Transport across membrane meets biophysics to unveil the mechanism of action of a novel gH625 analogue

Cell-penetrating peptides are widely used in drug delivery for their ability to facilitate the transport of nanomaterials inside the cell. We previously studied the gH-625 for its ability to cross cell membranes, delivering various cargos into different cell types. In this study, since gH-625 suffers from low proteolytic stability, we identified the main cleavage sites after incubation with the enzyme chymotrypsin, and l-amino acids at these sites were replaced with their d-enantiomers, which share similar physicochemical properties but have distinct biological roles. Four peptides, namely gH-w10, gH-l7, gH-y13, and gH-combi, were designed and synthesized. Their biosafety profiles were evaluated in both normal and cancer cell lines and no significant toxic effects were revealed at the tested concentrations. Subsequently, we assessed their cell-penetrating ability by evaluating cellular uptake through fluorescence microscopy and investigated their mechanism of action in a model system of liposomes, measuring fusogenic activity, peptide insertion into the lipid bilayer, and leakage activity. The impact of the d-amino acid substitution on secondary structure was explored by circular dichroism and nuclear magnetic resonance studies. Finally, in vitro safety profiling data of the gH-625 and its most promising derivative gH-combi were further confirmed in vivo using a chicken embryo model.