Soluble and insoluble polymer delivery systems for cellular targeting and remote drug delivery

Abstract

This dissertation is divided into two distinct parts connected by the theme of polymer-based drug delivery. Part 1 focuses on a series of studies aimed at improving polymer drug delivery systems prepared with poly[N-(2-hydroxy propyl) methacrylamide] aimed to deliver large therapeutics into the cytoplasm; part 1 also provides insight into the currently unknown mechanism of membrane translocation with cell-penetrating peptides. Specifically, the ability to synthesize poly(HPMA) conjugates, with the Tat cell-penetrating peptide, and subsequent cellular trafficking of the construct were investigated. This novel bioconjugate was designed and synthesized to mask the Tat peptide between two macromolecules (i.e., poly(HPMA)-Tat-poly(ethylene glycol)) minimizing non-specific cell-surface interactions resulting from free Tat peptide, allowing traditional endocytotic trafficking of internalized conjugate. Programmed release points (hydrazones) within the polymer carrier, allowing degradation and release of exposed free Tat peptide to interact with lysosome membrane components. Subcellular trafficking studies of fluorescent labeled conjugates indicates endocytotic uptake of poly(HPMA)-Tat-PEG conjugate at early time points and the Tat-PEG component gains cytoplasmic entry after extended incubations, while the poly(HPMA) carrier remains mostly entrapped within lysosomes. This suggests a mechanism involving active or selective transport of compounds covalently attached to Tat peptide. More detailed studies of specific chemistries required for polymer hydrazone chemistries were performed to delineate the requirements needed for forming hydrazones stable in circulation (pH 7.4), but which degrade at the pH of the target site (pH 5.0). It was found that stability was affected by the chemistries contained on the imine carbon, with hydrazones formed from π-conjugated methyl ketone groups showing the best combination of stability and pH 7.4 and lability at pH 5.0. Part 2 demonstrates practical application of PEG hydrogels for delivery of live Brucella abortus strain RB51 vaccine ballistically to bison in Yellowstone National Park. Hydrogels loaded into the payload of hydroxypropyl cellulose degradable biobullets showed similar ballistic properties compared to commercially available counterparts when fired out of an air-rifle. The live vaccine showed excellent viability in both hydrated and lyophilized gels and generates the desired immune response when administered to bison. These results are currently being considered by the National Parks Service in for long-term strategies to eradicate brucellosis in wild bison herds.