Poloxamers Have Vaccine-Adjuvant Properties by Increasing Dissemination of Particulate Antigen at Distant Lymph Nodes.

Vaccine technology is still facing challenges regarding some infectious diseases, which can be addressed by innovative drug delivery systems. In particular, nanoparticle-based vaccines combined with new types of adjuvants are actively explored as a platform for improving the efficacy and durability of immune protection. Here, biodegradable nanoparticles carrying an antigenic model of HIV were formulated with two combinations of poloxamers, 188/407, presenting or not presenting gelling properties, respectively. The study aimed to determine the influence of poloxamers (as a thermosensitive hydrogel or a liquid solution) on the adaptive immune response in mice. The results showed that poloxamer-based formulations were physically stable and did not induce any toxicity using a mouse dendritic cell line. Then, whole-body biodistribution studies using a fluorescent formulation highlighted that the presence of poloxamers influenced positively the dissemination profile by dragging nanoparticles through the lymphatic system until the draining and distant lymph nodes. The strong induction of specific IgG and germinal centers in distant lymph nodes in presence of poloxamers suggested that such adjuvants are promising components in vaccine development.

Polylactide Nanoparticles as a Biodegradable Vaccine Adjuvant: A Study on Safety, Protective Immunity and Efficacy against Human Leishmaniasis Caused by Leishmania Major.

Leishmaniasis is the 3rd most challenging vector-borne disease after malaria and lymphatic filariasis. Currently, no vaccine candidate is approved or marketed against leishmaniasis due to difficulties in eliciting broad immune responses when using sub-unit vaccines. The aim of this work was the design of a particulate sub-unit vaccine for vaccination against leishmaniasis. The poly (D,L-lactide) nanoparticles (PLA-NPs) were developed in order to efficiently adsorb a recombinant L. major histone H2B (L. major H2B) and to boost its immunogenicity. Firstly, a study was focused on the production of well-formed nanoparticles by the nanoprecipitation method without using a surfactant and on the antigen adsorption process under mild conditions. The set-up preparation method permitted to obtain H2B-adsorbed nanoparticles H2B/PLA (adsorption capacity of about 2.8% (w/w)) with a narrow size distribution (287 nm) and a positive zeta potential (30.9 mV). Secondly, an in vitro release assay performed at 37 °C, pH 7.4, showed a continuous release of the adsorbed H2B for almost 21 days (30%) from day 7. The immune response of H2B/PLA was investigated and compared to H2B + CpG7909 as a standard adjuvant. The humoral response intensity (IgG) was substantially similar between both formulations. Interestingly, when challenged with the standard parasite strain (GLC94) isolated from a human lesion of cutaneous leishmaniasis, mice showed a significant reduction in footpad swelling compared to unvaccinated ones, and no deaths occurred until week 17th. Taken together, these results demonstrate that PLA-NPs represent a stable, cost-effective delivery system adjuvant for use in vaccination against leishmaniasis.

Surface charge modulation of rifampicin-loaded PLA nanoparticles to improve antibiotic delivery in Staphylococcus aureus biofilms.

BACKGROUND: After the golden age of antibiotic discovery, bacterial infections still represent a major challenge for public health worldwide. The biofilm mode of growth is mostly responsible for chronic infections that current therapeutics fail to cure and it is well-established that novel strategies must be investigated. Particulate drug delivery systems are considered as a promising strategy to face issues related to antibiotic treatments in a biofilm context. Particularly, poly-lactic acid (PLA) nanoparticles present a great interest due to their ability to migrate into biofilms thanks to their submicronic size. However, questions still remain unresolved about their mode of action in biofilms depending on their surface properties. In the current study, we have investigated the impact of their surface charge, firstly on their behavior within a bacterial biofilm, and secondly on the antibiotic delivery and the treatment efficacy. RESULTS: Rifampicin-loaded PLA nanoparticles were synthetized by nanoprecipitation and characterized. A high and superficial loading of rifampicin, confirmed by an in silico simulation, enabled to deliver effective antibiotic doses with a two-phase release, appropriate for biofilm-associated treatments. These nanoparticles were functionalized with poly-L-lysine, a cationic peptide, by surface coating inducing charge reversal without altering the other physicochemical properties of these particles. Positively charged nanoparticles were able to interact stronger than negative ones with Staphylococcus aureus, under planktonic and biofilm modes of growth, leading to a slowed particle migration in the biofilm thickness and to an improved retention of these cationic particles in biofilms. While rifampicin was totally ineffective in biofilms after washing, the increased retention capacity of poly-L-lysine-coated rifampicin-loaded PLA nanoparticles has been associated with a better antibiotic efficacy than uncoated negatively charged ones. CONCLUSIONS: Correlating the carrier retention capacity in biofilms with the treatment efficacy, positively charged rifampicin-loaded PLA nanoparticles are therefore proposed as an adapted and promising approach to improve antibiotic delivery in S. aureus biofilms.

