Efficient intracellular delivery of proteins remains a significant challenge despite its immense therapeutic potential for enzyme therapies and genome-editing. Proteins often require active strategies for intracellular delivery due to their membrane impermeability. In addition, accessing the cytosol or subcellular compartments requires internalized proteins to escape the endosome to evade lysosome degradation or exocytosis. Past efforts to deliver proteins to intracellular targets have often relied on cell-penetrating peptides and lipid nanoparticles, which have shown promise, but often suffer from low potency, limited <i>in vivo</i> activity, and a lack of generalization. Endosomolytic peptides, which are able to fuse with the endosomal membrane following a pH-triggered conformational change, have shown promise in nanoparticle systems, but only a handful of these peptides have been tested for cytosolic protein delivery. Further studies are necessary to understand the full potential of these peptides for protein delivery and determine how to use these peptides more generally for delivering proteins that vary in size and composition.<br/>Our previous work site-specifically conjugated epidermal growth factor (EGFR)-targeting peptides to proteins to explore the effect of ligand valency and spacing on protein internalization in EGFR-overexpressing breast cancer cells. High EGFR ligand valency and clustering was associated with robust internalization as compared to unmodified protein, however, the internalized protein remained entrapped in the endosome leading to its eventual degradation. In this study, EGFR ligands and multiple endosomolytic peptides were site-specifically conjugated to the protein to enhance endosomal escape. Two endosomolytic peptides with differing mechanisms of endosomal disruption, e.g., the pore-forming and the carpet-like mechanisms, were explored for their ability to deliver a model maltose-binding protein to the cytosol. Both peptides decreased lysosomal protein degradation and enhanced endosomal disruption, although the carpet-like sequence was more effective. Furthermore, this approach was used to deliver p19 siRNA binding protein, for siRNA mediated gene silencing, and caspase, for enhanced cell apoptosis, demonstrating the modularity of the approach. These results, combined with the growing body of literature, may provide insight for future delivery systems so that intracellular protein therapies become more clinically relevant.

Cytosolic delivery of therapeutically-relevant proteins by site-specific incorporation of endosomolytic peptides for the treatment of breast cancer cells

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.