Author ORCID Identifier

https://orcid.org/0000-0002-1330-7381

Date Available

12-8-2021

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department/School/Program

Chemistry

First Advisor

Dr. Jason DeRouchey

Abstract

Gene therapy aims to treat patients by altering or controlling gene expression. Today, most clinical approaches are viral-based due to their inherent gene delivery activity. However, there is still a significant interest in nonviral alternatives for gene delivery, particularly synthetic lipids and polymers, that do not suffer the immunogenicity, high cost, or mutagenesis concerns of viral vectors. Polymeric vectors are of particular interest due to the ability to further tune the polymer properties through the incorporation of additional functional units such as targeting ligands or shielding domains. Polyethylenimine (PEI), a highly cationic polymer, is often considered a benchmark for polymer-based gene delivery and thus serves as an excellent model for investigating gene delivery mechanisms. One reason PEI, especially branched PEI, is thought to outperform many other cationic polymers is due to the presence of secondary and tertiary amines. These amines are thought to help facilitate escape from endocytic vesicles via a 'proton-sponge' mechanism. Despite its successful use for in vitro gene delivery, PEI was initially developed for use in common processes such as water purification. As such, the properties of PEI should not be expected to be optimal for gene delivery. In this dissertation, my research efforts focused on the incorporation of negatively charged succinyl groups to the PEI backbone to create succinylated zwitterion-like PEI (zPEIs). Specifically, we focused on the synthesis and characterization of zPEIs as well as the impact of zPEI on DNA condensation and gene expression.

This dissertation will discuss the results of three projects. In project (1), we studied the suitability of minimally modified zPEIs for gene expression. In this work, we reveal that modification of PEI as low as 2% amines was sufficient to provide significant improvements in gene delivery particularly in the presence of serum proteins. In project (2), we investigate the self-assembly of DNA induced by modified and unmodified branched PEIs using small-angle X-ray scattering (SAXS). Modified PEIs included both succinylated zPEI and acetylated PEIs (acPEI) both modified from 0-40%. We demonstrate that changing the degree of modification significantly alters the packing density of the resulting polyplexes. While acPEI shows a continuous decrease in DNA packaging efficiency with increasing degree of modification, zPEI shows a crossover behavior where DNA-DNA interhelical spacings increase at low succinylation but decrease at higher degrees of succinylation. Studies on the pH dependence on the inter-DNA spacing also show that lowering the pH leads to tighter DNA packaging for all PEIs studied. These findings shed light on the complex correlation between DNA packaging density and gene expression ability of PEI and modified PEI mediated gene delivery systems. In project (3), we studied the efficacy of zPEI polyplexes at varying protein concentrations ranging from 0-10 mg/mL of bovine serum albumin (BSA). These high protein concentrations are comparable to in vivo protein concentrations. We show that while PEI/DNA transgene expression decreases with higher protein concentrations, the zPEI studied stayed approximately constant over the protein range studied. To test if these conditions may lead to the formation of a protein corona on the nanoparticles, which was recently shown to enhance serum-free transfection in unmodified bPEI/DNA, we also measured the transgene expression of polyplexes pre-treated to form a protein corona on the polyplexes.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.016

Funding Information

This material is based upon work supported in part by the National Science Foundation under the Faculty Early Career Development (CAREER) Program grant no. MCB- 1453168 (Summar 2019, May 2020 to August 2021).

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