased knowledge

on how DCs initiate immune responses, we have begun to

unfold the cellular and molecular mechanisms underlying the

immune potentiating functions of adjuvants. Simplified,

adjuvants can work by two modes of action: or they directly

activate DCs or other innate immune cells, or they enhance

antigen uptake and presentation by DCs.20 The identification

of the crucial role of PRR triggering in DC activation and in

the subsequent induction and steering of effector T cell

responses has strongly boosted research in developing PRR

agonists that mimic the immune stimulatory properties of

natural PAMPs but display reduced toxicity during the last

decade.21–25 Most of the PAMP mimics currently tested are

agonists for diverse TLRs, and one of them, monophosphoryl

lipid A, is now included in the human papillomavirus vaccine

Cervarix (against cervical cancer)26 and in the improved

hepatitis B vaccine Fendrix,27 demonstrating the tremendous

potential of this approach. In contrast to aluminium salts,

TLR agonists have the capacity to stimulate DCs to secrete

IL-12 and type I interferons, thereby allowing the induction of

Th1 and CTL responses, which might finally bring also certain

intracellular pathogens in range.28–31

In addition to DC activation, strategies that enhance

antigen targeting towards DCs and increase or modulate

subsequent antigen presentation also bear the capacity to

increase adaptive immune responses. Antigen targeting

towards DCs can be obtained by coupling the antigens to

antibodies or ligands specific for DC surface receptors,32 or

alternatively by delivering antigens associated with particles in

the 0.1–10 mm range.33,34 Particulates in this size range indeed

mimic the dimensions of bacteria and viruses, to which DCs

have evolved to react. Examples of particulate adjuvants are

emulsions, liposomes, mineral salts, saponins, virus-like

particles and polymeric carriers, which will be the main focus

of this review.

4. Microparticulate antigen delivery

The new insights gathered in immunology have challenged

drug delivery scientists to develop a myriad of delivery systems

with the purpose of enhancing or modulating the induced

immune response. As discussed in more detail in the sections

below, there are several rationales to the formulation of

antigen into a delivery system. Single shot formulations aim

to replace the multiple booster injections often required to

generate adequate immunity by a single administration.35

Other formulation strategies intend to enhance antigen targeting

to DCs and to alter the way in which the processed antigen is

subsequently presented to T-cells.36,37 In addition, systems are

being designed to activate DCs by delivering immune

potentiators together with antigens. Such strategies allow

one to modulate the induced immune response through a

rational design of one or multiple components of the delivery

system. Predominantly, these systems involve microparticles

which offer stable antigen encapsulation and subsequently

gradually release the antigen, or allow the antigen to be

processed by intracellular proteases following internalization

by DCs.36 Although it is virtually impossible to go into detail

on all different types of carrier systems that have been

developed for vaccine delivery, we present here an overview

of the main antigen encapsulation strategies that have been

reported so far in literature.

4.1 Generating microparticles for antigen delivery

4.1.1 Solvent evaporation. Traditionally, microparticles are

generated by emulsification of two or more immiscible liquid

phases followed by a solidification step which leads to the

entrapment of the antigen into the emulsion droplets that

can subsequently be collected as solid microparticles. The

solidification steps can roughly involve solvent evaporation,

chemical cross-linking or ionic cross-linking.

Solvent evaporation is typically used in those cases where

hydrophobic polymers are used as matrix particles.38,39

In a first step, an aqueous phase containing the antigen is

emulsified in an immiscible organic solvent in which a non-

water soluble polymer is dissolved. The obtained liquid is

then emulsified a second time in an external aqueous phase

containing a stabilizer. In this way a so-called double or

water-in-oil-in-water emulsion is obtained which is subsequently

stirred at a temperature above the boiling point of the organic

solvent, allowing the solvent to evaporate and solid micro-

particles to form. The antigen that was in the innermost

aqueous phase becomes in this way entrapped within a hydro-

phobic matrix. For such encapsulation procedures, different

polymers have been used so far. One constant however

is that they should be able to release their content upon

administration to the body and, most preferably, readily

release their payload upon internalization by antigen present-

ing cells. Therefore, polymers that are prone to erosion

through hydrolytic degradation or that can respond to

physico-chemical difference between the endolysosomal or

cytoplasmatic compartments (i.e. slightly acidic and reductive

medium) compared to the extracellular medium are highly

desired.38 Further on in this review we will go more into detail

on which type of polymers are the most suited for intracellular

antigen release.

Although often applied, water-in-oil-in-water emulsification

suffers from important drawbacks. The most significant one is

the rather low encapsulation efficiency. Typically around

5% of the initial amount of antigen is retained within the

microparticles due to leakage of the internal aqueous phase

into the external aqueous phase upon emulsification.

Moreover, the use of high shear forces, often produced by

high energy ultrasound as well as the intimate contact between

antigen and organic solvents might lead to protein denaturation

which will evidently reduce the immune-activity of the antigen.

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 325

Last but not least, an important issue which hampers large

scale clinical applications is the difficulty to completely remove

solvent traces from the microparticles as these are often

retained within the hydrophobic polymer matrix.

An alternative approach to solvent evaporation which

circumvents the use of organic solvents is spray-drying from

an aqueous phase. Commonly, aqueous spray-drying involves

spraying of an aqueous solution containing drug molecules in

combination with one or more additional excipients which

ensure stabilization of the drug molecules or which enhance

the physico-chemical properties of the obtained dry powder.

As mostly all the components are easily water soluble, a clear

solution is obtained upon reconstitution in aqueous medium.

Recently we have introduced a new concept involving

spray-drying of stimuli-responsive polymers that are water

soluble or which form tiny microscopic aggregates under the

process conditions prior to atomization.40 After atomization

and evaporation of the water, solid microparticles are

obtained which retain their integrity upon reconstitution in

water. Polymers well suited for such applications are, for

example, thermosensitive polymers that are water soluble at

low temperature but which precipitate at body temperature.41

Similar systems based on enzymatic degradable polymers

could be anticipated as well.

4.1.2 Physico-chemical cross-linking. Instead of solvent

evaporation as the solidification step for antigen-loaded

emulsion droplets, physico-chemical cross-linking is a viable

option. Two major subdivisions which can be discriminated

are covalent and ionic cross-linking. Covalent cross-linking

involves the use of a polymer with specific functional groups in

combination with commonly a low molecular weight reactive

cross-linker.42 Alternatively, microparticles can also be formed

by in situ polymerisation of a hydrophilic monomer in the

presence of antigen inside pre-formed emulsion droplets.43 To

allow antigen processing upon cellular uptake by APCs, the

mesh size (i.e. the density of the polymer network) should

allow inwards diffusion of proteases and subsequently

outwards diffusion of processed peptide fragments. Another

option to ensure antigen release is the use of degradable cross-

links, involving a hydrolytically liable ester,39 acetal bonds44

or reduction sensitive disulfide bonds.45

The use of chemical synthesis is certainly a powerful tool

to design new cross-linking methods and unique types of micro-

particles. It offers a tight control over the physico-chemical

properties of the obtained microparticles, providing them with

stimuli-responsive properties, tailored surface chemistry or well

controlled antigen release rates. However, there are also serious

disadvantages as side reactions between antigen and cross-linking

moieties might occur as well. More specifically, amine and

carboxylic acid moieties of the antigen arginine, glutamic acid,

and aspartic acid residues might be affected by carbodiimide or

aldehyde based cross-linking strategies. Also Michael addition

between amines or thiols and (meth)acrylate moieties upon

radical polymerisation are to be feared. To circumvent these

issues the use of orthogonal chemistries such as ‘click’ chemistry

might be a promising option.46

A milder approach to cross-link emulsion droplets is ionic

cross-linking which commonly involves emulsification of the

antigen together with a water-soluble polymer followed by the

addition of a low or high molecular weight cross-linker. The

most widespread example of ionic cross-linking is the calcium

alginate system.47,48 Alginate is a polysaccharide consisting of

alternating manuronic and glucuronic units, making the

polymer abundantly substituted with carboxylic acid

moieties. Addition of divalent calcium (Ca2+) ions induces

instantaneous gelation through a so-called ‘zipper’-mechanisms

involving the formation of ionic cross-links between the Ca2+

ions and carboxylic acid pairs, yielding a stable hydrogel

network that can entrap proteins. These reaction conditions

are very mild and therefore well suited for encapsulation of

labile protein antigens. Decomposition of calcium alginate

hydrogel microspheres takes place when they are transferred

from a Ca2+-rich medium to a medium with physiological salt

concentrations (e.g. 150 mM NaCl) by exchange of divalent

Ca2+ ions by monovalent Na+ ions, which are not capable of

retaining the network structure of the alginate gels. The

simplicity of the calcium alginate system however has as

major drawback that protein release rates are tough to be

controlled. Release immediately starts upon contact with the

physiological medium, thus prior to uptake of the micro-

particles by APCs, which means a considerable loss of antigen.

Moreover, this remains an emulsion based encapsulation

method involving the use of organic solvents as external phase

as well as the use of high shear forces.

