Linker-dependent variations in the tumoricidal activity of DZ-1-dihydroartemisinin conjugate formats.

BACKGROUND

Cancer remains a leading cause of death worldwide, and many therapies are limited by poor tumor specificity, systemic toxicity, and drug resistance. Tumor-targeted strategies that enhance drug accumulation while minimizing off-target effects are therefore essential. We hypothesized that conjugating dihydroartemisinin (DHA) to the tumor-targeting near-infrared dye DZ-1 would improve tumor-selective accumulation, promote mitochondrial localization, and enhance reactive oxygen species (ROS)-mediated cytotoxicity. We also investigated how linker chemistry influences drug stability and therapeutic efficacy.

METHODS

Three types of linkers (ether, ester, and carbamate) were used to conjugate DZ-1 with dihydroartemisinin (DHA). The tumoricidal efficacy of these DZ-1-DHA conjugates was evaluated in HCT116 and BxPC3 cells. Cytotoxicity was assessed using the trypan blue exclusion assay. Apoptosis was analyzed by TUNEL staining and immunoblotting. Drug accumulation was compared among the conjugates. Mitochondrial membrane potential was assessed using JC-1 staining, and ROS generation was measured using DCF, MitoSOX, and Mito-TEMPO assays.

RESULTS

Ester-linked DZ-1-DHA showed the most potent, dose-dependent cytotoxicity in both HCT116 and BxPC3 cells. Apoptosis was confirmed by immunoblotting and TUNEL assays. Fluorescence microscopy revealed that the ester-linked conjugate effectively accumulated in mitochondria, efficiently generated reactive oxygen species (ROS), and disrupted mitochondrial membrane potential.

CONCLUSIONS

Our findings demonstrate that linker chemistry critically influences DZ-1-DHA accumulation and mitochondrial ROS generation, which in turn determine tumoricidal efficacy. Among the conjugates, the ester-linked DZ-1-DHA exhibited superior anticancer activity. These results provide mechanistic insight and highlight linker optimization as a key design principle for the development of next-generation tumor-targeted artemisinin therapeutics.