Nanoporous polyelectrolyte vaccine microcarriers. A formulation platform for enhancing humoral and cellular immune responses.

In this paper we report on the design, characterization and immuno-biological evaluation of nanoporous polyelectrolyte microparticles as vaccine carrier. Relative to soluble antigen, formulation of antigen as a sub-10 µm particle can strongly enhance antigen-specific cellular immune responses. The latter is crucial to confer protective immunity against intracellular pathogens and for anti-cancer vaccines. However, a major bottleneck in microparticulate vaccine formulation is the development of generic strategies that afford antigen encapsulation under benign and scalable conditions. Our strategy is based on spray drying of a dilute aqueous solution of antigen, oppositely charged polyelectrolytes and mannitol as a pore-forming component. The obtained solid microparticles can be redispersed in aqueous medium, leading to leaching out of the mannitol, thereby creating a highly porous internal structure. This porous structure enhances enzymatic processing of encapsulated proteins. After optimizing the conditions to process these microparticles we demonstrate that they strongly enhance cross-presentation in vitro by dendritic cells to CD8 T cells. In vivo experiments in mice confirm that this vaccine formulation technology is capable of enhancing cellular immune responses.

Just spray it--LbL assembly enters a new age.

Over the past two decades the Layer-by-Layer (LbL) assembly of multilayer thin films has witnessed an explosive growth. However, this has so far not been translated into numerous industrial applications mainly owing to the time-consuming multistep assembly procedure which was originally based on dipping of a substrate into a solution. More recently the use of spray-based approaches, both for planar films as well as for the construction of polymeric particles, has emerged. Here we highlight these recent advances that have the potential to move the LbL field forward.

Spray-dried polyelectrolyte microparticles in oral antigen delivery: stability, biocompatibility, and cellular uptake.

During the past decade, extensive research has undeniably improved the formulation and delivery of oral vaccines. Nevertheless, several factors, such as the harsh gastrointestinal environment together with tolerance induction to exogenous antigens, have thus far impeded the optimal effectiveness and clinical application of oral delivery systems. The current study encompasses an initial evaluation of the stability, biocompatibility, and cellular uptake of two promising candidate systems for oral antigen delivery, that is, calcium carbonate- (CP) and mannitol-templated (MP) porous microspheres. Both spray-dried formulations were efficiently internalized by human intestinal epithelial cells (Caco-2 and HT-29) and degraded into phagolysosomal intracellular compartments. In addition, cellular particle uptake and processing significantly up-regulated the expression of (HLA) class-II and costimulatory molecules on intestinal epithelial cells. Even though the high surface-area-to-volume ratio of the microspheres was expected to favor protease access, antigen release was remarkably limited in simulated intestinal fluid and was even absent under gastric conditions. Finally, neither CP nor MP exerted cytotoxicity upon prolonged in vitro incubation with high antigen concentration. Altogether, these data support the potential of CP and MP for oral antigen delivery and motivate the further development of these promising carrier systems in in vivo studies.

ß-Glucan microparticles are good candidates for mucosal antigen delivery in oral vaccination.

Continuously improving the developmental process and the efficacy of oral vaccines is essential in the fight against intestinal pathogens. A promising strategy for vaccination applying safe, biodegradable and non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. The current study evaluates the potential of ß-glucan particles (GP) as an oral antigen delivery system and their adjuvant characteristics. GP are efficiently internalized by human intestinal epithelial cell lines (Caco-2 and HT-29 cells), without exerting negative effects on cell viability. GP triggered the expression of pro-inflammatory cytokines IL-23p19, IL-8 and the ß-glucan receptors dectin-1 and TLR2 by activated Caco-2 cells, and CCL20 in HT-29 cells. In contrast, the expression level of TGF-ß, an important mediator of oral tolerance, was significantly downregulated in HT-29 cells. Additionally, adoptive transfer experiments showed proliferating ovalbumin (OVA)-specific CD4(+) T cells mainly in the spleens of GP-OVA-fed mice. Furthermore, we detected a significantly increased IL-17 and a trend towards increased IFN-γ production in the spleen of GP-OVA-fed mice upon antigen restimulation. Oral administration of GP-OVA induced increased OVA-specific IgA, secretory-IgA (S-IgA) and secretory component (SC) production in intestinal fluids. Our data show that GP vehicles are able to deliver OVA via an oral route allowing efficient antigen presentation alongside adaptive immune activation, resulting in a Th17-biased response and the production of OVA-specific IgA, secretory-IgA and secretory component antibodies.