4.1.3 Self assembly. Perhaps one of the mildest antigen

encapsulation strategies is by exploiting self-assembly, either

between specific molecules and the antigen itself of by

entrapping the antigen within a self-assembled structure or

other molecules. Driving forces for self-assembly can comprise

hydrophobic interactions, electrostatics, H-bonding, bio-

specific ligation etc. The most widespread example of

self-assembled drug delivery systems are liposomes which are

100 nm–10 mm sized lipid bilayer vesicles that spontaneously

form in aqueous medium upon hydration of a film of amphiphilic

lipids through inwards ordering of their hydrophobic domains

with the polar head groups pointing to both inner and outer

aqueous phase.49,50 A synthetic analogue to these liposomes

are polymersomes which consist of amphiphilic block

copolymers.51 When both of these vesicular structures are

formed in the presence of antigen, a part of the antigen will

become encapsulated within the hollow void of the vesicles. It

is however clear that this procedure can only offer very low

encapsulation efficiencies. Moreover, vesicles tend to be

thermodynamically unstable upon prolonged storage in water

and therefore is doubtful that vesicles are able to retain their

payload encapsulated for several days upon subcutaneous or

intramuscular injection prior to being internalized by APCs.

On the other hand, chemists have put major efforts in the

synthesis of both liposomes and polymersomes with enhanced

stability and stimuli responsive properties that make them

excellent carriers for intracellular drug delivery, at least

in vitro.52 Primarily intended for application in gene delivery,

it could be anticipated that such ‘smart’ vesicles also hold

promise for antigen delivery. Especially systems that overcome

the endolysosomal barrier and release their payload in the

326 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

cellular cytoplasm would be interesting to induce TH1 and

cellular immune responses.

Besides vesicles which are self-assembled through hydro-

phobic interaction, electrostatic interaction is another

strategy which has been intensively elaborated for antigen

encapsulation. The most simple case is the inclusion of antigen

in a polyelectrolyte complex through mixing the antigen with a

polyelectrolyte bearing an opposite net charge. A well studied

polymer for such application is chitosan which is a naturally

occurring cationic polysaccharide that has been reported to

open the tight junctions in the nasal-associated lymphoid tissue

(NALT).53 Ovalbumin (OVA) is ubiquitous in numerous

immunological studies and is due to its isoelectric point of

4 bearing a net anionic charge at physiological pH, which

allows it to form electrostatic complexes, of several hundreds

of nanometre in size, with the oppositely charged chitosan.

This electrostatic self-assembly principle is not only restricted

to anionic antigens as by carefully choosing the ratio

between chitosan and polyphosphate molecules one is able

to precipitate antigens regardless of their charge. However

indisputedly attractive for its conceptual simplicity,

such electrostatic precipitation procedure requires careful

optimization for every case and definitely lacks a tight control

over size and surface properties of the obtained microparticles.

A more recent, highly versatile strategy to design

microparticles with an unmet degree of control over particle

composition and physic-chemical properties is the Layer-

by-Layer (LbL) technique.54 Sacrificial microparticles are

coated with several polyelectrolyte bilayers of opposite charge,

exploiting electrostatics as driving force for the deposition of

each polyelectrolyte layer. Subsequently the microparticulate

core templates are dissolved resulting in hollow capsules.55–57

As will be discussed in more detail further on in this review,

the LbL technique is not only restricted to electrostatic

interactions but also H-bonding has emerged as a highly

promising strategy to design LbL capsules for drug delivery

purposes.58,59 Both types of LbL capsules allow efficient

antigen encapsulation using porous inorganic microparticles,

such as calcium carbonate or silica, that are pre-filled with

antigen prior to LbL coating. Subsequently the inorganic

cores are extracted in an aqueous medium containing EDTA60

or HF,61 liberating the antigen into the hollow void of the

capsules. These capsules are excellent in protecting their

payload from degradation before reaching APCs as their

target, while using polymers in the LbL coating that are prone

to enzymatic or reductive degradation, release only after

cellular uptake is assured.

4.2 Single shot vaccines

Current vaccines often require multiple injections to induce

protective immunity. The need for multiple booster injections

unfortunately often leads to patient non-compliance and

logistic issues, especially in developing countries. As a result,

there has been a lot of interest in developing antigen delivery

systems that allow a controlled release of antigens in a

pulsatile fashion to replace the classic prime-boost

schedules. The polymer tested by far the most for such

application is poly(lactic-co-glycolic) acid (PLGA). PLGA is

a biodegradable and biocompatible polymer which has been

used now for many years as resorbable suture material in

humans. PLGA particles degrade by the non-enzymatic

hydrolysis of the ester bonds in the backbone of the polymer,

resulting in the release of lactic and glycolic acid, two acids

that can be metabolized via the citric acid cycle. Early studies

with PLGA as an encapsulation material focused mainly on

the controlled long-term release of hormones and growth

factors. These studies clearly demonstrated that the release

of peptides/proteins from PLGA microspheres depends on

several factors, such as the ratio lactic/glycolic acid, the

molecular mass and hydrophobicity of the polymer, the type

of emulsifier used and the size of the microspheres prepared.

As a result, by carefully choosing the polymer’s characteristics,

one should be able to tailor the protein release as desired. In

this view, a single injection with a mixture of hepatitis B

surface antigen (HBsAg) containing PLGA microspheres

with different degradation rates resulted in antibody titers

comparable to three classical HBsAg/Alum injections.62

Similar results have been obtained with tetanus toxoid

encapsulated in different PLGA microsphere preparations.63

Nevertheless, single shot PLGA vaccines suffer from significant

drawbacks impeding their clinical application. First of all,

release rates not only depend on particle intrinsic factors, but

also on protein-specific factors such as molecular weight,

hydrophobicity and charge.64 In addition, these large PLGA

microspheres are generally prepared using a double emulsion

process often resulting in low antigen encapsulation. Preparation

of the microspheres also involves the use of chemical solvents

such as dichloromethane or chloroform, which are not only

highly toxic but also negatively affect protein stability as has

been demonstrated for bacterial toxoids.65 Moreover, PLGA

degradation results in an acidification of the injection spot,

further contributing to protein denaturation. Even if protein

stability can be improved by incorporating poorly soluble

bases as Mg(OH)266,67 or protein stabilizers as trehalose,68

preparing antigen-loaded PLGA microspheres that release the

antigens in the desired way remains a time-consuming and

costly process that needs to be optimized for each antigen.69

Finally, also scaling-up has been proven difficult, further

preventing industrial application.

Besides PLGA, spherical hydrogels are also being explored

for the controlled release of proteins. Such hydrogels might

have significant value for the development of single shot

vaccines, as their production does not involve the use of

organic solvents and their degradation can be tightly

controlled by varying the number of degradable cross-links.

Whether such hydrogels can replace classical prime-boost

schedules using Alum adsorbed antigens remains to be

established. Recently, a novel approach to create single-shot

vaccines, called self-exploding capsules,70–72 was explored by

De Geest et al. These core–shell particles consisted of a

degradable hydrogel core surrounded by a semi-permeable

polyelectrolyte membrane. Upon degradation of the hydrogel

core, an osmotic pressure builds up which finally ruptures the

capsule membrane, resulting in a single release pulse. Fig. 4A

shows a series of confocal snapshots taken at different time

points during degradation. The capsules’ hydrogel core consists

of polymerized methacrylated dextran whose methacrylate

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 327

groups are connected to the dextran backbone through a

hydrolysable carbonate ester. By varying the degree of cross-

linking of the hydrogel core, one is able to alter the onset of

capsule explosion and thus the time point of the release pulse.

This is illustrated in Fig. 4B showing pulsed release of

50 nm latex beads, used as model antigen, from two types of

exploding capsules with a different cross-link density and thus

degradation kinetics. Theoretically, by injecting a mixture

of coated gels with different degradation rates, classical

prime-boost schedules could be mimicked with a single

injection. Nevertheless, following subcutaneous injection of

self-exploding capsules, the polyelectrolyte shell of the parti-

cles was prone to infiltration and degradation by recruited

inflammatory cells, which probably prohibits a controlled

release of their payload in a pulsatile manner.73 Consequently,

although promising, much progress concerning shell stability

and resistance to cellular infiltration needs to be made before

such approach can fulfil its potential in vivo.

Finally, it still remains to be established whether antigens

really need to be released in a pulsatile manner for optimal

induction of antibody responses. Several studies indicate that

both continuous and discontinuous antigen release can induce

similar antibody responses in small-animal models.74 In

addition, due to the slow-decay kinetics of antibody responses

in rodents, it has been suggested that such models might not

be ideal to address the real value of single shot vaccine

formulations.34

4.3 Micro-and nanoparticles: targeting antigens towards

different APCs

A plethora of studies now have demonstrated that particles in

the 50 nm to 5 mm range are efficiently taken up by DCs

in vitro and in vivo. Nevertheless, DCs form a heterogeneous

population, with multiple subtypes showing different functional

properties.75 Resident lymphoid DCs directly differentiate in

the lymphoid tissue after their emigration from the blood,

without first entering peripheral tissues. Resident lymphoid

DCs can be divided into CD8a� and CD8a+ subsets, which

differ in their expression pattern of endocytosis receptors and

in their capacity to present antigens, the CD8a+ subset being

far more efficient in cross-presentation and the induction of

CTL responses.76 CD8a� DCs in contrast have been reported

to be more potent in MHCII mediated antigen presentation,

and thus the priming of CD4 T cells.77 Besides resident

lymphoid DCs, lymph nodes also contain migratory DCs.

Migratory DCs differentiate in peripheral tissues where they

reside in an immature status and continuously sample

antigens. Such antigen sampling is illustrated in Fig. 5 showing

a transmission electron microscopy (TEM) image of a DC

stretching its dendrites to catch a microparticle. Even in the

absence of inflammation, migratory DCs appear to undergo a

functional maturation program, making them migrate to the

draining lymph nodes. Antigen presentation by these mature

(but not activated) DCs is of crucial importance to maintain

peripheral tolerance to self-antigens. In case of infection,

Fig. 5 Transmission electron microscopy (TEM) image of a dendritic

cell stretching out its dendritic to capture a hollow microparticle.

