The position of hydrophobic residues impacts cellular uptake and intracellular localization of cell penetrating peptides.

Abstract

Cell-penetrating peptides (CPPs) are widely used to deliver cargo into mammalian cells, yet efficient cellular uptake and endosomal escape remain key challenges. In this study, we evaluated how hydrophobic (4S)-4-cyclohexylproline (ChPro) residues and their spatial arrangement influence cellular uptake, endosomal escape, and subcellular distribution of conformationally constrained cationic peptides consisting of (4S)-guanidiniumproline (Gup). The evaluation revealed a greater effect on uptake and endosomal escape by positioning the hydrophobic block at the C- rather than the N-terminus. The amphipathic peptides with a C-terminal (ChPro)₃ sequence accumulated in mitochondria and the endoplasmic reticulum. These insights are relevant for optimizing cellular uptake, intracellular localization, and endosomal escape of CPPs.