Protein Binding and Release by Polymeric Cell-Penetrating Peptide Mimics

• Published in: Biomacromolecules Vol. 23; no. 1; pp. 57 - 66
• Main Authors: Davis, Hazel C, Posey, Nicholas D, Tew, Gregory N
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10-01-2022
• Subjects: Cell-Penetrating Peptides - chemistry; Guanidine - chemistry; Hydrophobic and Hydrophilic Interactions; Polymers - chemistry; Protein Binding
• ISSN: 1525-7797; 1526-4602
• DOI: 10.1021/acs.biomac.1c00929

There is significant potential in exploiting antibody specificity to develop new therapeutic treatments. However, intracellular protein delivery is a paramount challenge because of the difficulty in transporting large, polar molecules across cell membranes. Cell-penetrating peptide mimics (CPPMs) are synthetic polymers that are versatile materials for intracellular delivery of biological molecules, including nucleic acids and proteins, with superior performance compared to their natural counterparts and commercially available peptide-based reagents. Studies have demonstrated that noncovalent complexation with these synthetic carriers is necessary for the delivery of proteins, but the fundamental interactions dominating CPPM–protein complexation are not well understood. Beyond these interactions, the mechanism of release for many noncovalent carriers is not well established. Herein, interactions expected to be critical in CPPM–protein binding and unbinding were explored, including hydrogen bonding, electrostatics, and hydrophobic interactions. Despite the guanidinium-rich functionality of these polymeric carriers, hydrogen bonding was shown not to be a dominant interaction in CPPM–protein binding. Fluorescence quenching assays were used to decouple the effect of electrostatic and hydrophobic interactions between amphiphilic CPPMs and proteins. Furthermore, by conducting competition assays with other proteins, unbinding of protein cargoes from CPPM–protein complexes was demonstrated and provided insight into mechanisms of protein release. This work offers understanding toward the role of carrier and cargo binding and unbinding in intracellular outcomes. In turn, an improved fundamental understanding of noncovalent polymer–protein complexation will enable more effective methods for intracellular protein delivery.