Hydrogen bonded polymeric multilayer films assembled below and above the cloud point temperature.

Polymeric multilayer films assembled via hydrogen-bonding are witnessing increased interest from the scientific community. Here we report on hydrogen bonded multilayers of tannic acid and neutral poly(2-oxazoline)s. Importantly we demonstrate, to the best of our knowledge, for the first time that a temperature responsive polymer, in this case poly(2-(n-propyl)-2-oxazline), can be assembled below and above its TCP with distinctly different growth mechanisms.

One-step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells.

Vaccination is regarded as the most efficient and cost-effective way to prevent infectious diseases. Vaccine design nowadays focuses on the implementation of safer recombinant subunit vaccines. However, these recombinant subunit antigens are often poor immunogens and several strategies are currently under investigation to enhance their immunogenicity. The encapsulation of antigens in biodegradable microparticulate delivery systems seems a promising strategy to boost their immunogenicity. Here, we evaluate the capacity of polyelectrolyte complex microparticles (PECMs), fabricated by single step spray-drying, to deliver antigens to porcine dendritic cells and how these particles affect the functional maturation of dendritic cells (DCs). As clinically relevant model antigen F4 fimbriae, a bacterial adhesin purified from a porcine-specific enterotoxigenic Escherichia coli strain was chosen. The resulting antigen-loaded PECMs are efficiently internalised by porcine monocyte-derived DCs. F4 fimbriae-loaded PECMs (F4-PECMs) enhanced CD40 and CD25 surface expression by DCs and this phenotypical maturation correlated with an increased secretion of IL-6 and IL-1ß. More importantly, F4-PECMs enhance both the T cell stimulatory and antigen presentation capacity of DCs. Moreover, PECMs efficiently promoted the CD8(+) T cell stimulatory capacity of dendritic cells, indicating an enhanced ability to cross-present the encapsulated antigens. These results could accelerate the development of veterinary and human subunit vaccines based on polyelectrolyte complex microparticles to induce protective immunity against a variety of extra- and intracellular pathogens.

Interaction between polymeric multilayer capsules and immune cells.

Polymeric multilayer capsules are emerging carrier systems in the field of drug delivery. These materials are fabricated by set-wise assembly of interaction species onto a sacrificial template followed by the decomposition of this template, yielding hollow capsules. Using bio-responsive polymers that can be triggered by pH, enzymes or reduction, several groups are exploring these systems for intracellular drug delivery. In this review we focus on the recent efforts made in investigating the in vitro and in vivo interaction between these capsules and cells of the immune system.

Single-step formation of degradable intracellular biomolecule microreactors.

Here we present a single-step all-aqueous approach to encapsulate biomolecules such as enzymes and proteins into stable microreactors. Key in this method is the use of spray-drying of the biomolecules of interest in combination with oppositely charged polyelectrolytes and mannitol as the sacrificial template. Remarkably, upon spray-drying in the presence of polyelectrolyte, mannitol crystallization is suppressed and the obtained amorphous mannitol offers enhanced preservation of the biomolecules' activity. Moreover, the use of mannitol allows the formation of nanopores within the microparticles upon rehydration of the microparticles in aqueous medium and subsequent dissolution of the mannitol. The oppositely charged polyelectrolytes provide a polymeric framework which stabilizes the microparticles upon rehydration. The versatility of this approach is demonstrated using horseradish peroxidase as the model enzyme and ovalbumin as the model antigen.