Biodegradable microparticles and nanoparticles on oral vaccine delivery: Investigation of critical determinants.

Abstract

Orally administered antigens are known to induce mucosal and systemic immune responses. Biodegradable nanoparticles/microparticles have been investigated as carrier systems for antigens in oral immunization. Optimization of various factors influencing uptake of particles by the gut-associated lymphoid tissue (GALT) is thus important in achieving an optimal immune response. The objective of this study was to investigate key determinants in the mechanism of nanoparticle facilitated immunization. In this study biodegradable microparticles of graded diameters, 100 nm, 500 nm, 1 $\mu$m and 10 $\mu$m were formulated using polylactic polyglycolic acid copolymer (PLGA, 50:50) by a water-in-oil-in-water emulsification solvent evaporation technique. Bovine serum albumin was used as a model antigen in the preliminary studies. Microparticles were characterized for protein loading, size distribution, degradation characteristics, and in vitro protein release kinetics. The effects of particle size and other factors affecting uptake were investigated in vitro using Caco-2 cell monolayer, and in situ using rat intestinal loop model. A time release capsule (PORT System$\sp{\rm TM})$ was formulated to deliver antigen containing nanoparticles to the intestine, protecting it from gastric pH and enzymes. Immunization studies were done in rabbits using staphylococcal enterotoxin B (SEB) toxoid by an oral (capsule) and subcutaneous (SC) routes of immunization. The results of these studies demonstrated that 100 nm diameter particles are taken up more efficiently by the GALT compared to larger size microparticles. In addition, the Peyer's patch, a lymphoidal rich region of the small intestinal submucosa had comparatively higher uptake of nanoparticles than the nonlymphoidal intestinal tissue. SEB-toxoid incorporated into microparticles retained it's antigenicity as determined by ELISA assay and demonstrated sustained release over 60 days. The immune response to SEB-toxoid nanoparticles was almost comparable to alum-SEB conjugate by SC immunization. Oral immunization with SEB nanoparticles enclosed in an enteric capsule demonstrated elevated antibody titers following a SC alum-SEB booster, indicating that animals were sensitized to the antigen. Thus the overall results support the hypothesis that biodegradable nanoparticles could serve as an effective carrier system for oral immunization.