Optimizing amphipathic switching in hydrocarbon stapled peptides for enhancing endosome escape

The characteristics of the cell membrane pose a major barrier to delivering cell-impermeable, negatively charged molecules into cells. Herein, we report the synthesis and biological characterization of a novel series of hydrocarbon-stapled peptides that are rationally designed and engineered to form a large hydrophobic surface composed of aromatic amino acid residues, aiming to systematically compare their cellular uptake, endosomal escape, and molecular transporter efficiencies. Guided by the helical-wheel projection of the α-helical peptide A4 previously reported by our research group, we substituted the amino acid residues at four hydrophobic face positions (1, 2, 4, and 8) of this peptide with the hydrophobic and aromatic L-2-naphthylalanine (Φ), tryptophan (Trp), and phenylalanine (Phe). The experimental results demonstrated that P2 and P12 exhibited the most remarkable enhancement in activity, with their cellular uptake and endosomal escape efficiencies both increasing by nearly 4-6-fold relative to the parent peptide A4. Importantly, our findings provide direct experimental evidence that a large hydrophobic surface formed by aromatic amino acid residues and its specific localization serve as the key structural basis for efficient cellular uptake and subsequent endosomal escape. Ultimately, the superior transporter efficiency of P2 and P12 was marked by a 7-fold (P2) and 8-fold (P12) enhancement in GpTEEI uptake in HeLa cells, compared to the cargo alone. In the GFP knockdown assay, P2 and P12 induced a significant reduction in fluorescence intensity compared with the untreated control group, a result that confirms their ability to escape the endosome and thereby mediate efficient gene silencing. Collectively, these findings underscore its promise as both a cell-penetrating peptide and a versatile molecular transporter.