Strategy of oral vaccination by polymeric nanoparticles : M cell targeting or bioadhesion

Oral vaccines delivery is an attractive alternative to parenteral route for vaccination but is limited by destruction and low absorption of the antigen in the gastro-intestinal tract. Development of new formulations encapsulating antigens in polymeric biodegradable nanoparticles is a promising strategy to counter these drawbacks. The objective of this thesis was to compare two delivery strategies: M cell targeted or bioadhesive nanoparticles. The first strategy consisted in encapsulating antigens in nanoparticles targeting M cells by presenting specific M cell ligands at nanoparticle surface. M cells, localised in the follicle associated epithelium on the Peyer’s Patches, are part of the mucosal immune system and specialized in antigen uptake. The core of the nanoparticles was constituted of PLGA, and PLGA-PEG and PCL-PEG were included in the formulation to stabilize it by sterical repulsion and to graft on PEG residue a ligand targeting M cells. As β1 integrins are over-expressed at the apical pole of human M cells, we studied the potential of some ligands of the integrin family to increase nanoparticle transport across the epithelium. The second strategy was to encapsulate the antigen in bioadhesive polymers, like chitosan and trimethylchitosan, to improve the intestinal uptake of antigens by increasing adsorption of the nanoparticles on mucus and intestinal cells. Nanoparticles were formed by ionic gelation between cationic polymers and anionic tripolyphosphate. An in vitro model of the human follicle associated epithelium was developed and used to compare the transport of targeting and bioadhesive nanoparticles. The potential of the different antigen-loaded nanoparticles to induce immune responses was also tested through intra-duodenal immunization in vivo. Bioadhesive chitosan nanoparticles appeared to be more transported through the epithelium than PLGA nanoparticles, with or without targeting ligands, but were not M cells specific. PLGA nanoparticles exposing a RGD peptidomimetic molecule as ligand significantly increased the transport through M-like cells in comparison to normal enterocytes-like cells. Oral immunization induced higher immune response with PLGA nanoparticles targeting M cells β integrins with RGD peptidomimetic. However, presence of chitosan could influence immune response type and played a role of immunopotentiator by enhancing Th1 cytokines secretion. Depending on the type of immune response that needs to be elicited, oral vaccine by PLGA based nanoparticles targeting M cells could be more useful to induce an humoral response whereas chitosan might be more useful to stimulate a cellular immune response oriented Th1. Then a combination of different strategies could be an asset for oral vaccine delivery and it seems necessary to add some mucosal adjuvants and immunopotentiators like ligands targeting the antigen presenting cells to boost the immune responses.(FARM 3) -- UCL, 201

Fate of polymeric nanocarriers for oral drug delivery

This review will focus on two polymeric nanocarriers: nanoparticles and micelles that have been studied for oral drug delivery at preclinical level. Their potential for oral drug delivery will first be illustrated. Then their mechanisms of uptake and their fate after oral delivery will be discussed. Future directions for oral delivery with nanocarriers will be analyzed with a special emphasis on optimal properties. The recent advances highlight the need to tune and to control their design with a good balance in their physicochemical properties and suggest that more sophisticated nanosystems will be developed for the oral delivery of drugs, biopharmaceuticals and vaccines, thanks to (i) the development of biocompatible polymers with tailored properties for oral drug delivery and formulation of nanocarriers, (ii) the understanding of cellular uptake mechanisms of polymeric nanocarriers, (iii) the novel techniques to study the fate of nanocarriers, polymers and drugs in the body and (iv) the identification of new ligands for targeted oral delivery. Major recent advances Recent advances in the (i) development of biocompatible polymers with tailored properties for oral drug delivery and nanocarrier formulation, (ii) the understanding of cellular uptake mechanisms of polymeric nanocarriers (iii) the new techniques to study fate of nanocarriers, polymers and drugs in the body and (iv) the identification of new ligands for targeted oral delivery have promoted the development of novel polymeric carriers for the oral delivery of drugs, biopharmaceuticals and vaccines

Bioadhesive nanoparticles of fungal chitosan for oral DNA delivery

Chitosan is an ideal candidate for oral DNA delivery due to its mucoadhesive properties. Chitosan (CS) produced under GMP conditions from fungal source was used to encapsulate a plasmid DNA coding for a reporter gene. Nanoparticles made by complex coacervation of CS and DNA had a size around 200nm, a positive zeta potential, a high association of DNA and protected the plasmid against nuclease degradation. Their transfection ability was assessed in differentiated intestinal Caco-2 cells. A N/P ratio of 4 and a DNA concentration of 8mug/ml were the optimal condtions leading to a transfection efficiency similar to the one reached with PEI-DNA complexes without cytotoxicity. M cells in monolayers influenced DNA uptake up to 8mug of DNA/ml when complexed with CS. Fungal trimethylchitosan was also tested but the complexes interactions were too strong to induce transfection in vitro. Confocal microscopy studies showed that CS/DNA and PEI/DNA nanoparticles were found at the apical surface of cell monolayers and DNA was co-localized within the nucleus. Quantification seemed to show that more DNA was associated with the cells when incubated with CS nanoparticles and that the presence of M cells slightly influenced DNA uptake when complexed with CS. In conclusion, we developed a new nanocarrier made of fungal CS promising for oral gene delivery and oral DNA vaccination

