Molecular design of nanoparticle-based delivery vehicles for pneumonic plague
The work described in this dissertation focuses on the design of polyanhydride nanoparticles that function as both adjuvants and long-term antigen delivery vehicles in order to improve vaccination, specifically for biodefense-related applications. Chapter 1 is an introduction into the threat of bioterrorism, polymer-based controlled delivery systems and challenges associated with vaccine design. Chapter 2 is a detailed literature review of topics related to the research conducted in this dissertation. Areas covered include basic immunology, vaccine design, degradable polymer-based adjuvant engineering, plague (Yersinia pestis) biology, and vaccines that confer protection against plague. Chapter 3 overviews the research objectives and the specific aims of this work. Chapter 4 describes the effect polymer chemistry has on uptake of polyanhydride nanoparticles by THP-1 human monocytic cells. Nanoparticles of similar size, regardless of poly[1,6-bis(p-carboxyphenoxy)hexane-co-sebacic acid] (CPH:SA) copolymer chemistry, were fabricated using a novel anti-solvent precipitation technique. Confocal microscopy revealed that less hydrophobic nanoparticles (SA-rich) were more readily internalized and trafficked by monocytes. Interestingly, exposure to nanoparticles of any chemistry enhanced soluble protein uptake by monocytes over cells exposed to soluble protein alone. Chapter 5 utilizes the combination of population and individual analyses in order to better understand the effect chemistry has on nanoparticle uptake and subsequent activation of dendritic cells. Nanoparticles composed of CPH:SA and poly[1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane-co-CPH] (CPTEG:CPH) were used for this study in order to investigate a wide range of chemistries. Nanoparticles composed of less hydrophobic chemistry were able to activate cell surface marker expression where as nanoparticles composed of more hydrophobic chemistry were able to cause the enhanced secretion of cytokines. Using confocal microscopy it was determined that less hydrophobic nanoparticles were more readily internalized and degraded where as more hydrophobic nanoparticles maintained their size intracellularly. 50:50 CPTEG:CPH nanoparticles possessed characteristics of both less hydrophobic and more hydrophobic chemistries. Also, 50:50 CPTEG:CPH nanoparticles were intracellularly aggregated in vesicles which is similar to how dendritic cells treat bacteria. This pathogen-like behavior may explain the activation capacity of nanoparticles of this chemistry. Chapter 6 describes the capacity of a single-dose, antigen-loaded 50:50 CPTEG:CPH nanoparticle-based vaccine to convey long-lived protection against live Yersinia pestis challenge. While the combination of a commercial adjuvant (MPLA) or unloaded 50:50 nanoparticles with soluble antigen was able to convey some protection at 6 weeks post-vaccination, only a combination of soluble antigen with antigen-loaded 50:50 CPTEG:CPH nanoparticles was able to convey 100% protection at 6 and 23 weeks post-vaccination. For mice vaccinated with soluble antigen plus antigen-loaded nanoparticles, bacteria burden and histopathological analyses showed no presence of bacteria and no pathological damage, respectively. Chapter 7 details the conclusions of this dissertation and the future directions of this research. Antigen modification, nanoparticle optimization, novel polymer chemistry and new intracellular imaging tools are all topics covered in this chapter.
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