Fig. 4 (A) Confocal microscopy images taken at different time intervals of self-exploding capsules. The red fluorescent membrane consists of 4

bilayers dextran sulfate /poly-L-arginine. The interior is a degradable hydrogel bead loaded with green fluorescent 50 nm latex beads. (B)

Cumulative release curves of 50 nm latex beads from self-exploding capsules, using dex-HEMA hydrogel beads with different degradation kinetics.

(Reprinted with permission from ref. 73. Copyright 2009, Elsevier.)

328 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

peripheral DCs get however activated by PRR triggering and

become potent APCs capable of priming effector T cell

responses. In addition, in case of infection inflammatory

monocytes are recruited to the site of inflammation, which

subsequently differentiate into DCs that are capable of

priming CD4 and CD8 T cell responses.78,79 Differential

targeting of these different DC subpopulations with their

distinct properties most likely will also strongly impact the

type of immune response elicited.

Ultrasmall nanoparticles (20-50 nm) and particles in the

lower mm range (0.5–5 mm) significantly differ in their in vivo

fate following subcutaneous injection. Nanoparticles rapidly

reach the lymph nodes through passive drainage via the

lymphatics, making them interesting tools to directly target

the lymphoid DC population. Targeting resident lymph node

DCs to induce both cellular and humoral immune responses

has been successfully applied by Reddy et al., who used 25 nm

sized antigen-coupled polypropylene sulfide nanoparticles.80

In contrast to these ultrasmall nanoparticles, particles in the

lower micron range are more tightly retained in the interstitial

space and require active transport by migratory DCs to reach

the lymph nodes. Recently, using polystyrene beads of

different sizes, Manolova et al. have analyzed the effects of

particle size on DC targeting following subcutaneous

injection. Nanoparticles were shown to enter the lymph nodes

via the subcapsular sinus, and to be subsequently taken up not

only by resident lymphoid DCs but also by subcapsular

macrophages and B cells. Approximately half of the DCs

containing 20 nm particles were resident CD8a+ DCs,

specialized in cross-presentation. Microparticles on the other

hand were exclusively retrieved in CD8a� CD40low DCs, a

phenotype consistent with DCs derived from phagocytic

monocytes.81 These observations clearly demonstrate that

micro- and nanoparticles target different APC populations

in vivo. The functional repercussions of this differential

targeting between micro- and nanoparticles remain currently

unclear. Although microparticles do not target resident

lymphoid CD8a+ DCs thought to be specialized in cross-

presentation, many studies have now demonstrated their

strong potential in inducing CD8 T cell responses in addition

to CD4 T cell responses.82 This is likely due to their capacity

to recruit inflammatory monocytes following injection.

Following antigen uptake, these inflammatory monocytes

differentiate into DCs that not only have the capacity to

induce Th1 polarized CD4 T cell responses but are also highly

efficient in cross-priming CD8 T cells. Consequently,

micro- and nanoparticles can both elicit CD4 and CD8 T cell

responses. Fig. 6 illustrates this for the particular case of

antigen loaded polyelectrolyte capsules, demonstrating that

antigen (ovalbumin; OVA) loaded capsules are far more

potent inducers of T cell proliferation then merely soluble

antigen. The particle size that produces the optimal immune

response remains to be established, and might be even different

depending on the pathogen one wants to target.

Following subcutaneous injection, most of the micro-

particles injected are not transported by DCs to the lymph

node but remain at the site of injection where they are taken

up by other phagocytic cells (e.g. macrophages). As a

result, microparticles generally target fewer DCs compared

to nanoparticles that are passively drained to the lymph node.

Strategies that increase microparticle targeting towards DCs

following injection thereby might also enforce the strength of

the induced immune response. Several approaches have been

explored to enhance particle targeting to DCs or specific DC

subsets. Particulate carriers can be modified with antibodies or

ligands specific for DC surface markers and endocytosis

receptors. In this view, functionalisation of liposomes with

anti-CD11c antibody derivatives has been demonstrated to

promote DC targeting and to provoke more potent immunity

in a B16 melanoma model.83 Similarly, Kwon et al. have

functionalized 1 mm sized pH sensitive particles with

anti-DEC205 antibodies, resulting in an increased percentage

of DEC205+ lymph node DCs containing the particles

compared to non-functionalized particles. This increased DC

targeting also resulted in an augmented CTL response,

confirming the potential of this approach.84 Microparticulate

uptake by peripheral DCs and inflammatory monocytes might

also benefit from the incorporation of components that

promote the selective recruitment of DCs and monocytes to

the injection site. Carriers that have been used to gradually

release DC attracting chemokines include polyester particles,85

and alginate based hydrogels.86,87 Alternatively, injection of

the biomaterial itself might provoke the in situ production of

chemokines and cytokines as has been demonstrated for

aluminium hydroxide and the oil-in-water adjuvant MF59.88

4.4 Particulate carriers for increased cross-presentation

The most appealing part of using particulate carriers for

antigen delivery is their capacity to promote cross-presentation.

While endocytosed antigens generally enter the MHCII

antigen processing pathway, DCs have the capacity to

cross-present exogenous antigens via the MHCI route. For

soluble antigens, this process appears to be dependent on their

routing to stable early endosomes specialized for cross-

presentation.89,90 Targeting of antigens to these specialized

compartments is mediated by binding to specific endocytotic

receptors, such as DEC205,32 the mannose receptor89 and

langerin.91 Intriguingly, these receptors are mainly expressed

on CD8a+ DCs, which have been reported to be specialized

in cross-presentation.92 Nevertheless, although receptor-

mediated endocytosis of soluble antigens can result in cross-

presentation, the efficiency of this process is rather inefficient

and requires high amounts of antigen. Antigens delivered to

DCs in a particulate form in contrast, such as viruses

and bacteria, are internalized via macropinocytosis or

phagocytosis, and are far more efficiently cross-presented to

CD8 T cells. The particulate nature of viruses and bacteria can

be mimicked by encapsulating antigens in polymeric carriers

with similar dimensions. Multiple studies have now

demonstrated a strong increase in cross-presentation and the

induction of CTL responses after antigen encapsulation in

PLGA microparticles.93–95 However, the capacity to promote

cross-presentation is certainly not restricted to PLGA, as

many other particulate carriers including polystyrene beads,96

hydrogel particles97 and also polyelectrolyte microcapsules

(Fig. 6)98 have been shown to promote cross-presentation,

allowing cross-presentation at 100 to 1000-fold lower antigen

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 329

doses. How particles mediate cross-presentation is still a

matter of debate, and depending on the material tested multi-

ple mechanisms have been proposed.

As cross-presentation can be blocked in most cases by

inhibitors of the proteasome, a mechanism has been

proposed where antigen is exported from the phagosome or

macropinosome, and subsequently enters the classical MHCI

processing route similar to cytosolic proteins (Fig. 7A).

Phagosomal escape has indeed been reported following uptake

of PLGA nanoparticles, resulting in an increased presence of

antigen in the cystosol and efficient cross-presentation.93,99 In

contrast, Walter et al. failed to detect phagosomal escape using

PLGA microparticles.100 These discrepancies might be due to

differences in polymer hydrophobicity and charge, or in

particle size, with the smaller nanoparticles being able to

escape through membrane disruptions more easily than the

bigger microparticles. The phagosomal escape hypothesis has

driven researchers to develop microparticles capable of

rupturing phagosomal membranes, in order to release the

antigen directly into the cytosol. The main approach explored

to achieve this goal has been the use of pH responsive particles

that degrade or disassemble upon phagosomal acidification.

Hydrazide or acetal containing microparticles are known to

readily undergo acid hydrolysis and are well suited for this

purpose as their single components exert an osmotic pressure

on the phagosomal membrane leading to its rupture.97,101,102

On the other hand, amine-containing polymers often exhibit a

so-called proton sponge effect which means that they buffer

the phagosomal compartment, preventing the phagosomal

acidification process. The subsequent influx of protons trying

to force the acidification, induces an osmotic pressure which is

also able to rupture the phagosomal membrane. Fig. 8 gives an

excellent example of cytosolic antigen delivery mediated by pH

responsive particles.103

Phagosomal disruption may however not be necessary to

promote antigen cross-presentation. Recently, it has been

proposed that phagosomes and macropinosomes are fully

competent organelles for cross-presentation themselves and

recruit all the necessary machinery for MHCI-mediated

antigen presentation, possibly by fusing with ER

membranes,104–106 although this is still a matter of ongoing

debate. A mechanism has been suggested in which antigens are

exported from the phagosomal lumen, processed by recruited

immunoproteasomes, and re-imported into the same

phagosome for loading onto MHCI molecules (Fig. 7B).107

Recent evidence indicates that antigen processing and loading

onto MHCI and MHCII actually occur in the same

phagosome, but at distinct time intervals following particle

Fig. 6 (A) Polyelectrolyte microcapsule synthesis. Antigen (yellow) is mixed with CaCl2 and Na2CO3, resulting in the generation of

macromolecule-filled CaCO3 microparticles (gray), which are subsequently coated with alternating layers of dextran sulfate and poly-L-arginine

(red, blue). Dissolution of the CaCO3 core by EDTA results in the generation of a hollow microcapsule composed of macromolecules surrounded

by the polyelectrolyte shell. (B) Scanning electron and (C) confocal microscopy image of polyelectrolyte capsules. (D) Antigen presentation by

BM-DCs after uptake of soluble and encapsulated ovalbumin. Proliferation of OT-I cells was used as a measure for MHC-I-mediated

cross-presentation of ovalbumin (left graph), proliferation of OT-II cells as a measure for MHC-II mediated presentation (right). The open

symbols represent soluble ovalbumin while the solid symbols represent encapsulated ovalbumin.

