Enzyme-Triggered Charge and Conformation Switching for Tumor-Selective Peptide–Drug Conjugates

Functionalizing anticancer drugs with activatable peptides offers a versatile strategy to achieve tumor-selective delivery while minimizing systemic toxicity. Here, we report a γ-glutamyltransferase (GGT)-responsive cell-penetrating peptide ( P -EE ) generated by γ-glutamyl masking of penetratin for tumor-selective activation in hepatocellular carcinoma. Upon GGT-mediated cleavage, P -EE restores its cationic charge and helical conformation, enabling efficient membrane translocation and selective accumulation in GGT-overexpressing tumor cells. P -EE showed enhanced internalization and approximately 5-fold higher cytotoxic potency in HepG2 cells compared with NHDF normal cells. Covalent conjugation of camptothecin (CPT) to P -EE yielded a peptide–drug conjugate ( CPT-P -EE ) with greatly improved aqueous solubility, potent antitumor activity, and over 50-fold reduced toxicity to normal cells. In vivo, CPT-P -EE significantly suppressed tumor growth and prolonged survival without inducing systemic toxicity. This study establishes a generalizable enzyme-activatable peptide–drug conjugate platform that integrates conformational switching with targeted delivery, offering a promising direction for precision chemotherapy against GGT-positive malignancies.