Platform Reagent Development for the Intracellular Delivery of Therapeutic Peptides

B.C. Evans, C. Lander, C.L. Duvall
Moerae Matrix, Inc.,
United States

Keywords: peptide, polymer, intracellular delivery, endosomal escape, cell penetrating peptide, polyplex


Peptides hold many advantages over the small molecule drugs that dominate the current healthcare market in terms of potency, specificity, and biocompatibility, and they are faster and easier to implement than genetic modifications for completion of hypothesis-driven studies. Peptides can inhibit enzymes and alter protein-protein interactions to have profound effects on cellular behavior. Despite these advantages, peptides suffer from poor intracellular uptake and entrapment within intracellular compartments, thereby preventing access to their intracellular target and limiting their potential as research tools and therapeutics. Positively charged cell-penetrating peptides (CPPs) are commonly used to improve peptide uptake by cells. We previously developed a polyplex peptide delivery technology comprising electrostatically complexed, endosomolytic nano-polyplexes (NPs) containing the pH-responsive polymer poly(propylacrylic acid) (PPAA) for the enhanced delivery of cell penetrant, intracellular-acting MAPKAP Kinase 2 inhibitory peptide (MK2i-NPs This NP system has been applied for peptide delivery to vascular tissue to inhibit vascular graft intimal hyperplasia and pathological vasoconstriction. We explored the generalizability of this polyplex approach as a user-friendly reagent that further enhances CPP peptide uptake and dramatically improves potency and durability of action by enabling escape from intracellular compartments. The general utility of this approach was investigated with a library of therapeutic peptides that contain different CPP sequences. We first investigated formulation optimization based upon flow cytometric analysis of peptide cellular uptake, demonstrating that optimal peptide uptake was dependent on the concentration of the polymeric delivery reagent alone (i.e., peak uptake at 2.5 ┬ÁM PPAA in vascular smooth muscle cells) and independent of peptide dose or the ratio of peptide to polymer. To further elucidate the mechanism of enhanced uptake, we performed uptake studies comparing co-delivery of the peptide and delivery reagent to sequential delivery where cells are treated with the delivery reagent, washed, and then treated with the peptide. Sequential delivery also enhanced uptake, suggesting that the delivery reagent coats cell membranes through hydrophobic and electrostatic interactions, resulting in a net increase in the net negative charge at the cell surface that increases cell surface affinity for cationic CPPs. We then demonstrated the ability of the delivery reagent to enhance peptide uptake across a range of cell types including vascular smooth muscle cells, endothelial cells, and macrophages. Control experiments comparing uptake of a peptide with and without a CPP sequence demonstrated that the presence of a CPP is necessary for polymer-mediated enhancement of uptake. Finally, we compared PPAA-mediated peptide delivery to seven commercially available protein/peptide delivery reagents, revealing that PPAA was superior in enhancing peptide uptake. Future studies will investigate the intracellular retention, endosomal escape, and biocompatibility of a range of peptides delivered with our polyplex technology compared to commercially available reagents. In conclusion, this nanopolyplex approach holds promise as a superior alternative for intracellular peptide delivery that is optimized specifically for delivery of cationic cargo (e.g., most CPP-based peptides). As such, this technology demonstrates potential to fill a current gap in the delivery reagent market, bolstering peptide use, accelerating scientific advancement, and enabling peptide therapeutic approaches for intracellular targets.