Stepwise progressive dual-regulated micelles enhance the therapeutic efficacy of hepatocellular carcinoma by remodeling the tumor microenvironment.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by severely immunosuppressive tumor microenvironment (TME), high recurrence and metastasis, and unsatisfactory clinical outcomes. Tenacissoside H (Ten H) shows remarkable anti-HCC activity, while ginsenoside Rh2 (GRh2) effectively regulates TME and reverses immunosuppression. However, their poor water solubility, low in vivo bioavailability and lack of tumor-targeting ability limit clinical application. To address these drawbacks, we developed glycyrrhetinic acid (GA)-modified stepwise progressive dual-regulatory micelles co-loaded with GRh2 and Ten H (GRh2-GA-Ten H-Ms) for synergistic anti-HCC therapy via TME remodeling and precise tumor eliminating. METHODS: GRh2-GA-Ten H-Ms were prepared by thin-film dispersion and physicochemically characterized. In vitro antitumor effects were evaluated in Hepa1-6 cells, including cellular uptake, cytotoxicity, apoptosis, proliferation, migration, immunogenic cell death (ICD), as well as M2-to-M1 macrophage repolarization in tumor-macrophage co-culture systems. In vivo antitumor, anti-metastatic and anti-recurrence efficacy was validated in subcutaneous, pulmonary metastasis and postoperative recurrence HCC mouse models, accompanied by biosafety assessment. RESULTS: GRh2-GA-Ten H-Ms showed smooth spherical morphology, uniform size, favorable stability, blood compatibility, high encapsulation efficiency (EE) of more than 90%, reactive oxygen species (ROS)-responsive release and GA-mediated liver targeting. In vitro, they inhibited proliferation, migration with an IC50 value of approximately 67 μM, induced apoptosis with an apoptosis rate of approximately 30%, ICD and macrophage repolarization. In vivo, they enhanced tumor accumulation, suppressed primary tumor growth with a tumor growth inhibition rate of roughly 75%, reduced pulmonary metastatic nodules, delayed postoperative recurrence, and remodeled the TME without obvious toxicity. CONCLUSION: GRh2-GA-Ten H-Ms exhibit sequential synergistic effects combining GRh2-mediated TME remodeling and Ten H-mediated tumor cell elimination. With experimentally validated tumor-targeting capacity and biocompatibility, this system provides a multifunctional traditional Chinese medicine (TCM) nanodelivery strategy for HCC therapy.