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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Polymer Science and Engineering

Year Degree Awarded

2019

Month Degree Awarded

February

First Advisor

Gregory N. Tew

Second Advisor

Maria Santore

Third Advisor

Richard Vachet

Subject Categories

Biochemistry | Biophysics | Polymer Chemistry

Abstract

Delivering functional proteins and antibodies into cells can allow researchers to probe the intracellular environment, discover new cellular pathways, and pioneer new therapeutics. However, the entry of exogenous, charged molecules, like proteins, into the cell is usually restricted by the membrane, thereby hindering intracellular delivery. Membrane permeable molecules such as cell penetrating peptides (CPPs) and protein transduction domains (PTDs) can be used to bypass the cell membrane and deliver protein into the cell, but these peptides involve iterative and laborious syntheses and are limited in terms of their chemical diversity.

This dissertation work overall focuses on the design and synthesis of polymeric CPP or PTD mimics (CPPMs or PTDMs), which are easier to synthesize and more effective at non-covalently binding and delivering protein cargo into cells. (From here on and for consistency, these mimics will be referred to as PTDMs only.) Specifically, ring-opening metathesis polymerization was employed in this work to create highly tuned and optimized amphiphilic block copolymer PTDMs which can non-covalently bind and deliver protein cargo into difficult-to-transfect human T cells. From previous work on PTDMs, we learned that PTDMs require a hydrophobic block for effective protein delivery and that this block could be optimized for enhanced delivery. We also received early indications that the protein binding ability of PTDMs might be correlated with the ability to deliver protein cargo.

Herein, in an initial study, the hydrophobicity of PTDMs were carefully modulated by fine degrees to ascertain how precise changes in polymer hydrophobicity affected the ability to bind and deliver one model, protein cargo. Overall, changing the hydrophobicity in a systematic and significant way did not impact the ability to bind protein cargo, but it did reveal a requisite optimal hydrophobicity for maximal delivery in accordance with the results of related studies. Additionally, binding ability and delivery ability were uncorrelated in this study unlike the results of past research.

In a second study, the relationship between binding and delivery was explored further by first measuring the binding strength of PTDMs to various types of protein cargo. The results of this study established that PTDMs indeed exhibited preferential protein binding depending on the attributes of the protein. These preferences were manifested in measured dissociation constants (Kd’s) that differed by orders of magnitude, from weak to very tight binding. These substantial differences in binding ability were uncorrelated to the delivery outcomes further highlighting the importance of other factors, like PTDM hydrophobicity.

In a third study, the ability of a model PTDM to self-assemble at protein binding application relevant concentrations was probed in an effort to determine the importance of PTDM self-assembly to the protein cargo binding process. This study suggested that low molecular weight PTDMs act as weak surfactants and do not self-assemble, but can form detectable aggregates by dynamic light scattering (DLS) in the presence of protein. In further efforts to study the binding process, beyond the quantification of Kd’s and self-assembly, various PTDM-protein binding conditions, such as the presence of increased salt and urea, were explored to elucidate the underlying causes of binding. Future work in this field will fundamentally explore how PTDMs bind and unbind proteins, how these complexes are formed and organized, and how binding impacts the functionality of the protein.

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