Folate receptor-targeted cantharidin and staurosporine dual-drug nanoliposomes for multi-target synergistic therapy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) exhibits limited response to monotherapy owing to its complex signalling pathways and high heterogeneity. Combination therapy is therefore a promising strategy to improve HCC treatment. This study constructed folate (FA)-modified cantharidin (CTD) and staurosporine (STS) co-loaded targeted nanoliposomes (FA/CTD-STS/NLP) for multi-target synergistic therapy of HCC. The optimal CTD: STS mass concentration ratio was determined to be 588:1 using the Chou-Talalay method, and DSPE-PEG2000-FA functionalized nanoliposomes were prepared via a combined thin-film dispersion and embedding technique. The prepared FA/CTD-STS/NLP exhibited spherical morphology with a particle size of 116.4 nm, a PDI of 0.228, and high encapsulation efficiencies (90.1% for CTD, 93.4% for STS), along with good stability during storage and in serum. In vitro studies demonstrated that FA/CTD-STS/NLP enters H22 cells via folate receptor (FR)-mediated endocytosis, significantly inhibiting cell proliferation and promoting apoptosis. In the H22 tumor-bearing mouse model, FA/CTD-STS/NLP demonstrated enhanced tumor accumulation, achieving an 83.24% tumor inhibition rate even at reduced doses, with minimal systemic toxicity. Mechanistic studies indicate that FA/CTD-STS/NLP synergistically inhibit key target proteins in the mTOR/HIF-1α/VEGF signaling pathway, increase the Bax/Bcl-2 ratio to activate the mitochondrial apoptosis pathway, and upregulate CD4 and CD8 expression in tumor tissues, suggesting a potential immunomodulatory effect. This study developed FA/CTD-STS/NLP co-delivery system for the first time, achieving efficient tumor-targeted delivery through optimized drug combination ratio. It coordinately regulated the mTOR/HIF-1α/VEGF signaling pathway and the tumor immune microenvironment, providing a novel strategy for efficient and low-toxicity treatment of HCC.