Cell penetrating peptides (CPPs) are short peptides that traverse the eukaryotic cell membrane without causing significant damage. They have emerged as promising delivery vehicles to transport a wide variety of cell-impermeable cargos (such as proteins and nucleic acids) into mammalian cells. At low concentrations (&lt;10 µM), most CPPs enter the cell in membrane-bound vesicles called endosomes; subsequent endosomal escape governs their cytosolic delivery efficiency. Most CPPs face the challenge of poor endosomal escape efficiency, which is compounded by the lack of mechanistic understanding of the endosomal escape process. We aim to bridge this significant knowledge gap, by studying the uptake of CPPs in live cells. Previous research from our lab led to the proposal of a novel hypothesis of vesicle budding and collapse to explain the mechanism by which CPPs escape the endosome. To test this hypothesis in live cells, we designed a series of confocal microscopic experiments to visualize endosomal escape using a pH-sensitive fluorescent dye. We also used a PIK-Fyve inhibitor to obtain a swollen vesicle phenotype, to observe the localization of the CPP inside the endosome. We captured budding and collapse events occurring on the endosomal membrane, which provided conclusive evidence of this mechanism. Our findings resolve a long-lasting mystery in the field of CPPs and provide a toolbox of experimental techniques for probing endosomal escape.

Cell penetrating peptides: Mechanism of action

ACS Fall 2021

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