Date of Graduation

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor/Mentor

Zaharoff, David A.

Committee Member

Erf, Gisela F.

Second Committee Member

Beitle, Robert R. Jr.

Third Committee Member

Wolchok, Jeffrey C.

Keywords

Applied sciences; Antigen delivery systems; Chitosan; Immunotherapy; Vaccine delivery

Abstract

Particle-based vaccine delivery systems are under exploration to enhance antigen-specific immunity against safe but poorly immunogenic polypeptide antigens. Chitosan is a promising biomaterial for antigen encapsulation and delivery due to its ability to form nano- and microparticles in mild aqueous conditions thus preserving the antigenicity of loaded polypeptides. The objective of this work is to develop a chitosan particle based antigen delivery system for enhanced vaccine response. Chitosan particle sizes, which ranged from 300 nm to 3 ìm, were influenced by chitosan concentration, chitosan molecular weight and addition rate of precipitant salt. The composition of precipitant salt played a significant role in particle formation with upper Hofmeister series salts containing strongly hydrated anions yielding particles with a low polydispersity index (PDI) while weaker anions resulted in aggregated particles with high PDIs. Sonication power had minimal effect on mean particle size, however, it significantly reduced polydispersity. Protein loading efficiencies in chitosan nano/microparticles, which ranged from 14.3% to 99.2%, was inversely related to the hydration strength of precipitant salts, and protein molecular weight and directly related to the concentration and molecular weight of chitosan. Protein release rates increased with particle size and were generally inversely related to protein molecular weight. In vitro studies showed that the uptake of antigen loaded chitosan particles (AgCPs) by dendritic cells and macrophages was found to be dependent on particle size, antigen concentration and exposure time. Flow cytometry analysis revealed that compared to soluble antigen, uptake of AgCPs enhanced upregulation of surface activation markers on APCs and increased the release of pro-inflammatory cytokines. Lastly, antigen-specific T cells exhibited higher proliferative responses when stimulated with APCs activated with AgCPs versus soluble antigen. These data suggest that encapsulation of antigens in chitosan particles enhances uptake, activation and presentation by APCs with 1 µm mean particle size being optimal. Similarly, in vivo studies showed that immunizing mice with AgCPs enhanced both humoral and cell mediated immune response. Compared to PLGA nanoparticle and standard alum adjuvants, AgCPs induced more potent humoral immune responses as evidenced by the high total antigen specific IgG titer.

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