A visualizable mitochondrial-targeted Chlorambucil prodrug for enhancing anti-hepatoma efficacy

Mitochondria play essential roles in tumor biology, influencing apoptosis, redox homeostasis, ion balance, and genetic stability; disruptions in these processes often dictate the balance between tumor progression and suppression. Consequently, mitochondria-targeted cancer therapy has emerged as a promising strategy for improving therapeutic selectivity. Chlorambucil (CLB), a bifunctional alkylating agent, is clinically limited by its lack of tumor specificity and substantial toxicity to normal tissues, including the risk of inducing secondary malignancies. To overcome these limitations, we designed a structurally derivatized CLB prodrug-BPA-CLB that incorporates fluorescence for visualization and a mitochondrial-targeting moiety to enhance intratumoral selectivity. It was constructed by modifying the 1,8-naphthalimide scaffold to improve delocalization and lipophilicity and subsequently conjugating it to CLB. This design leverages organelle-specific distribution and real-time imaging to enable precise drug delivery at the subcellular level. Using advanced confocal imaging and LC-MS analysis, we confirmed that BPA-CLB accumulates selectively in mitochondria. In vitro and in vivo evaluations demonstrated that BPA-CLB markedly reduces toxicity to normal cells while maintaining potent antitumor activity. Mechanistically, BPA-CLB disrupts mitochondrial morphology and function, increases reactive oxygen species (ROS) production, induces apoptosis, and suppresses tumor-cell migration. This process is related to the combination of VDAC2. In conclusion, BPA-CLB provides a visually traceable and mitochondria-specific delivery strategy that minimizes off-target interactions and enhances CLB’s therapeutic selectivity. By precisely perturbing mitochondrial function, BPA-CLB significantly improves antitumor efficacy against hepatocellular carcinoma, highlighting its potential as a new class of subcellularly targeted chemotherapeutics.