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.