Polylactide-Based Reactive Micelles as a Robust Platform for mRNA Delivery.

PURPOSE: mRNA has recently emerged as a potent therapeutics and requires safe and effective delivery carriers, particularly prone to address its issues of poor stability and escape from endosomes. In this context, we designed poly(D,L-lactide) (PLA)-based micelles with N-succinimidyl (NS) ester decorated hydrophilic hairy corona to trap/couple a cationic fusogenic peptide and further complex mRNA. METHODS: Two strategies were investigated, namely (i) sequential immobilization of peptide and mRNA onto the micelles (layer-by-layer, LbL) or (ii) direct immobilization of peptide-mRNA pre-complex (PC) on the micelles. After characterization by means of size, surface charge, peptide/mRNA coupling/complexation and mRNA serum stability, carrier cytotoxicity and transfection capacity were evaluated with dendritic cells (DCs) using both GFP and luciferase mRNAs. RESULTS: Whatever the approach used, the micellar assemblies afforded full protection of mRNA in serum while the peptide-mRNA complex yielded complete mRNA degradation. In addition, the micellar assemblies allowed to significantly reduce the toxicity observed with the peptide-mRNA complex. They successfully transfected hard-to transfect DCs, with a superior efficiency for the LbL made ones (whatever mRNAs studied) showing the impact of the elaboration process on the carrier properties. CONCLUSIONS: These results show the relevance and potential of this new PLA/peptide based micelle platform to improve mRNA stability and delivery, while offering the possibility of further multifunctionality through PLA core encapsulation.

Tuning the Hydrophilic/Hydrophobic Balance to Control the Structure of Chitosan Films and Their Protein Release Behavior.

The control over the crystallinity of chitosan and chitosan/ovalbumin films can be achieved via an appropriate balance of the hydrophilic/hydrophobic interactions during the film formation process, which then controls the release kinetics of ovalbumin. Chitosan films were prepared by solvent casting. The presence of the anhydrous allomorph can be viewed as a probe of the hydrophobic conditions at the neutralization step. The semicrystalline structure, the swelling behavior of the films, the protein/chitosan interactions, and the release behavior of the films were impacted by the DA and the film processing parameters. At low DAs, the chitosan films neutralized in the solid state corresponded to the most hydrophobic environment, inducing the crystallization of the anhydrous allomorph with and without protein. The most hydrophilic conditions, leading to the hydrated allomorph, corresponded to non-neutralized films for the highest DAs. For the non-neutralized chitosan acetate (amorphous) films, the swelling increased when the DA decreased, whereas for the neutralized chitosan films, the swelling decreased. The in vitro release of ovalbumin (model protein) from chitosan films was controlled by their swelling behavior. For fast swelling films (DA = 45%), a burst effect was observed. On the contrary, a lag time was evidenced for DA = 2.5% with a limited release of the protein. Furthermore, by blending chitosans (DA = 2.5% and 45%), the release behavior was improved by reducing the burst effect and the lag time. The secondary structure of ovalbumin was partially maintained in the solid state, and the ovalbumin was released under its native form.

Zinc-Stabilized Chitosan-Chondroitin Sulfate Nanocomplexes for HIV-1 Infection Inhibition Application.