330 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

internalization. Active alkalization of the phagosome by

recruitment of the NADPH oxidase NOX2 appears to be

crucial for MHCI loading by preventing activation of

lysosomal proteases and consequently rescuing antigens from

fast degradation. This alkalization is however transient, and

several hours after microparticle uptake NOX2 activity

decreases causing the phagosomes to gradually acidify thus

allowing the activation of lysosomal proteases that process the

antigens into peptides for MHCII loading.108,109 These in-

sights clearly have significant implications for the design of

new particulate antigen carriers. If cross-presentation indeed

occurs solely in the first hours after particle uptake, fast

degrading particles should allow a more efficient cross-

presentation compared to slow degrading ones such as PLGA.

A recent study by Broaders et al. indicates that this

indeed might be the case, as shown in Fig. 9. These authors

encapsulated ovalbumin in acetylated dextran beads, which

rapidly decompose upon phagosomal acidification. Degradation

of the beads is depended on the degree of acetylation, with

heavy acetylated particles degrading significantly slower. Fast

degrading particles performed approximately ten times

better in stimulating cross-presentation than slower ones, also

including PLGA microparticles.110

Nevertheless, in these cases particle degradation still

depends on a drop in pH, which might counteract cross-

presentation and rather stimulate MHCII mediated presentation

as it also results in the activation of lysosomal proteases.

pH-independent strategies, in order to trigger rapid antigen

release following particle uptake, might further improve

MHCI loading. An elegant strategy to allow microcapsule

decomposition in a pH-independent fashion might be the use

of disulfide bonding stabilized particles. Zelikin et al.

produced such biodeconstructible capsules by modifying

poly(methacrylic acid) (PMA) with thiol groups (Fig. 10).111

Fig. 7 Proposed mechanisms for antigen cross-presentation mediated by particulate carriers (A) Phagosome-to-cytosol route for cross-

presentation. Cross-presentation is dependent on the ability of the particulate carrier to disrupt phagosomal membranes and to release the

antigen directly into the cytosol, where it is processed by the proteasome, imported via TAP transporters into the ER and subsequently loaded onto

MHCI molecules. (B) Phagosomes as fully competent organelles for cross-presentation. Upon internalization, the antigen gets released from the

particulate carrier into the phagosome, which contains all machinery for MHCI presentation, possibly by fusing with ER membranes. The antigen

is exported from the phagosome, cleaved by immunoproteasomes associated to the phagosome, re-imported into the same phagasome via TAP

transporters and loaded onto MHCI molecules in the phagosomal membrane.

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 331

In an oxidative environment, these capsules are stabilized by

disulfide linkages between the polymer layers. However,

in a more reductive environment, such as present in early

endosomes and probably also in early phagosomes, the

capsules are expected to decompose and to release their cargo.

Use of thiolated PMA allowed the encapsulation of the

cysteine-modified KP9 peptide into PMA particles by disulfide

linkages. When placed in a reductive environment (5 mM

GSH), over 80% of the peptide was released from the capsules

within one hour.112 Proof of principle that such a strategy can

indeed promote CD8 T cell responses was demonstrated in a

non-human primate model of SIV infection.112,113 In a recent

paper, Sexton et al. have extended this approach to protein

antigens. Encapsulation of ovalbumin in disulfide stabilized

PMA particles significantly enhanced antigen presentation to

both CD4 and CD8 T cells in vitro when compared to soluble

ovalbumin. Nevertheless, in vitro CD8 T cell proliferation was

increased with merely a factor 3.8 to 7.9, which is at best

moderate compared to other particles such as PLGA or acid

degradable particles. CD4 T cell proliferation in contrast was

enhanced 5.7–42 fold, indicating these particles mainly

promoted the MHCII route of antigen presentation. Similar

observations were made in vivo, with OVA loaded PMA

particles increasing predominantly CD4 T cell responses

(70-fold increase) and to a lesser extent CD8 T cell responses

(6-fold increase).114 A possible reason why this approach only

moderately stimulates CD8 T cell responses might be the

subcellular localization of the microcapsules. Following

Fig. 8 pH-sensitive core–shell nanoparticles deliver OVA to the cytosol of primary dendritic cells and promote CD8+ T cell priming. (A–D)

CLSM images: (A, C) bright-field images; (B, D) fluorescence overlays of OVA (green) and nanoparticles (red). (A, B) BMDCs incubated with

OVA adsorbed to PDEAEMA core–shell nanoparticles. (C, D) Cells incubated with OVA adsorbed to PMMA core–shell nanoparticles. Scale bars

10 mm. (E) BMDCs were incubated with medium alone (no OVA), soluble OVA, OVA-coated PDEAEMA-core nanoparticles, or OVA-coated

PMMA-core nanoparticles, then washed and mixed with naı̈ve OT-1 OVA-specific CD8+ T cells. IFN-g secreted by the T cells in response to

antigen presentation by the DCs was measured by ELISA after 72 h. Error bars represent standard deviation of triplicate samples. (Reprinted with

permission from ref. 103. Copyright 2009, American Chemical Society.)

332 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

uptake, microparticles tend to end up in phagosomal

compartments, which have a far less reducing environment

compared to the cytosol or the nucleus, resulting in inefficient

disulfide reduction115 and slow microcapsule decomposition.

Similar to PMA capsules, a layer-by-layer technique can

also be applied to produce polyelectrolyte microcapsules. In

contrast to PMA particles such polyelectrolyte microcapsules

are not stabilized by covalent disulfide bridges, but by

electrostatic interaction.54,56,57,116 Recently, we have

demonstrated that polyelectrolyte microcapsules composed

of dextran-sulfate/poly-L-arginine strongly promoted antigen

presentation of encapsulated ovalbumin to both CD4 and

CD8 T cells.98 In contrast to the PMA particles, these

microcapsules however stimulated predominantly CD8 T cell

proliferation, an observation we have now also confirmed

in vivo (unpublished data). These discrepancies might again

be due to the kinetics of antigen release and/or availability for

processing. Using DQ-OVA, a BODIPY labelled ovalbumin,

which is self-quenched in its native state but becomes brightly

fluorescent after enzymatic degradation, De Koker et al.

showed that following uptake by DCs, dextran-sulfate/

poly-L-arginine encapsulated ovalbumin becomes readily

available for enzymatic processing, even before visual rupturing

of the microcapsules’ shell 24 h after ingestion, as shown in

Fig. 11.98 In this view, hollow microcapsules with the antigen

only being surrounded by a thin shell might offer significant

benefits compared to entire polymeric particles such as PLGA.

In the case of hollow microcapsules, mere shell erosion

or local rupturing is enough to make the entire microcapsule

content available for processing, while in the case of

‘filled’ polymeric particles, polymer erosion starts at the

border making only these proteins that are located near the

particle border rapidly available for enzymatic processing.

Using DQ-OVA, Heit et al. showed that degradation of

ovalbumin encapsulated in PLGA microspheres only starts

six hours after cellular uptake. Moreover, degradation

was initiated at the border of the particle and then

gradually proceeded towards the inner core of the PLGA

microsphere.117

Another matter of intensive debate is whether there exists an

optimal particle size to promote cross-presentation. In a recent

study, Tran and Shen nicely demonstrated that, at least

in vitro, DCs cross-present antigen bounded to 0.5–3 mmpolystyrene beads far more efficient than antigen bounded to

50 nm beads.96 Importantly, as 50 nm nanoparticles were

rapidly shuttled to acidic compartments while larger micro-

particles remained in a more neutral environment, these data

are in accordance with the earlier mentioned observation that

fast phagosomal acidification actually inhibits cross-presentation.

Nevertheless, several other studies have claimed that nano-

particles are more potent in inducing CD8 T cell responses

in vivo compared to microparticles.118 Differences in particle

Fig. 9 (A) Synthesis of acetal modified dextran (Ac-DEX) and particle formation: (i) 2-methoxypropene, pyridinium-p-toluenesulfonate, DMSO

(ii) solvent evaporation based particle formation (scale bar is 2 mm). (B) Relative MHCI presentation from BMDCs for OVA-containing

particles made from Ac-DEX5, Ac-DEX10, Ac-DEX30, and Ac-DEX60 (corresponding to degradation half-lives of 0.27, 1.7, 11, and 16 h)

(n = 3, mean � SD). (C) Relative MHCI presentation from BMDCs for OVA-containing particles made from Ac-DEX10, Ac-DEX60, PA

particles, PLGA, and iron oxide. Quickly degrading materials (Ac-DEX10 and PA particles) show presentation at significantly lower protein

concentrations (n= 3, mean� SD). (D) Relative MHCII presentation from BMDCs for OVA-containing particles used in B (n= 3, mean� SD).

(Reprinted with permission from ref. 110. Copyright 2009, National Academy of Sciences.)

