Chitosan-mediated vaccine delivery through the intestinal epithelium

Chitosan is one of the most widely studied materials in the field of drug delivery. Chitosan nanoparticles produced by ionic gelation can encapsulate macromolecular drug cargo. This work explores the potential of a specific chitosan molecule, ultrapure chitosan chloride, for oral vaccine delivery in vitro using polarised Caco-2 epithelial cells and in vivo using Balb/C mice. The chosen antigen used in the chitosan nanoparticles was ovalbumin (OVA). Work revealed an optimum concentration of 1 mg/ml of chitosan and OVA (i.e. 1:1 mass ratio), with addition of tripolyphosphate (TPP), for production of nanoparticles with a size of 196.5 nm. The chitosan:OVA nanoparticles showed a zeta potential of +9.68 mv. SDS gel studies showed that OVA stability was not compromised following interaction with chitosan. Nanoparticle exposure to hydrochloric acid (HCl) and trypsin resulted in breakdown of OVA with HCl, while some protection was apparent with trypsin. Chitosan:OVA nanoparticles at 1 mg/ml displayed a good toxicity profile, measured through 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS) and lactate dehydrogenase (LDH) assays. Transepithelial electrical resistance (TEER) data also support these findings, with TEER reversibility following application apparent. Application of chitosan:OVA nanoparticles to Caco-2 cell monolayers showed a reversible decrease in TEER, but a similar effect was seen with OVA solution and therefore a nanoparticle-specific effect could not be established in terms of tight junction opening. Chitosan:OVA nanoparticles however significantly and notably enhanced fluorescein isothiocyanate ovalbumin (FITC-OVA) permeability across Caco-2 monolayers when applied at 0.1 mg/ml (1:1 chitosan:OVA mass ratio). Studies which used Balb/c mice whereby chitosan:OVA nanoparticles were administered orally, did not show a convincing immune response. Specifically, IgG1, IgG2a and IgA showed no response, while total IgG showed a small response with and without cholera toxin. This work therefore shows that a specific chitosan molecule is able to complex with OVA as a model antigen, producing sub 200 nm nanoparticles with a positive zeta potential. These systems showed clear effects in vitro, enhancing OVA permeability significantly, but in vivo response was not clear. This study illustrates that macromolecular absorption enhancement seen in vitro in intestinal models does not reliably predict in vivo immune response of vaccine delivery systems.