Subcellular targeted therapy using HMCD-artemisinin conjugates for metastatic castration-resistant prostate cancer

Background: Resistance to androgen-deprivation therapy leads to metastatic castration-resistant prostate cancer (mCRPC), a lethal stage with limited treatment options. Artemisinin (ART) derivatives exert anti-tumor activity through reactive oxygen species (ROS) generation and mitochondrial disruption but lack tumor selectivity. Our laboratory identified a near-infrared (NIR) heptamethine carbocyanine dye (HMCD) that preferentially accumulates in tumors via organic anion-transporting polypeptides (OATPs) upregulated under hypoxia, enabling tumor-specific delivery. Methods: To achieve subcellularly targeted chemotherapy, four HMCD-ART conjugates with distinct linkers were synthesized: HMCD-ART1 (artesunate linker), HMCD-ART2 (carbamate linker), HMCD-ART3 (ether linker), and HMCD-ART4 (ester linker). These were evaluated in androgen-independent prostate cancer models (22Rv1, PC-3, C4-2B, and resistant derivatives). Cytotoxicity, selectivity, and chemosensitization were analyzed in vitro, while subcellular localization, ROS generation, and cell-death mechanisms were examined by fluorescence microscopy, flow cytometry, and Western blotting. Tumor targeting and therapeutic efficacy were determined by NIR imaging and 22Rv1 xenograft models. Results: Among the conjugates, HMCD-ART1 showed the greatest potency and selectivity, effectively killing taxane-, abiraterone-, and enzalutamide-resistant prostate cancer cells while sparing normal prostate epithelial cells. HMCD-ART1 enhanced sensitivity to chemotherapeutics, inhibited colony formation and migration, and demonstrated prolonged tumor retention for up to eight weeks. Mechanistic analyses revealed mitochondrial and lysosomal co-localization, ROS induction, cytochrome c release, DNA damage (pATM, gammaH2AX), and mitochondrial-fission-dependent necrotic death. In vivo, HMCD-ART1 significantly suppressed xenograft tumor growth without observable systemic toxicity. Conclusion: HMCD-ART1 integrates NIR-guided tumor targeting with mitochondrial-specific cytotoxicity, providing a novel therapeutic platform to overcome drug resistance in mCRPC. This strategy represents a next-generation precision chemotherapeutic approach combining targeted delivery, subcellular selectivity, and enhanced safety.