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 333

uptake and especially DC targeting may underlie this apparent

paradox: while microparticles might be intrinsically superior

in promoting antigen processing towards the MHCI route,

they are less efficient in targeting DCs in vivo compared to

nanoparticles, which can passively drain to the lymph

nodes via the lymphatics. If so, strategies that can increase

microparticle targeting to DCs or DC subpopulations might

also result in superior cross-presentation, as has been

demonstrated by Kwon et al. who used acid-labile 1 mmparticles couple to the DC-specific antibody DEC205.84

Further studies are necessary to address these issues, and to

resolve which particle size is indeed the most optimal.

4.5 Intrinsic immune activating properties of particulate

carriers

To induce potent adaptive immune responses, mere antigen

delivery to DCs is insufficient and can even lead to tolerance.

Indeed, in order to induce effector T cell responses, DCs also

need to become activated to upregulate co-stimulatory ligands

and to secrete inflammatory cytokines. The most direct assay to

evaluate the immunopotentiator properties of new biomaterials

is assessing their capacity to activate DCs in vitro. Several

studies have demonstrated a moderate upregulation of the

co-stimulatory ligands CD83 and CD86 and an increased

release of IL-12 and TNF-a by DCs following uptake of PLGA

particles.119 Other groups however failed to detect any DC

maturation following incubation with PLGA.120,121 These

discrepancies might be attributed to differences in polymer

and stabilizer used, but also to differences in bacterial

contaminants such as LPS adsorbed to the microspheres’

surface. Similarly, incubation with poly-L-lactic acid (PLA),122

poly-(g-glutamic acid)123 or poly-b-amino-ester containing

particles124 has been reported to promote DC maturation.

Recently, our group also observed a slight increase in the

expression of CD40, CD86 and MHCII on bone marrow

derived DCs after incubation with polyelectrolyte micro-

capsules composed of dextran-sulfate and poly-L-arginine

(unpublished results). How particulate antigen carriers promote

DC maturation has however largely remained a mystery, as

they do not contain any known ligands for PRRs. Only very

recently, a number of publications have shed a new light on how

particulates might exert their adjuvant properties. First, it was

recognized that at least part of the adjuvant properties

of aluminium hydroxide components can be attributed to

formation of the NALP3 inflammasome and the subsequent

release of the potent pro-inflammatory cytokine IL-1b.19,125–128

NALP3 is a member of the nucleotide binding domain (NOD)

family, a group of cytosolic PRRs that can be triggered

by various endogenous and microbial danger signals.

Inflammasome formation results in caspase-1 activation,

pro-IL-1b cleavage and IL-1b release.129 Subsequently, these

observations have been extended to particulate adjuvants

including chitosan, QuilA,127 polystyrene and PLGA

microparticles,130 which all appear to activate the NALP3

inflammasome. Sharp et al. demonstrated that following uptake

of polystyrene and PLGA particles, NALP3 activation depended

on phagosomal acidification and the lysosomal cysteine protease

cathepsin B, indicating a possible role for phagosomal disruption

in NALP activation.130 Although IL-1b release by DCs in

response to particulates requires a pre-stimulation step of the

DCs with LPS to produce pro-IL-1b in vitro, in vivo this appears

not to be necessary as injection of PLGA particles in the absence

of TLR agonists did induce local IL-1b at the injection site.

Likely, injection of the particles resulted in the release of

endogenous danger signals by inflicting tissue damage, which

could replace for microbial PAMPs to trigger pro-IL-1bformation. Remarkably, although NALP3 activation was crucial

for initiating cell-mediated immune responses, it was completely

dispensable for initiating humoral immune responses, indicating

that something else that remains to be unravelled must be

involved in the adjuvant properties of particulates.

Fig. 10 (A) Encapsulation of Cys-KP9 into degradable polymeric

capsules: (i) conjugation of oligopeptides to an anchoring PMASH

polymer; (ii) adsorption of conjugates onto an amine-functionalized

silica particle; (iii) assembly of a thin polymer film prepared via the

alternating deposition of PVPON and PMASH and oxidation of

PMASH thiol groups into bridging disulfide linkages; (iv) removal

of the core particle to result in a stable polymer capsule; (v) degradation

of the capsule, releasing Cys-KP9. (B) Confinement of KP9 within

polymer capsules through conjugating the oligopeptide to a carrier

polymer. To achieve this, a sample of PMA was modified with thiol

groups (i), which were subsequently activated for thiol-disulfide

exchange using Ellman’s reagent (ii). The resulting polymer was

reacted with an N-terminal cysteine-modified KP9 to yield a

PMA–KP9 conjugate wherein the PMA serves as an anchor for

successful encapsulation. The disulfide linkage ensures a reversible

nature of the linkage. (Reprinted with permission from ref. 112.

Copyright 2009, Elsevier.)

334 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

4.6 Enhancing immune responses by co-delivery of PRR

agonists

As most recombinant antigens fail to activate DCs, addition of

microbial components or their synthetic derivatives (e.g. TLR

ligands) can strongly enhance their immunogenicity by

stimulating DC activation. Similarly, although the particulate

nature of microparticles might be sufficient to be recognized by

the immune system as intrinsically dangerous via inflammasome

activation, most particulates are only poor activators of DCs. As

a result, co-delivery of particulate antigen formulations with

PRR agonists might well work synergistic in evoking potent

immune responses by combining the antigen presentation

promoting capacities of particulates with the DC activating

properties of PRR agonists. Such co-administration of antigen

carrier and DC activator can be achieved either by mere

co-injection, or by physical linkage of the DC activation stimulus

to the carrier via surface adsorption and encapsulation. Recent

data clearly indicate that the latter strategy is superior in inducing

strong effector T cell responses. Blander et al. have demonstrated

that antigen processing and loading onto MHCII molecules is

regulated at the level of the individual phagosome, and that

antigen and TLR agonist need to be present in the same

endocytic compartment to ensure optimal antigen presentation

to CD4 T cells.131,132 Combining particles with TLR agonists

might be of particular interest when using agonists for TLRs

localized in phagolysosomal compartments such as TLR3, 7, 8

and 9. Schlosser et al. have used biodegradable PLGA

microspheres to co-encapsulate ovalbumin and the TLR9 ligand

CpG.133 Importantly, co-encapsulation of both antigen and CpG

generated far superior CTL responses compared to a mixture of

OVA containing microspheres with CpG containing micro-

spheres, thus further extending the observations made by

Blander et al. concerning the induction of CD4 T cell responses

Fig. 11 (A) Processing of dextran sulfate/poly-L-arginine microcapsule encapsulated OVA was analyzed using DQ-OVA. Confocal microscopy

images of BM-DCs incubated with DQ-OVA-microcapsules for 0, 4 and 48 h (overlay of green fluorescence and DIC). Insets: flow cytometry

analysis of green fluorescence intensity (FL1-H). An overlay of DC green autofluorescence (black line) and green fluorescence intensity of

DQ-OVA microcapsules (gray line) is given in the first histogram. The second and third histograms show the green fluorescence intensity

after incubating BM-DCs with DQ-OVA microcapsules for 4 and 48 h, respectively. (B) TEM images of BM-DCs that have internalized dextran

sulfate/poly-L-arginine microcapsules at the indicated time intervals. Microcapsule shell: dotted arrows; membranes surrounding the micro-

capsules: open arrows. In the encircled area, microcapsule rupture and cytoplasmic invagination are clearly distinguishable. Lysosomes,

endoplasmatic reticulu (ER), and a mitochondrion are indicated by the solid arrows. (Reprinted with permission from ref. 98. Copyright 2009,

Wiley.)

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 335

to cross-presentation and the induction of CD8 CTL responses.

Similar results have been obtained by Heit et al., who demon-

strated that co-encapsulating antigen and CpG in PLGA micro-

spheres was far superior in inducing CD4 and CD8 T cell

responses compared to a mixture of soluble CpG with soluble

antigen. PLGA encapsulating both ovalbumin and CpG was

able to confer protection against a lethal challenge with

ovalbumin-expressing Listeria monocytogenes, while micro-

spheres containing ovalbumin alone failed to protect.

Unfortunately, in this study no comparison was made

with a mixture of ovalbumin-containing PLGA and soluble

CpG.117

Increased humoral immune responses and levels of IFN-gsecreting T cells were also observed after co-encapsulation of

ovalbumin and polyU, a synthetic TLR7/8 agonist mimicking

ssRNA, inside polylactic particles.134 Enhancement of

immune responses by co-delivery of TLR agonists is certainly

not restricted to agonists that trigger endosomal TLRs.

Kazzaz et al. made similar observations using PLGA and

the TLR4 agonist monophosphoryl lipid A (MPL), a

detoxified LPS analogue recently approved for human use in

the EU.135 Encapsulation of MPL together with the naturally

expressed tumor antigen tyrosinase related protein-2 (Trp-2)

in PLGA nanoparticles could even induce therapeutic

immunity against the highly aggressive B16 melanoma, further

emphasizing the strength and potential of this approach.136

Although these initial experiments have clearly demonstrated

the benefits of encapsulating antigen and immune potentiatior

Fig. 12 Polyhydroxylated nanoparticle surfaces activate complement. (a) Synthesis and stabilization with two different forms of Pluronic allowed

the generation of polyhydroxylated- or polymethoxylatednanoparticles. (b) The a,o-terminal OH groups on Pluronic could be converted to OCH3

groups. (c) The proposed mechanism where OH groups on the polyhydroxylated nanoparticles can bind to the exposed thioester of C3b to activate

complement by the alternative pathway. (d) Nanoparticle-induced complement activation, as measured through C3a presence in human serum

after incubation with nanoparticles, was demonstrated to be high with polyhydroxylated nanoparticles but low with polymethoxylated

nanoparticles (OH– and CH3O–NPs, respectively). Results are normalized to control of serum incubation with PBS. Values are means of three

independent experiments; error bars correspond to standard error of mean, s.e.m. (Reprinted with permission from ref. 80. Copyright 2007, Nature

Publishing Group.)