Polyelectrolyte complexes (PECs) constituted of chitosan and chondroitin sulfate (ChonS) were formed by the one-shot addition of default amounts of polyanion to an excess of polycation. Key variables of the formulation process (e.g., degree of depolymerization, charge mixing ratio, the concentration, and pH of polyelectrolyte solutions) were optimized based on the PECs sizes and polydispersities. The PECs maintained their colloidal stability at physiological salt concentration and pH thanks to the complexation of polyelectrolytes with zinc(II) ion during the nanoPECs formation process. The PECs were capable of encapsulating an antiretroviral drug tenofovir (TF) with a minimal alteration on the colloidal stability of the dispersion. Moreover, the particle interfaces could efficiently be functionalized with anti-OVA or anti-α4ß7 antibodies with conservation of the antibody biorecognition properties over 1 week of storage in PBS at 4 °C. In vitro cytotoxicity studies showed that zinc(II) stabilized chitosan-ChonS nanoPECs were noncytotoxic to human peripheral blood mononuclear cells (PBMCs), and in vitro antiviral activity test demonstrated that nanoparticles formulations led to a dose-dependent reduction of HIV-1 infection. Using nanoparticles as a drug carrier system decreases the IC50 (50% inhibitory concentration) from an aqueous TF of 4.35 µmol·L(-1) to 1.95 µmol·L(-1). Significantly, zinc ions in this system also exhibited a synergistic effect in the antiviral potency. These data suggest that chitosan-ChonS nanoPECs can be promising drug delivery system to improve the antiviral potency of drugs to the viral reservoirs for the treatment of HIV infection.

A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo.

Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models.

Preparation and in vitro evaluation of imiquimod loaded polylactide-based micelles as potential vaccine adjuvants.

PURPOSE: Activation of immune cells through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) or NOD-like receptors (NLRs), has been identified as a key issue in the development of new efficient vaccine adjuvants. We report here on the elaboration and immunostimulatory potential of polylactide (PLA)-based micelles core-loaded with imiquimod TLR7 ligand and able to be further surface-functionalized with antigenic protein (HIV-1 Gag p24) for antigen delivery purpose. METHODS: Micelles prepared from poly(D,L-lactide)-b-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) amphiphilic copolymer were incubated in the presence of imiquimod, leading to 1.2 wt% loading, and further conjugated to p24 antigen through reaction of p24 lysines and N-terminal amine with the N-succinimidyl pendant groups of the micelle corona. The impact of imiquimod encapsulation in the micelles on its immunostimulatory properties was investigated in vitro, by monitoring: (i) the NF-κB and mitogen-activated protein kinases (MAPK) pathways through experiments with RAW-Blue™ cells, a mouse macrophage cell line encoding an NF-κB/AP-1-inducible reporter construct; (ii) human dendritic cells (DCs) maturation markers by flow cytometry. RESULTS: RAW-Blue™ cells based experiments showed that imiquimod encapsulated in the micelles was much more efficient to activate the NF-κB and MAPK pathways than free imiquimod. Furthermore, encapsulated imiquimod was found to induce much higher maturation of DCs than the free analog. Finally, these immunostimulatory properties of the loaded imiquimod were shown to be conserved when the p24 antigen was coupled at the micelle surface. CONCLUSIONS: Taken together, these data regarding improved immunostimulatory efficiency suggest the strong potential of our micelle-based nano-system for vaccine delivery.

An innovative Fuller's earth-based film-forming formulation for skin decontamination, through removal and entrapment of an organophosphorus compound, paraoxon-ethyl.

Organophosphorus compounds (OPs), such as VX, pose a significant threat due to their neurotoxic and hazardous properties. Skin decontamination is essential to avoid irreversible effects. Fuller's earth (FE), a phyllosilicate conventionally employed in powder form, has demonstrated decontamination capacity against OPs. The aim of this study was to develop a formulation that forms a film on the skin, with a significant OP removal capacity (>95 %) coupled with sequestration capabilities, favorable drying time and mechanical properties to allow for easy application and removal, particularly in emergency context. Various formulations were prepared using different concentrations of polyvinyl alcohol (PVA), FE and surfactants. Their removal and sequestration capacity was tested using paraoxon-ethyl (POX), a chemical that simulates the behavior of VX. Formulations with removal capacity levels surpassing 95 % were mechanically characterized and cell viability assays were performed on Normal Human Dermal Fibroblast (NHDF). The four most promising formulations were used to assess decontamination efficacy on pig ear skin explants. These formulations showed decontamination levels ranging from 84.4 ± 4.7 % to 96.5 ± 1.3 %, which is equivalent to current decontamination methods. These results suggest that this technology could be a novel and effective tool for skin decontamination following exposure to OPs.

