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