336 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

in PLGA microparticles, the encapsulation process itself is far

from standard practice, and requires lots of optimization for

each antigen and antigen–immune potentiator combination

being applied. Given their capacity to efficiently encapsulate

protein antigen and the high versatility of the layer-by-layer

technique used to produce them, polyelectrolyte microcapsules

might be an interesting alternative for PLGA microspheres.

As these microcapsules are generated by the deposition of

polyelectrolytes, their surface charge can be easily made

cationic by depositing a positively charged polyelectrolyte as

outer layer, which allows an easy binding of negatively charged

TLR agonists such as CpG oligonucleotides and the dsRNA

analogue polyI:C via mere electrostatic interaction (unpublished

results).

Linking functional ligands to microparticles not only may

enhance the general strength of the immune response, but

might also allow us to steer the immune response towards the

desired direction. For example, adding CpG to particulates

skews the immune response towards a Th1 type. By using

other ligands, for example b-glucans that promote IL-23

secretion by DCs, one might be able to induce a more Th17

skewed response.

In addition to potentiating immune responses by

co-delivering antigen and DC activation stimulus, particulate

delivery vehicles also have the significant benefit of reducing

the inflammatory toxicity generally associated with the use of

an immune potentiator by limiting its free diffusion and

focusing its effects on the target cell, being the DC. In this

respect, combining CpG oligonucleotides with poly-L-

arginine, a polycationic amino acid, has been demonstrated

to enhance CpG uptake by DCs in vivo, while inhibiting the

systemic release of inflammatory cytokines observed after

injection of free CpG.137

Finally, although co-delivery of antigens and immune

potentiators such as TLR agonists within or associated to

the same particle clearly has tremendous benefits in augmenting

the strength of the induced immune response, co-encapsulating

them remains a complex and challenging task. Developing

polymeric particles with strong intrinsic immune activating

properties could alleviate these complex procedures and might

constitute a major breakthrough to pave the way for a broader

clinical applicability of polymeric carriers in vaccines. One way to

achieve this goal might be the use of biomaterials that activate

the complement system. Complement activation not only

constitutes a direct biochemical defense mechanism to kill and

opsonize microorganisms, but also has been shown to modulate

the DC activation status and to promote antigen-specific immune

responses. Biomaterials containing high levels of free hydroxyls

and amine nucleophiles strongly activate the complement

cascade by binding to the exposed thioester of the complement

factor C3b. Recently, Reddy et al. have developed antigen

coupled polyhydroxylated nanoparticles composed of Pluronic

stabilized polypropylene sulfide to exploit complement activation

as activating stimulus, as shown in Fig. 12.80 Injection

of these particles resulted in a fast and strong activation

of APCs, followed by the generation of humoral and

cellular immune responses, including the induction of IFN-gsecreting CD8 T cells, clearly showing the potential of this

approach.

5. Conclusions and future perspectives

During the last decade, particulate antigen delivery using

polymeric carriers has clearly demonstrated its strong

benefits in enhancing antigen immunogenicity in vaccination.

Importantly, particulates strongly promote cross-presentation,

thereby allowing the induction of CTL responses, a feature

hardly achievable when using soluble antigens. In addition,

particulate carriers can also be used to target PRR agonists to

DCs, which not only works synergistic in enforcing immunity,

but also should allow one to steer immune responses to a certain

direction and to reduce inflammatory side effects. Nevertheless,

much progress remains to be made. Novel insights in the

mechanisms underlying cross-presentation can provide drug

delivery scientists with clues regarding the optimal size and

composition of particles for cross-presentation. For example, if

cross-presentation indeed occurs exclusively in the first hours

after particle uptake and before the phagosome acidifies, particles

that rapidly decompose and release their antigen following

internalization should offer a significant benefit. In addition,

modifying particles with certain ligands that target them to

specific cellular compartments following receptor mediated

internalization or to specific DC subsets, will also affect the

way the antigen is presented and should allow one to further tune

the immune response induced.

Also many practical hurdles remain to be taken before

polymeric carriers will become widespread antigen delivery

vehicles in human vaccines. For most of the experimental

carriers developed to date, antigen encapsulation is a complex

and challenging process, involving multi-steps often resulting

in low antigen encapsulation. Given the limited availability

and cost of many recombinant antigens, there is consequently

a strong need to develop new and more efficient encapsulation

strategies. Nevertheless, cost might not be an insurmountable

issue if particulate delivery can yield effective therapeutic

vaccines against incurable diseases as cancer or HIV.

Designing particle-based vaccines for large-scale preventive

immunization schedules in contrast will certainly require the

development of new strategies enabling an efficient, cheap and

easy encapsulation of antigens and immune potentiators,

preferably involving single step processes yielding a dry

powder such as spray-drying. Achieving this will allow us to

develop a next generation of vaccines, tailored towards 21st

century needs.

Acknowledgements

S.D.K. thanks Ghent University (BOF-GOA) for funding.

B.D.G. acknowledges the FWO for a postdoctoral

scholarship.

Notes and references

1 R. Rappuoli, H. I. Miller and S. Falkow, Science, 2002, 297,937–939.

2 R. Rappuoli, Nat. Biotechnol., 2007, 25, 1361–1366.3 B. Pulendran and R. Ahmed, Cell, 2006, 124, 849–863.4 P. E. Jensen, Nat. Immunol., 2007, 8, 1041–1048.5 J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque,

Y. J. Liu, B. Pulendran and K. Palucka, Annu. Rev. Immunol.,2000, 18, 767–811.

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 337

6 N. Luckashenak, S. Schroeder, K. Endt, D. Schmidt, K. Mahnke,M. F. Bachmann, P. Marconi, C. A. Deeg and T. Brocker,Immunity, 2008, 28, 521–532.

7 J. K. Tan and H. C. O’Neill, J. Leukocyte Biol., 2005, 78,319–324.

8 D. Kabelitz and R. Medzhitov, Curr. Opin. Immunol., 2007, 19,1–3.

9 R. Medzhitov, Nature, 2007, 449, 819–826.10 R. Medzhitov and C. Janeway, Jr., Immunol. Rev., 2000, 173,

89–97.11 C. Pasare and R. Medzhitov, Semin. Immunol., 2004, 16,

23–26.12 B. Pulendran, Immunol. Rev., 2004, 199, 227–250.13 B. Pulendran, K. Palucka and J. Banchereau, Science, 2001, 293,

253–256.14 K. Bottomly, Immunol. Today, 1988, 9, 268–274.15 W. Huang, L. Na, P. L. Fidel and P. Schwarzenberger, J. Infect.

Dis., 2004, 190, 624–631.16 L. E. Harrington, P. R. Mangan and C. T. Weaver, Curr. Opin.

Immunol., 2006, 18, 349–356.17 F. L. van de Veerdonk, M. S. Gresnigt, B. J. Kullberg, J. W. van

der Meer, L. A. Joosten and M. G. Netea, BMB Rep., 2009, 42,776–787.

18 E. De Gregorio, E. Tritto and R. Rappuoli, Eur. J. Immunol.,2008, 38, 2068–2071.

19 B. N. Lambrecht, M. Kool, M. A. M. Willart and H. Hammad,Curr. Opin. Immunol., 2009, 21, 23–29.

20 B. Guy, Nat. Rev. Microbiol., 2007, 5, 505–517.21 J. R. Baldridge, P. McGowan, J. T. Evans, C. Cluff, S. Mossman,

D. Johnson and D. Persing, Expert Opin. Biol. Ther., 2004, 4,1129–1138.

22 E. Celis, Cancer Res., 2007, 67, 7945–7947.23 D. H. Persing, R. N. Coler, M. J. Lacy, D. A. Johnson,

J. R. Baldridge, R. M. Hershberg and S. G. Reed, TrendsMicrobiol., 2002, 10, s32–37.

24 V. E. Schijns and W. G. Degen, Clin. Pharmacol. Ther., 2007, 82,750–755.

25 B. S. Thompson, P. M. Chilton, J. R. Ward, J. T.Evans and T. C. Mitchell, J. Leukocyte Biol., 2005, 78,1273–1280.

26 D. M. Harper, E. L. Franco, C. M. Wheeler, A. B. Moscicki,B. Romanowski, C. M. Roteli-Martins, D. Jenkins, A. Schuind,S. A. Costa Clemens and G. Dubin, Lancet, 2006, 367,1247–1255.