Sublingual protein delivery by a mucoadhesive patch made of natural polymers.

The sublingual mucosa is an appealing route for drug administration. However, in the context of increased use of therapeutic proteins, development of protein delivery systems that will protect the protein bioactivity is needed. As proteins are fragile and complex molecules, current sublingual formulations of proteins are in liquid dosage. Yet, protein dilution and short residence time at the sublingual mucosa are the main barriers for the control of the dose that is delivered. In this work, a simple delivery scaffold based on the assembly of two polysaccharides, chitosan and hyaluronic acid, is presented. The natural polymers were assembled by the Layer-by-Layer methodology to produce a mucoadhesive and oro-dispersible freestanding membrane, shown to be innocuous for epithelial human cells. The functionalization of the membrane with proteins led to the production of a bioactive patch with efficient loading and release of proteins, and suitable mechanical properties for manipulation. Sublingual administration of the patch in mouse evidenced the absence of inflammation and an extended time of contact between the model protein ovalbumin and the mucosa compared to liquid formulation. The delivery of fluorescent ovalbumin in mouse sublingual mucosa demonstrated the penetration of the protein in the epithelium 10 min after the patch administration. Moreover, a migration assay with a chemokine incorporated into the patch showed no decrease in bioactivity of the loaded protein after enzymatic release. This study therefore provides a promising strategy to develop a sublingual protein delivery system. STATEMENT OF SIGNIFICANCE: Although the oral route is largely used for drug delivery, it has limitations for the delivery of proteins that can be degraded by pH or gastric enzymes. The sublingual route therefore appears as an interesting approach for protein administration. In this work, a simple delivery scaffold is presented based on the assembly of two polysaccharides by the Layer-by-Layer methodology to produce a mucoadhesive patch. The produced patch allowed efficient loading and release of proteins, as well as protection of their bioactivity. An extended time of contact between the protein and the mucosa compared to liquid formulation was highlighted in mouse model. This study provides a promising strategy to develop a sublingual protein delivery system.

Biodistribution of surfactant-free poly(lactic-acid) nanoparticles and uptake by endothelial cells and phagocytes in zebrafish: Evidence for endothelium to macrophage transfer.

In the development of therapeutic nanoparticles (NP), there is a large gap between in vitro testing and in vivo experimentation. Despite its prominence as a model, the mouse shows severe limitations for imaging NP and the cells with which they interact. Recently, the transparent zebrafish larva, which is well suited for high-resolution live-imaging, has emerged as a powerful alternative model to investigate the in vivo behavior of NP. Poly(D,L lactic acid) (PLA) is widely accepted as a safe polymer to prepare therapeutic NP. However, to prevent aggregation, many NP require surfactants, which may have undesirable biological effects. Here, we evaluate 'safe-by-design', surfactant-free PLA-NP that were injected intravenously into zebrafish larvae. Interaction of fluorescent NPs with different cell types labelled in reporter animals could be followed in real-time at high resolution; furthermore, by encapsulating colloidal gold into the matrix of PLA-NP we could follow their fate in more detail by electron microscopy, from uptake to degradation. The rapid clearance of fluorescent PLA-NP from the circulation coincided with internalization by endothelial cells lining the whole vasculature and macrophages. After 30 min, when no NP remained in circulation, we observed that macrophages continued to internalize significant amounts of NP. More detailed video-imaging revealed a new mechanism of NP transfer where NP are transmitted along with parts of the cytoplasm from endothelial cells to macrophages.

Investigation of polylactic acid (PLA) nanoparticles as drug delivery systems for local dermatotherapy.