Drug delivery to inflamed colon by nanoparticles: comparison of different strategies

For inflammatory bowel disease (IBD) treatment, local delivery of molecules loaded in nanoparticles to the inflamed colon could be a promising strategy. The aim of this study was to investigate how drug-loaded polymeric nanoparticles target the site of inflammation and to analyse the influence of different colon-specific delivery strategies. Three different polymeric nanoparticles were formulated using ovalbumin (OVA) as a model drug. pH-sensitive nanoparticles were made with Eudragit(®) S100. Mucoadhesive nanoparticles were created with trimethylchitosan (TMC). A mix of polymers, PLGA, PEG-PLGA and PEG-PCL, were used to obtain a sustained drug delivery. Furthermore, ligands targeting immune cells (i.e. mannose) or the inflamed colon (i.e. a specific peptide) were grafted on the PEG chain of PCL. Interaction of nanoparticles with the intestinal epithelium was explored using Caco-2 monolayers designed to mimic an inflamed epithelium and then visualized using confocal laser microscopy. TMC nanoparticles had the highest apparent permeability for OVA in the untreated model. However, in the inflamed model, there were no difference between TMC, PLGA-based and Eudragit(®) nanoparticles. The uptake of nanoparticles in the inflamed mouse colon was assessed in a horizontal diffusion chamber. Mannose-grafted PLGA nanoparticles showed the highest accumulation of OVA in inflamed colon. Based on these results, active targeting of macrophages and dendritic cells may be a promising approach for targeting the colon in IBD

Mechanistic study of the adjuvant effect of biodegradable nanoparticles in mucosal vaccination

For oral vaccination, incorporation of antigens into nanoparticles has been shown to protect the antigen from degradation, but may also increase its uptake through the intestinal epithelium via M-cells. The aim of this study was to understand the mechanisms by which oral administration of antigen-loaded nanoparticles induces an immune response and to analyze the effect of the nanoparticle composition on these mechanisms. Nanoparticles made from chitosan (CS) and its N-trimethylated derivative, TMC, loaded with a model antigen ovalbumin (OVA) were prepared by ionic gelation with tripolyphosphate. Intraduodenal vaccination with OVA-loaded nanoparticles led to significantly higher antibody responses than immunization with OVA alone. TMC nanoparticles induced anti-OVA antibodies after only a priming dose. To explain these results, the interaction of nanoparticles with the intestinal epithelium was explored, in vitro, using a follicle associated epithelium model and visualized, ex vivo, using confocal laser scanning microscopy. The transport of FITC-OVA-loaded TMC nanoparticles by Caco-2 cells or follicle associated epithelium model was higher than FITC-OVA-loaded CS or PLGA nanoparticles. The association of nanoparticles with human monocyte derived dendritic cells and their effect on their maturation were determined with flow cytometry. TMC nanoparticles but not CS or PLGA nanoparticles had intrinsic adjuvant effect on DCs. In conclusion, depending on their composition, nanoparticles can increase the M-cell dependent uptake and enhance the association of the antigen with DC. In this respect, TMC nanoparticles are a promising strategy for oral vaccination

PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination

To improve the efficiency of orally delivered vaccines, PEGylated PLGA-based nanoparticles displaying RGD molecules at their surface were designed to target human M cells. RGD grafting was performed by an original method called "photografting" which covalently linked RGD peptides mainly on the PEG moiety of the PCL-PEG, included in the formulation. First, three non-targeted formulations with size and zeta potential adapted to M cell uptake and stable in gastro-intestinal fluids, were developed. Their transport by an in vitro model of the human Follicle associated epithelium (co-cultures) was largely increased as compared to mono-cultures (Caco-2 cells). RGD-labelling of nanoparticles significantly increased their transport by co-cultures. due to interactions between the RGD ligand and the I intregrins detected at the apical surface of co-cultures. In vivo studies demonstrated that RGD-labelled nanoparticles particularly concentrated in M cells. Finally, ovalbumin-loaded nanoparticles were orally administrated to mice and induced an IgG response, attesting antigen ability to elicit an immune response after oral delivery

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination

The presence of RGD on nanoparticles allows the targeting of β1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL–PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization