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.
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