(136e) Rational Design for Therapeutic Peptide-Amphiphile-Based Intracellular Delivery

Therapeutic peptides are highly promising tools to manipulate protein-protein interactions, because of their target selectivity and potential safety. Although, peptide amphiphile (PA) technology that combines a polar peptide with a hydrophobic tail efficiently delivers peptide payloads into cells and increases their stability in the circulation, obstacles remain for viable clinical translation. One major obstacle is that PAs frequently accumulate within endosomal compartments because of the greasy hydrophobic tail, which limits the ability of delivered peptides to target proteins within the cytosol. Thus, overcoming endosomal entrapment is a crucial step for the effective clinical transition of these peptide-based targeting agents. Additionally, intracellular trafficking and the ultimate fates of the hydrophobic tails and peptides are largely unknown.

In this study, we aim to overcome endosomal sequestration of peptide cargoes through a PA design that includes an endosomal cleavable linker between a diC16 hydrophobic tail and a previously validated biofunctional peptide sequence from the tumor suppressor p53 (p53_(14-29))_. We use a cathepsin cleavable sequence, combined with FRET compatible double fluorescence dyes, to study the internalization, trafficking, and endosomal escape of intact PAs as well as individual lipid and p53 peptide moieties. Single molecule FRET measurements show efficient enzyme activity between the lipid tail and p53_(14-29) while having no activity on the peptide itself. We find that cathepsin-cleavable PAs accumulate within the cell more rapidly than PAs lacking this sequence and very quickly loose intracellular FRET signaling indicating rapid cleavage of the p53_(14-29) from diC₁₆. Using confocal microscopy, we show that cleaved p53_(14-29) peptides and hydrophobic diC₁₆ tails migrate to different parts of the cytosol. While a portion of diC₁₆ tails appear to recycle to the cellular membrane, intracellular accumulation also exists. Conversely, PAs lacking a protease cleavage site retain FRET signaling over time, predominate within endosomes and exhibit far less intracellular accumulation over time.

We believe that cathepsin cleavable PAs will provide opportunities to functionally target numerous protein-protein interactions using a variety of therapeutic peptides while also providing a tool to better elucidate PA trafficking in real time within cells.