(729c) Enzyme-Cleavable Peptide Amphiphiles Enhance Intracellular Delivery
To address these questions, we have designed enzyme-cleavable PAs to enhance the intracellular delivery of the therapeutic peptides. The aim of this study is to prevent the endosomal sequestration of peptide cargoes using a PA design that includes an endosomal cleavable linker between the hydrophobic tail and a p53(14-29) peptide known to dissociate p53 from MDM2/MDMX. The PAs internalize through endocytosis, where they are later cleaved from their hydrophobic tail via cathepsin B. Combining fluorescence labeling of the therapeutic and the hydrophobic tail enabled the detection of intracellular trafficking in real time, allowing elucidation of co-localization behaviors using confocal microscopy. Fluorescence resonance energy transfer (FRET) analysis was used for the first time to follow enzymatic cleavage of PAs within the cell. We find that over time, the cleaved p53(14-29) peptide accumulates intracellularly while the lipid carrier is recycled outside of the cell within extracellular vesicles. Conversely, PAs without cleavable groups do not accumulate within the cell and are quickly moved through the cell and into similar extracellular vesicles.
Our novel PA design allows for increased intracellular delivery of biological peptides and elucidates how peptide and lipid carriers are recycled following delivery. These results shed light on methods to overcome limited biological effects using traditional non-cleavable PAs allowing for enhanced clinical translation.