Redox-cleavable disulfide linker enhances siRNA delivery by prodrug-type bifunctional cell-penetrating peptide

In prodrug strategies, introducing a cleavable linker enables activation within target tissues. We developed a novel prodrug-type cell-penetrating peptide (CPP), PI-linker-cRGD, incorporating a sterically refined redox-cleavable linker for intracellular siRNA delivery. Disulfide bonds are valuable in CPP-based prodrug designs because they are cleavable in the reductive environment of cancer cells. We previously designed a bifunctional CPP, PI-cRGD, by conjugating the membrane-permeable α-aminoisobutyric acid (Aib)-containing amphipathic helical peptide (PI) to the α_vβ₃ integrin-targeting ligand cRGD via a disulfide bond. The PI-cRGD/siRNA complex exhibited low cytotoxicity and significant cellular uptake via endocytosis. However, it failed to achieve efficient cytosolic siRNA delivery, likely due to steric hindrance that prevented the disulfide cleavage in the reductive cell surface and intracellular environments, which in turn inhibited the release of membrane-permeable PI. To address this issue, we demonstrate that inserting a sterically refined redox-cleavable linker between PI and cRGD sustains low toxicity and targeting ability while enabling cleavage of the disulfide bond through thiol–disulfide exchange at the cell surface, thereby activating the CPP, promoting membrane translocation, and ultimately achieving efficient cytosolic siRNA delivery. This simple cleavable-linker strategy holds broad applicability for diverse therapeutic agents.