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Author ORCID Identifier

https://orcid.org/0000-0002-4066-9548

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Polymer Science and Engineering

Year Degree Awarded

2021

Month Degree Awarded

May

First Advisor

Gregory N. Tew

Subject Categories

Biomaterials | Polymer Chemistry

Abstract

Protein drugs, including antibodies, are rapidly emerging as the top-selling pharmaceuticals worldwide owing to their unparalleled specificity and biocompatibility. However, none of the currently-approved protein therapeutics act intracellularly, despite the vast majority of potential drug targets residing within the cell. This is due mainly to the paramount challenge of transporting hydrophilic macromolecular cargoes across the plasma membrane. As such, effective protein carriers are essential for the advancement of modern medicine. Despite significant advances, many challenges still plague protein delivery. Following membrane transduction, delivery vectors must preserve the structure and activity of their cargoes while transporting them to the correct subcellular destination and release them in a pharmacokinetically appropriate manner. Ultimately, the intracellular availability (IA), or fraction of total internalized drug which reaches its target in an active state, must be maximized to achieve meaningful biological outcomes.

Our group has developed a library of synthetic cell-penetrating peptide mimics (CPPMs) for intracellular delivery. These polymers have enabled robust non-covalent binding and delivery of functional protein and nucleic acid cargoes, resulting in substantial manipulation of cell biology. This dissertation comprises four studies wherein polymer structure is modulated to boost protein IA. First, CPPMs were directly compared to other non-covalent carriers for quantity of protein delivered and found to overwhelmingly outperform the others across all conditions tested, most significantly in particularly difficult-to-transfect T cells, neurons, and stem cells. In a second study, new block copolypeptide CPPs were reverse-engineered from optimized CPPM structures. Several of these new peptides were able to non-covalently bind and deliver proteins, a rare ability for CPPs, and hydrophobicity was revealed to universally predict IA. The next chapter documents the competing effects of polymer length on cargo uptake and function, revealing longer amphiphiles to exhibit greater IA. Finally, the effects of CPPM structure on subcellular protein delivery to the mitochondrion were investigated. This work was enabled by the simultaneous development of a new flow cytometry technique for rapid quantification of intra-organellular drug delivery. In general, these findings indicate that uptake-driven optimization is a gross oversimplification of the delivery process. Future studies should seek to better understand the mechanisms underlying IA.

DOI

https://doi.org/10.7275/22485197.0

Available for download on Saturday, May 14, 2022

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