PURPOSE: The development of particle-based carriers for transepidermal drug delivery has become a field of major interest in dermatology. In this study, we investigated the suitability of biodegradable poly-lactic acid (PLA) particles loaded with fluorescent dyes as carriers for transepidermal drug delivery. METHODS: The penetration profiles of PLA particles (228 and 365 nm) and the release of dye from the particles were investigated in human skin explants using fluorescence microscopy, confocal laser scanning microscopy and flow cytometry. RESULTS: PLA particles penetrated into 50% of the vellus hair follicles, reaching a maximal depth corresponding to the entry of the sebaceous gland in 12-15% of all observed follicles. The accumulation of particles in the follicular ducts was accompanied by the release of dye to the viable epidermis and its retention in the sebaceous glands for up to 24 h. Kinetic studies in vitro as well as in skin explants revealed, that, although stable in aqueous solution, destabilization of the particles and significant release of incorporated dye occurred upon contact with organic solvents and the skin surface. CONCLUSIONS: These results suggest that particles based on PLA polymers may be ideal carriers for hair follicle and sebaceous gland targeting.

Self-assembled amphiphilic copolymers as dual delivery system for immunotherapy.

Subunit vaccines using recombinant antigens appear as the privileged vaccination technology for safety reasons but still require the development of carriers/adjuvants ensuring optimal immunogenicity and efficacy. Micelles from self-assembled amphiphilic copolymers have recently emerged as highly relevant and promising candidates owing to their ease of preparation, low size (entering in lymphatic capillaries for reaching lymph nodes), size/surface tunability and chemical versatility enabling introduction of stimuli (e.g. pH) responsive features and biofunctionalization with dedicated molecules. In particular, research efforts have increasingly focused on dendritic cells (DCs) targeting and activation by co-delivering (with antigen) ligands of pattern recognition receptors (PRRs, e.g. toll-like receptors). Such strategy has appeared as one of the most effective for eliciting CD 8+ T-cell response, which is crucial in the eradication of tumors and numerous infectious diseases. In this short review, we highlight the recent advances in such micelle-based carriers in subunit vaccination and how their precise engineering can be a strong asset for guiding and controlling immune responses.

Molecular modelling of TLR agonist Pam3CSK4 entrapment in PLA nanoparticles as a tool to explain loading efficiency and functionality.

Designing potent and safe-of-use therapies against cancers and infections remains challenging despite the emergence of novel molecule classes like checkpoint inhibitors or Toll-Like-Receptor ligands. The latest therapeutic perspectives under development for immune modulator administration exploits vectorization, and biodegradable delivery systems are one of the most promising vehicles. Nanoparticles based on Poly (D,L) Lactic Acid (PLA) as polymer for formulation are widely investigated due to its bioresorbable, biocompatible and low immunogen properties. We propose a PLA-based nanoparticle delivery system to vectorize Pam3CSK4, a lipopeptide TLR1/2 ligand and a potent activator of the proinflammatory transcription factor NF-κB that shows a self-assembling behavior from 30 µg/mL onwards. We demonstrate successful encapsulation of Pam3CSK4 in PLA nanoparticles by nanoprecipitation in a 40-180 µg/mL concentration range, with 99% of entrapment efficiency. By molecular modelling, we characterize drug/carrier interactions and conclude that Pam3CSK4 forms clusters onto the nanoparticle and is not encapsulated into the hydrophobic core. In silico predictions provide nanoprecipitation optimization and the mechanistic understanding of the particle dynamics. The loaded-Pam3CSK4 maintains bioactivity on TLR2, confirmed by in vitro experiments using reporter cell line HEK-Blue hTLR2. Our presented data and results are convincing evidence that Pam3CSK4-loaded in PLA nanoparticles represent a promising immune modulating system.

Poly(lactic acid) nanoparticles and cell-penetrating peptide potentiate mRNA-based vaccine expression in dendritic cells triggering their activation.

