Modification and reformulation of chitosan-DNA nanoparticles for vaginal DNA vaccination

Gene therapy applications have moved away from replacement or augmentation therapy to the treatment of acquired diseases in recent years. This shift in focus allows for a wider range of vectors since integration and permanent expression of genes are not necessarily required. Non-viral vectors for gene delivery offer biological safety and versatility that most viral vectors cannot afford. Chitosan has previously shown promise as a non-viral gene carrier, particularly in mucosal vaccination strategies. The goal of this work was to evaluate the potential of this gene carrier in vaginal DNA vaccination.; Chitosans over a range of degrees of deacetylation and molecular weights were synthesized by two different reactions: via heterogeneous acetylation of highly deacetylated chitosan and by homogeneous deacetylation of chitin. The synthesized chitosans were evaluated for the ability to effectively complex with DNA. After characterization of the size, zeta potential, and DNAse protection ability of the chitosan-DNA nanoparticles, high molecular weight (390 kDa) chitosan with degrees of deacetylation 90, 70 and 62% and +/- ratios 3.3, 5.0 and 9.0, respectively, were chosen for subsequent experimentation. While nanoparticle formulations formed by lower deacetylation chitosan failed to produce higher levels of gene expression in vitro as previously seen with 90% DA chitosan nanoparticles, gene expression levels in vivo were found to be significantly higher.; Since modifications in chitosan deacetylation alter the physical properties of the polymer itself; cellular attachment and trafficking experiments were performed to evaluate these changes, with both plain and transferrin-conjugated nanoparticles. FACScan experiments showed a decrease in cellular attachment as the degree of deacetylation of chitosan used to form the nanoparticles decreased. Confocal microscopy experiments revealed that, at later time points, nanoparticles of 70 and 62% deacetylation were more frequently co-localized to acidic and transferrin-containing vesicles versus nanoparticles formed from 90% deacetylated chitosan. Although transferrin conjugation imparted specificity to the nanoparticles, as observed via confocal microscopy, it did not necessarily translate to increased cellular attachment or gene expression. Inclusion of an endosomalytic polymer, PPAA, to nanoparticles formed with 90% deacetylated chitosan increased gene expression levels and resulted in increased intracellular release of DNA from the nanoparticles.; Biodistribution and cellular morphology were evaluated after instillation of chitosan-DNA nanoparticles into the vagina of mice. Nanoparticles formed with 90% DA chitosan were found to attach to the vaginal wall surface and cause disruption in intercellular tight junctions more readily than nanoparticles formed with 70 or 62% DA chitosan. Nanoparticles composed of chitosan with lower degrees of deacetylation more readily dissociated in the vaginal environment, while 90% DA chitosan nanoparticles remained intact and were transported away from the instillation site. The differences in the uptake and transport properties translated to differences in the gene expression in vivo . When these nanoparticles were evaluated for efficacy in eliciting immune response to HIV-gag1, however, no significant response occurred.; Although the immunization application did not produce a positive result, a firm groundwork for continued study has been laid. Initial gene expression results, as well as vaginal biodistribution and transport studies point to the possibility of the using these nanoparticle formulations in future application to the vaginal mucosal route.