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A FUNDAMENTAL STUDY OF CO-ENGINEERING PROTEIN- NANOPARTICLE FOR INTRACELLULAR PROTEIN DELIVERY
Author ORCID Identifier
Campus-Only Access for Five (5) Years
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Vincent M. Rotello
Michael J. Knapp
Richard W. Vachet
M. Sloan Siegrist
Biotechnology | Medicinal-Pharmaceutical Chemistry | Nanomedicine | Organic Chemistry | Polymer Chemistry
Direct cytosolic delivery of therapeutic proteins such as the CRISPR/Cas9 protein provides enormous opportunity in curing human diseases. The foremost approach to achieving this is through engineered nanomaterials. Intrinsic nanoscopic properties provide access to unique chemical and physical properties. In addition, the structural and functional diversity of gold and polymeric nanocarriers provide unrivaled control of nanostructural properties for effective transport of therapeutic cargos, overcoming barriers on the cellular and organismal level. In this thesis, I describe the application of several new nanomaterials for functional protein intracellular delivery. Initially, I generated a gold nanoparticle-based nanocomposite delivery system for delivery of engineered CRISPR-Cas9 ribonucleoprotein. Specifically, this system was used to alter macrophage DNA and knock out SIRP-α in macrophages to increase phagocytosis of cancer cells. After demonstrating effective CRISPR-Cas9 RNP delivery in vitro, I utilized the nanoassemblies to deliver CRISPR-Cas9 RNP into macrophages in vivo through systemic administration. Additionally, in a related system I generated a library of poly(oxanorbornene)imide (PONI) polymers consisting of a ‘semi-arthritic’ backbone necessary for delivery of engineered proteins. These polymers self-assemble with E-tagged proteins to form discrete polymer-protein nanocomposites (PPNCs) that are stable and capable of delivering functional proteins under physiologically relevant conditions. Finally, inspired by bioconjugate chemistry I employed the versatile and non-disruptive biotin-Streptavidin interaction as an approach to engineer the glutamic acids tags for intracellular delivery through non-covalent tethering of components into a single effective delivery vector. In summary, this thesis provides a fundamental understanding and evidence of the utility of next generation chemical and nanomaterial tools in intracellular protein-based therapeutics development.
Lee, Yi-Wei, "A FUNDAMENTAL STUDY OF CO-ENGINEERING PROTEIN- NANOPARTICLE FOR INTRACELLULAR PROTEIN DELIVERY" (2021). Doctoral Dissertations. 2192.
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