Beyond arginine count: Rational engineering of amphipathic macrocyclic CPPs with enhanced intracellular delivery and endosomal escape

Guanidinium-rich cell-penetrating peptides (CPPs) are powerful intracellular delivery vectors, yet their broad utility is limited by high arginine content, endosomal sequestration, and associated toxicity. Here, we report a rational peptide engineering strategy that integrates precise lactam macrocyclization with controlled modulation of amphipathicity to enhance cellular uptake and endosomal escape while reducing arginine burden. Using a rolling-loop scan approach, we identify lactam-stapled octa-arginine analogs that exhibit superior internalization relative to their linear counterparts, consistent with helix stabilization. Strategic incorporation and spatial clustering of lipophilic residues on a single helical face further yielded amphipathic macrocyclic CPPs with markedly enhanced cellular penetration and cytosolic delivery in HeLa cells. Notably, the lead compound 36a displays up to a 1.9-fold increase in uptake compared to R9, despite containing fewer arginine residues, and demonstrates partial endosomal escape as confirmed by live-cell confocal microscopy. Collectively, these findings establish that CPP efficiency is governed not solely by arginine number, but by the precise spatial organization of charge, hydrophobicity, and conformational constraint, providing a generalizable framework for the design of next-generation CPPs with improved intracellular performance and potential pharmacokinetic advantages.