Messenger RNA-based vaccines have the potential to trigger robust cytotoxic immune responses, which are essential for fighting cancer and infectious diseases like HIV. Dendritic Cells (DCs) are choice targets for mRNA-based vaccine strategies, as they link innate and adaptive immune responses and are major regulators of cytotoxic and humoral adaptive responses. However, efficient delivery of antigen-coding mRNAs into DC cytosol has been highly challenging. In this study, we developed an alternative to lipid-based mRNA delivery systems, using poly(lactic acid) nanoparticles (PLA-NPs) and cationic cell-penetrating peptides as mRNA condensing agent. The formulations are assembled in two steps: (1) formation of a polyplex between mRNAs and amphipathic cationic peptides (RALA, LAH4 or LAH4-L1), and (2) adsorption of polyplexes onto PLA-NPs. LAH4-L1/mRNA polyplexes and PLA-NP/LAH4-L1/mRNA nanocomplexes are taken up by DCs via phagocytosis and clathrin-dependent endocytosis, and induce strong protein expression in DCs in vitro. They modulate DC innate immune response by activating both endosome and cytosolic Pattern Recognition Receptors (PRRs), and induce markers of adaptive responses in primary human DCs in vitro, with prevalent Th1 signature. Thus, LAH4-L1/mRNA and PLA-NP/LAH4-L1/mRNA represent a promising platform for ex vivo treatment and mRNA vaccine development.

Recombinant Haemagglutinin Derived From the Ciliated Protozoan Tetrahymena thermophila Is Protective Against Influenza Infection.

Current influenza vaccines manufactured using eggs have considerable limitations, both in terms of scale up production and the potential impact passaging through eggs can have on the antigenicity of the vaccine virus strains. Alternative methods of manufacture are required, particularly in the context of an emerging pandemic strain. Here we explore the production of recombinant influenza haemagglutinin using the ciliated protozoan Tetrahymena thermophila. For the first time we were able to produce haemagglutinin from both seasonal influenza A and B strains. This ciliate derived material was immunogenic, inducing an antibody response in both mice and non-human primates. Mice immunized with ciliate derived haemagglutinin were protected against challenge with homologous influenza A or B viruses. The antigen could also be combined with submicron particles containing a Nod2 ligand, significantly boosting the immune response and reducing the dose of antigen required. Thus, we show that Tetrahymena can be used as a manufacturing platform for viral vaccine antigens.

Towards biocompatible vaccine delivery systems: interactions of colloidal PECs based on polysaccharides with HIV-1 p24 antigen.

This work reports on the interactions of a model protein (p24, the capside protein of HIV-1 virus) with colloids obtained from polyelectrolyte complexes (PECs) involving two polysaccharides: chitosan and dextran sulfate (DS). The PECs were elaborated by a one-shot addition of default amounts of one counterpart to the polymer in excess. Depending on the nature of the excess polyelectrolyte, the submicrometric colloid was either positively or negatively charged. HIV-1 capsid p24 protein was chosen as antigen, the ultrapure form, lipopolysaccharide-free (endotoxin-, vaccine grade) was used in most experiments, as the level of purity of the protein had a great impact on the immobilization process. p24 sorption kinetics, isotherms, and loading capacities were investigated for positively and negatively charged particles of chitosans and dextran sulfates differing in degrees of polymerization (DP) or acetylation (DA). Compared with the positive particles, negatively charged colloids had higher binding capacities, faster kinetics, and a better stability of the adsorbed p24. Capacities up to 600 mg x g(-1) (protein-colloid) were obtained, suggesting that the protein interacted within the shell of the particles. Small-angle X-rays scattering experiments confirmed this hypothesis. Finally, the immunogenicity of the p24-covered particles was assessed for vaccine purposes in mice. The antibody titers obtained with immobilized p24 was dose dependent and in the same range as for Freund's adjuvant, a gold standard for humoral responses.

Antibiotic incorporation in jet-sprayed nanofibrillar biodegradable scaffolds for wound healing.