27 M. Kundi, Expert Rev. Vaccines, 2007, 6, 133–140.28 A. Pashine, N. M. Valiante and J. B. Ulmer, Nat. Med., 2005, 11,

S63–68.29 R. S. Chu, O. S. Targoni, A. M. Krieg, P. V. Lehmann and

C. V. Harding, J. Exp. Med., 1997, 186, 1623–1631.

30 A. Heit, F. Schmitz, M. O’Keeffe, C. Staib, D. H. Busch,H. Wagner and K. M. Huster, J. Immunol., 2005, 174,4373–4380.

31 G. Napolitani, A. Rinaldi, F. Bertoni, F. Sallusto andA. Lanzavecchia, Nat. Immunol., 2005, 6, 769–776.

32 L. Bonifaz, D. Bonnyay, K. Mahnke, M. Rivera,M. C. Nussenzweig and R. M. Steinman, J. Exp. Med., 2002,196, 1627–1638.

33 D. T. O’Hagan and M. Singh, Expert Rev. Vaccines, 2003, 2,269–283.

34 D. T. O’Hagan, M. Singh and J. B. Ulmer, Methods, 2006, 40,10–19.

35 J. Hanes, J. L. Cleland and R. Langer, Adv. Drug Delivery Rev.,1997, 28, 97–119.

36 S. T. Reddy, M. A. Swartz and J. A. Hubbell, Trends Immunol.,2006, 27, 573–579.

37 D. T. O’Hagan and N. M. Valiante, Nat. Rev. Drug Discovery,2003, 2, 727–735.

38 C. Wang, Q. Ge, D. Ting, D. Nguyen, H. R. Shen, J. Z. Chen,H. N. Eisen, J. Heller, R. Langer and D. Putnam, Nat. Mater.,2004, 3, 190–196.

39 W. N. Haining, D. G. Anderson, S. R. Little, M. S. vonBerwelt-Baildon, A. A. Cardoso, P. Alves, K. Kosmatopoulos,L. M. Nadler, R. Langer and D. S. Kohane, J. Immunol., 2004,173, 2578–2585.

40 B. G. De Geest, S. De Koker, Y. Gonnissen, L. J. De Cock,J. Grooten, J. P. Remon and C. Vervaet, Soft Matter, 2010, 6,305–310.

41 O. Soga, C. F. van Nostrum and W. E. Hennink, Biomacro-molecules, 2004, 5, 818–821.

42 J. Ochoa, J. M. Irache, I. Tamayo, A. Walz, V. G. DelVecchioand C. Gamazo, Vaccine, 2007, 25, 4410–4419.

43 S. Jain, W. T. Yap and D. J. Irvine, Biomacromolecules, 2005, 6,2590–2600.

44 N. Murthy, M. C. Xu, S. Schuck, J. Kunisawa, N. Shastri and J.M. J. Frechet, Proc. Natl. Acad. Sci. U. S. A., 2003, 100,4995–5000.

45 J. K. Oh, D. J. Siegwart, H. I. Lee, G. Sherwood, L. Peteanu,J. O. Hollinger, K. Kataoka and K. Matyjaszewski, J. Am. Chem.Soc., 2007, 129, 5939–5945.

46 B. G. De Geest, W. Van Camp, F. E. Du Prez, J. Demeester,S. C. De Smedt and W. E. Hennink, Chem. Commun., 2008,190–192.

47 D. Lemoine, F. Wauters, S. Bouchend’homme and V. Preat, Int.J. Pharm., 1998, 176, 9–19.

48 W. R. Gombotz and S. F. Wee, Adv. Drug Delivery Rev., 1998, 31,267–285.

49 D. Christensen, K. S. Korsholm, I. Rosenkrands,T. Lindenstrom, P. Andersen and E. M. Agger, Expert Rev.Vaccines, 2007, 6, 785–796.

50 J. Kunisawa, S. Nakagawa and T. Mayumi, Adv. Drug DeliveryRev., 2001, 52, 177–186.

51 B. M. Discher, Y. Y. Won, D. S. Ege, J. C. M. Lee, F. S. Bates,D. E. Discher and D. A. Hammer, Science, 1999, 284,1143–1146.

52 H. Lomas, I. Canton, S. MacNeil, J. Du, S. P. Armes, A. J. Ryan,A. L. Lewis and G. Battaglia, Adv. Mater., 2007, 19,4238.

53 L. Illum, I. Jabbal-Gill, M. Hinchcliffe, A. N. Fisher andS. S. Davis, Adv. Drug Delivery Rev., 2001, 51, 81–96.

54 G. Decher, Science, 1997, 277, 1232–1237.55 F. Caruso, R. A. Caruso and H. Mohwald, Science, 1998, 282,

1111–1114.56 E. Donath, G. B. Sukhorukov, F. Caruso, S. A. Davis and

H. Mohwald, Angew. Chem., Int. Ed., 1998, 37,2201–2205.

57 G. B. Sukhorukov, E. Donath, S. Davis, H. Lichtenfeld,F. Caruso, V. I. Popov and H. Mohwald, Polym. Adv. Technol.,1998, 9, 759–767.

58 J. F. Quinn, A. P. R. Johnston, G. K. Such, A. N. Zelikin andF. Caruso, Chem. Soc. Rev., 2007, 36, 707–718.

59 V. Kozlovskaya, E. Kharlampieva, I. Erel and S. A. Sukhishvili,Soft Matter, 2009, 5, 4077–4087.

60 D. V. Volodkin, A. I. Petrov, M. Prevot and G. B. Sukhorukov,Langmuir, 2004, 20, 3398–3406.

61 A. M. Yu, Y. J. Wang, E. Barlow and F. Caruso, Adv. Mater.,2005, 17, 1737.

62 L. Feng, X. R. Qi, X. J. Zhou, Y. Maitani, S. C. Wang, Y. Jiangand T. Nagai, J. Controlled Release, 2006, 112, 35–42.

63 P. Johansen, F. Estevez, R. Zurbriggen, H. P. Merkle,R. Gluck, G. Corradin and B. Gander, Vaccine, 2000, 19,1047–1054.

64 M. Sandor, D. Enscore, P. Weston and E. Mathiowitz,J. Controlled Release, 2001, 76, 297–311.

65 S. P. Schwendeman, H. R. Costantino, R. K. Gupta, G. R. Siber,A. M. Klibanov and R. Langer, Proc. Natl. Acad. Sci.U. S. A.,1995, 92, 11234–11238.

66 G. Zhu, S. R. Mallery and S. P. Schwendeman, Nat. Biotechnol.,2000, 18, 52–57.

67 G. Zhu and S. P. Schwendeman, Pharm. Res., 2000, 17,351–357.

68 H. Tamber, P. Johansen, H. P. Merkle and B. Gander, Adv. DrugDelivery Rev., 2005, 57, 357–376.

69 W. Jiang, R. K. Gupta, M. C. Deshpande andS. P. Schwendeman, Adv. Drug Delivery Rev., 2005, 57, 391–410.

70 B. G. De Geest, C. Dejugnat, M. Prevot, G. B. Sukhorukov,J. Demeester and S. C. De Smedt, Adv. Funct. Mater., 2007, 17,531–537.

71 B. G. De Geest, C. Dejugnat, G. B. Sukhorukov, K. Braeckmans,S. C. De Smedt and J. Demeester, Adv. Mater., 2005, 17, 2357.

338 Chem. Soc. Rev., 2011, 40, 320–339 This journal is c The Royal Society of Chemistry 2011

72 B. G. De Geest, C. Dejugnat, E. Verhoeven, G. B. Sukhorukov,A. M. Jonas, J. Plain, J. Demeester and S. C. De Smedt,J. Controlled Release, 2006, 116, 159–169.

73 B. G. De Geest, S. De Koker, J. Demeester, S. C. De Smedt andW. E. Hennink, J. Controlled Release, 2009, 135,268–273.

74 R. K. Gupta, M. Singh and D. T. O’Hagan, Adv. Drug DeliveryRev., 1998, 32, 225–246.

75 W. R. Heath, G. T. Belz, G. M. Behrens, C. M. Smith,S. P. Forehan, I. A. Parish, G. M. Davey, N. S. Wilson,F. R. Carbone and J. A. Villadangos, Immunol. Rev., 2004, 199,9–26.

76 J. M. den Haan, S. M. Lehar and M. J. Bevan, J. Exp. Med.,2000, 192, 1685–1696.

77 M. L. Lin, Y. Zhan, J. A. Villadangos and A. M. Lew, Immunol.Cell Biol., 2008, 86, 353–362.

78 E. Segura and J. A. Villadangos, Curr. Opin. Immunol., 2009, 21,105–110.

79 B. Leon and C. Ardavin, Immunol. Cell Biol., 2008, 86,320–324.

80 S. T. Reddy, A. J. van der Vlies, E. Simeoni, V. Angeli,G. J. Randolph, C. P. O’Neil, L. K. Lee, M. A. Swartz andJ. A. Hubbell, Nat. Biotechnol., 2007, 25, 1159–1164.

81 V. Manolova, A. Flace, M. Bauer, K. Schwarz, P. Saudan andM. F. Bachmann, Eur. J. Immunol., 2008, 38, 1404–1413.

82 Y. Waeckerle-Men, E. U. Allmen, B. Gander, E. Scandella,E. Schlosser, G. Schmidtke, H. P. Merkle and M. Groettrup,Vaccine, 2006, 24, 1847–1857.

83 C. L. van Broekhoven, C. R. Parish, C. Demangel, W. J. Brittonand J. G. Altin, Cancer Res., 2004, 64, 4357–4365.

84 Y. J. Kwon, E. James, N. Shastri and J. M. Frechet, Proc. Natl.Acad. Sci. U. S. A., 2005, 102, 18264–18268.

85 X. Zhao, S. Jain, H. Benjamin Larman, S. Gonzalez andD. J. Irvine, Biomaterials, 2005, 26, 5048–5063.

86 Y. Hori, A. M. Winans, C. C. Huang, E. M. Horrigan andD. J. Irvine, Biomaterials, 2008, 29, 3671–3682.

87 Y. Hori, A. M. Winans and D. J. Irvine, Acta Biomater., 2009, 5,969–982.

88 A. Seubert, E. Monaci, M. Pizza, D. T. O’Hagan and A. Wack,J. Immunol., 2008, 180, 5402–5412.

89 S. Burgdorf, A. Kautz, V. Bohnert, P. A. Knolle and C. Kurts,Science, 2007, 316, 612–616.

90 S. Burgdorf, C. Scholz, A. Kautz, R. Tampe and C. Kurts, Nat.Immunol., 2008, 9, 558–566.

91 J. Idoyaga, C. Cheong, K. Suda, N. Suda, J. Y. Kim, H. Lee,C. G. Park and R. M. Steinman, J. Immunol., 2008, 180,3647–3650.

