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Designing Polymer-Protein Complexes for Intracellular Delivery

Previous work in the Tew group has demonstrated polymer cell-penetrating peptide mimics (CPPMs) as effective transporters of biological agents, including proteins and antibodies. These synthetic polymers non-covalently bind to cargo, offering a mechanism to deliver proteins in a way that does not alter protein secondary structure. However, correlations of the protein binding-delivery relationship or the role of polymer-protein complexation on intracellular activity of protein cargo are understudied. The work presented herein connects a fundamental understanding of polymer-protein complexation with intracellular internalization and cargo activity. Characterization and quantification of polymer-protein binding relationships were established using fluorescence quenching assays. In particular, the identification of dominant interactions of electrostatics and hydrophobicity was observed given the sensitivity of binding assays to salt disruption and the manipulation of block polymer copolymer architecture. New assays to investigate competition of polymer-cargo complexes by intracellular proteins were introduced and revealed that competition of xii polymer-cargo is influenced by initial binding strength. Furthermore, limitations of current assays to quantify polymer-protein binding were discussed and a new method of Covalent- Labeling Mass Spectrometry was introduced to quantify non-covalent polymer interactions with model protein surface patches. This represents the first use of this method for identifying non-covalent polymer-protein interactions. This method allowed for increased understanding of the binding interactions between polymer-based CPPMs and anionic and hydrophobic surface patches on protein cargo. The structural diversity afforded by polymer-based synthetic CPPMs was additionally leveraged to design a series of polymers with previously optimized hydrophobic:cationic block ratios of 2:1. These polymers were further investigated for their self-assembly properties. The role of polymer self-assembly was observed to impact not only initial binding interactions with protein cargo, but also internalization and intracellular activity. Finally, future directions for designing next generation cell-penetrating peptide mimics as carriers of protein cargo were discussed. Studies that facilitate understanding in CPPM-mediated intracellular protein delivery provide unprecedented insight for how non-covalently bound carriers deliver cargo. This understanding can be applied beyond cell penetrating peptide mimics to the design of smart carriers that are capable of binding to a range of novel cargo, have controllable cargo release properties, and even preferentially binding to proteins.
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