In view of preparing antibiotic-loaded structures that can be used as dressing to prevent or contain wound infections, this study evaluates biodegradable nanofibrillar matrices obtained by jet-spraying and containing ciprofloxacin (CIF). The matrices were prepared from different blends of poly-(ε-caprolactone) (PCL) and poly-d,l-(lactic acid) (PDLLA) in view of controlling mechanical properties, biodegradation and antibiotic release rate. The effect of CIF incorporation was assessed in regard of matrices fiber diameter, mechanical properties and degradation while antibiotic release from the polymer blends of different PCL/PDLLA ratios was measured in buffers of different pH to better mimic the wound context. Finally, antibiotic activity of the nanofibrillar matrices and their ability to be colonized by skin cells were evaluated. Non-woven nanofibrillar matrices could be obtained from various polymer blends by jet-spraying and CIF crystals incorporation was easily obtained. The crystals were dispersed in the fibers, without complete embedding. Antibiotic incorporation resulted in a slight increase of fiber diameter and did not modified the mechanical properties of the various matrices composed of different polymer blends. Unlike fiber diameter, degradation and mechanical properties of the fibrillar matrices, CIF release profiles were not controlled by the polymer blend ratios. However, sustained release was observed over more than 23days. Due to the antibiotic pH-dependent solubility, burst release was more prominent in acidic conditions, which mimic the pH of undamaged skin. Finally the incorporated antibiotic was efficient in inhibiting bacterial growth of E. coli and B. subtilis whereas human fibroblasts were able to colonize the CIF-loaded matrices.

Coadsorption of HIV-1 p24 and gp120 proteins to surfactant-free anionic PLA nanoparticles preserves antigenicity and immunogenicity.

Biodegradable micro- or nanoparticles with surface adsorbed antigens represent a promising method for in vivo delivery of vaccines. Most vaccines, licensed or under development, are based on combined delivery of multiple antigens. Thus, we investigated the feasibility of combining two vaccine antigens, HIV-1 p24 and gp120 proteins, on the surface of surfactant-free anionic PLA nanoparticles obtained by an improved solvent diffusion method. The analysis of adsorption isotherms has shown that both proteins had similar and high affinities for the nanoparticles. Coadsorption of p24 and gp120 onto the same PLA particle was evidenced by sandwich ELISA, using antibodies directed against one protein for particle capture and the other one for detection. To assess structural integrity, the antigenicity of free and PLA-adsorbed antigens was compared by competition ELISA, using a set of 6 anti-p24 and 7 anti-gp120 antibodies, as well as soluble CD4. The antigenicity of proteins on the nanoparticle surface was well preserved, adsorbed either individually or in combination. Furthermore, both antigens maintained their immunogenicity, since high antibody titres (10(6) for p24 and 10(5) for gp120) were elicited in mice with monovalent and divalent PLA formulations. Taken together our results show that development of multivalent vaccines based on anionic PLA nanoparticles is possible. Moreover, coadsorption of a ligand for cell-specific targeting or of an immunostimulatory molecule will further extend the field of application of delivery systems based on charged micro- and nanoparticles.

Surfactant-free anionic PLA nanoparticles coated with HIV-1 p24 protein induced enhanced cellular and humoral immune responses in various animal models.

Microparticles and nanoparticles prepared with poly(D,L-lactide-co-glycolide) (PLGA) or poly(D,L-lactide) (PLA) polymers represent a promising method for in vivo delivery of encapsulated peptide, protein or DNA antigens. However, one major issue that limits the potential of these delivery systems is the instability or the degradation of the entrapped antigen. Charged microparticles carrying surface adsorbed antigen were developed to resolve this problem and appear more suitable for vaccine applications. We describe here new anionic PLA nanoparticles obtained by the dialysis method that are absolutely surfactant-free, which makes them more appropriate for use in humans. The potency of this delivery system as a vaccine carrier was tested in various animal models using HIV-1 p24 protein. p24-coated PLA nanoparticles (p24/PLA) induced high antibody titres (>10(6)) in mice, rabbits and macaques. Moreover, p24/PLA nanoparticles elicited strong CTL responses and a Th1-biased cytokine release (IFNgamma, IL-2) in mice. p24 protein seemed to generate a more Th1-oriented response when administered coated onto the surface of PLA nanoparticles than adjuvanted with Freund's adjuvant. Most importantly, the ability of p24/PLA particles to induce Th1 responses was also confirmed in the macaque model, since high levels of IFNgamma-producing CD4+ T cells and CD8+ T cells could be detected by the ELISPOT assay. This protein delivery system confirms the potential of charged nanoparticles in the field of vaccine development.