92 D. Dudziak, A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz,C. Trumpfheller, S. Yamazaki, C. Cheong, K. Liu, H. W. Lee,C. G. Park, R. M. Steinman and M. C. Nussenzweig, Science,2007, 315, 107–111.

93 H. Shen, A. L. Ackerman, V. Cody, A. Giodini, E. R. Hinson,P. Cresswell, R. L. Edelson, W. M. Saltzman and D. J. Hanlon,Immunology, 2006, 117, 78–88.

94 C. D. Partidos, P. Vohra, D. H. Jones, G. Farrar andM. W. Steward, J. Controlled Release, 1999, 62, 325–332.

95 Y. Ataman-Onal, S. Munier, A. Ganee, C. Terrat, P. Y. Durand,N. Battail, F. Martinon, R. Le Grand, M. H. Charles, T. Delairand B. Verrier, J. Controlled Release, 2006, 112,175–185.

96 K. K. Tran and H. Shen, Biomaterials, 2009, 30, 1356–1362.97 N. Murthy, M. Xu, S. Schuck, J. Kunisawa, N. Shastri and

J. M. Frechet, Proc. Natl. Acad. Sci. U. S. A., 2003, 100,4995–5000.

98 S. De Koker, B. G. De Geest, S. K. Singh, R. DeRycke, T. Naessens, Y. Van Kooyk, J. Demeester, S. C.De Smedt and J. Grooten, Angew. Chem., Int. Ed., 2009, 48,8485–8489.

99 J. Panyam, W. Z. Zhou, S. Prabha, S. K. Sahoo andV. Labhasetwar, FASEB J., 2002, 16, 1217–1226.

100 E. Walter, D. Dreher, M. Kok, L. Thiele, S. G. Kiama, P. Gehrand H. P. Merkle, J. Controlled Release, 2001, 76,149–168.

101 W. N. Haining, D. G. Anderson, S. R. Little, M. S. von Bergwelt-Baildon, A. A. Cardoso, P. Alves, K. Kosmatopoulos,

L. M. Nadler, R. Langer and D. S. Kohane, J. Immunol., 2004,173, 2578–2585.

102 N. Murthy, Y. X. Thng, S. Schuck, M. C. Xu and J. M. Frechet,J. Am. Chem. Soc., 2002, 124, 12398–12399.

103 Y. Hu, T. Litwin, A. R. Nagaraja, B. Kwong, J. Katz, N. Watsonand D. J. Irvine, Nano Lett., 2007, 7, 3056–3064.

104 A. L. Ackerman, C. Kyritsis, R. Tampe and P. Cresswell, Proc.Natl. Acad. Sci. U. S. A., 2003, 100, 12889–12894.

105 P. Guermonprez, L. Saveanu, M. Kleijmeer, J. Davoust, P. VanEndert and S. Amigorena, Nature, 2003, 425, 397–402.

106 M. Houde, S. Bertholet, E. Gagnon, S. Brunet, G. Goyette,A. Laplante, M. F. Princiotta, P. Thibault, D. Sacks andM. Desjardins, Nature, 2003, 425, 402–406.

107 S. Burgdorf and C. Kurts, Curr. Opin. Immunol., 2008, 20,89–95.

108 A. Savina, C. Jancic, S. Hugues, P. Guermonprez, P. Vargas,I. C. Moura, A. M. Lennon-Dumenil, M. C. Seabra, G. Raposoand S. Amigorena, Cell, 2006, 126, 205–218.

109 C. Jancic, A. Savina, C. Wasmeier, T. Tolmachova, J. El-Benna,P. M. Dang, S. Pascolo, M. A. Gougerot-Pocidalo, G. Raposo,M. C. Seabra and S. Amigorena, Nat. Cell Biol., 2007, 9,367–378.

110 K. E. Broaders, J. A. Cohen, T. T. Beaudette, E. M. Bachelderand J. M. Frechet, Proc. Natl. Acad. Sci. U. S. A., 2009, 106,5497–5502.

111 A. N. Zelikin, J. F. Quinn and F. Caruso, Biomacromolecules,2006, 7, 27–30.

112 S. F. Chong, A. Sexton, R. De Rose, S. J. Kent, A. N. Zelikin andF. Caruso, Biomaterials, 2009, 30, 5178–5186.

113 R. De Rose, A. N. Zelikin, A. P. R. Johnston, A. Sexton,S. F. Chong, C. Cortez, W. Mulholland, F. Caruso andS. J. Kent, Adv. Mater., 2008, 20, 4698.

114 A. Sexton, P. G. Whitney, S. F. Chong, A. N. Zelikin,A. P. Johnston, R. De Rose, A. G. Brooks, F. Caruso andS. J. Kent, ACS Nano, 2009, 3, 3391–3400.

115 C. D. Austin, X. Wen, L. Gazzard, C. Nelson, R. H. Scheller andS. J. Scales, Proc. Natl. Acad. Sci. U. S. A., 2005, 102,17987–17992.

116 D. V. Volodkin, N. I. Larionova and G. B. Sukhorukov,Biomacromolecules, 2004, 5, 1962–1972.

117 A. Heit, F. Schmitz, T. Haas, D. H. Busch and H.Wagner, Eur. J.Immunol., 2007, 37, 2063–2074.

118 T. Fifis, A. Gamvrellis, B. Crimeen-Irwin, G. A. Pietersz, J. Li,P. L. Mottram, I. F. McKenzie and M. Plebanski, J. Immunol.,2004, 173, 3148–3154.

119 M. Yoshida and J. E. Babensee, J. Biomed. Mater. Res., 2004,71a, 45–54.

120 S. Fischer, E. Uetz-von Allmen, Y. Waeckerle-Men,M. Groettrup, H. P. Merkle and B. Gander, Biomaterials, 2007,28, 994–1004.

121 Y. Waeckerle-Men, E. Scandella, E. Uetz-Von Allmen,B. Ludewig, S. Gillessen, H. P. Merkle, B. Gander andM. Groettrup, J. Immunol. Methods, 2004, 287, 109–124.

122 A. Westwood, G. D. Healey, E. D. Williamson and J. E. Eyles,Vaccine, 2005, 23, 3857–3863.

123 T. Uto, X. Wang, K. Sato, M. Haraguchi, T. Akagi, M. Akashiand M. Baba, J. Immunol., 2007, 178, 2979–2986.

124 S. R. Little, D. M. Lynn, Q. Ge, D. G. Anderson, S. V. Puram,J. Chen, H. N. Eisen and R. Langer, Proc. Natl. Acad. Sci.U. S. A., 2004, 101, 9534–9539.

125 M. Kool, T. Soullie, M. van Nimwegen, M. A. M. Willart,F. Muskens, S. Jung, H. C. Hoogsteden, H. Hammad andB. N. Lambrecht, J. Exp. Med., 2008, 205, 869–882.

126 H. Li, S. Nookala and F. Re, J. Immunol., 2007, 178, 5271–5276.127 H. Li, S. B. Willingham, J. P. Ting and F. Re, J. Immunol., 2008,

181, 17–21.128 S. C. Eisenbarth, O. R. Colegio, W. O’Connor, F. S. Sutterwala

and R. A. Flavell, Nature, 2008, 453, 1122–1126.129 M. Kool, V. Petrilli, T. De Smedt, A. Rolaz, H. Hammad, M. van

Nimwegen, I. M. Bergen, R. Castillo, B. N. Lambrecht andJ. Tschopp, J. Immunol., 2008, 181, 3755–3759.

130 F. A. Sharp, D. Ruane, B. Claass, E. Creagh, J. Harris,P. Malyala, M. Singh, D. T. O’Hagan, V. Petrilli, J. Tschopp,L. A. O’Neill and E. C. Lavelle, Proc. Natl. Acad. Sci. U. S. A.,2009, 106, 870–875.

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 320–339 339

131 J. M. Blander, Trends Immunol., 2007, 28, 19–25.132 J. M. Blander and R. Medzhitov, Nature, 2006, 440,

808–812.133 E. Schlosser, M. Mueller, S. Fischer, S. Basta, D. H. Busch,

B. Gander and M. Groettrup, Vaccine, 2008, 26, 1626–1637.134 A. Westwood, S. J. Elvin, G. D. Healey, E. D. Williamson and

J. E. Eyles, Vaccine, 2006, 24, 1736–1743.

135 J. Kazzaz, M. Singh, M. Ugozzoli, J. Chesko, E. Soenawan andD. T. O’Hagan, J. Controlled Release, 2006, 110, 566–573.

136 S. Hamdy, O. Molavi, Z. Ma, A. Haddadi, A. Alshamsan,Z. Gobti, S. Elhasi, J. Samuel and A. Lavasanifar, Vaccine,2008, 26, 5046–5057.

137 K. Lingnau, A. Egyed, C. Schellack, F. Mattner, M. Buschle andW. Schmidt, Vaccine, 2002, 20, 3498–3508.