Amphipathic Proline-Rich Cell Penetrating Peptides for Targeting Mitochondria

Cell-penetrating peptides (CPPs) offer a platform for targeted intracellular delivery. Here, we developed amphipathic oligoprolines for targeting mitochondria. The rigid peptides feature cationic guanidinium and hydrophobic cyclohexyl groups aligned along the edges of the polyproline II (PPII) helical backbone. Systematic variations of the hydrophobicity through C-terminal and backbone modifications provided CPPs with enhanced cellular uptake and mitochondrial selectivity. Comparative studies with conformationally more flexible analogs revealed the benefit of aligned cationic and hydrophobic residues on a rigid backbone for mitochondria targeting. Notably, the amphipathic peptides undergo time-dependent intracellular redistribution, leading to selective and prolonged mitochondrial residency. Our findings established design principles for optimizing CPPs to target mitochondria.