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Author ORCID Identifier

https://orcid.org/0000-0002-1502-4289

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2021

Month Degree Awarded

May

First Advisor

Vincent M. Rotello

Second Advisor

Michael J. Knapp

Third Advisor

Richard W. Vachet

Fourth Advisor

M. Sloan Siegrist

Subject Categories

Biotechnology | Medicinal-Pharmaceutical Chemistry | Nanomedicine | Organic Chemistry | Polymer Chemistry

Abstract

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.

DOI

https://doi.org/10.7275/21251696.0

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Saturday, May 14, 2022

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