Dual-ligand-modified chitosan nanoliposomes for the targeted delivery of germacrone: Effects on mitochondrial apoptosis and ROS-mediated endoplasmic reticulum stress in hepatocellular carcinoma.

Germacrone (GER), a major bioactive constituent isolated from Curcumae Rhizoma volatile oil, exhibits favorable anticancer activity, but poor aqueous solubility and low bioavailability limit its therapeutic efficacy against hepatocellular carcinoma (HCC). In this study, we developed a novel dual-ligand nanoliposome based on chitosan co-modified with lactobionic acid (LA) and folic acid (FA) to achieve targeted delivery of GER (GER@GC-FA-LIP) to liver cancer foci. Transmission electron microscopy, Fourier-transform infrared spectroscopy, and proton nuclear magnetic resonance confirmed the successful conjugation of FA and LA onto chitosan, and the dual-ligand liposomes exhibited a spherical morphology. Characterization of GER@GC-FA-LIP showed that it had a particle size of 166.1 ± 2.9 nm (zeta potential of +28.7 ± 1.0 mV), an encapsulation efficiency of 87.36 ± 1.19%, a drug loading capacity of 5.679 ± 0.076%, and good stability over 7 days. Cellular uptake results showed that GER@GC-FA-LIP exhibited approximately 2.54-fold stronger cellular internalization than free GER. GER@GC-FA-LIP exhibited stronger cytotoxicity, reduced mitochondrial membrane potential, and up-regulated activating transcription factor 4 (ATF4) and CHOP homologous protein, potentially via sustained reactive oxygen species(ROS) production and endoplasmic reticulum stress, and was associated with subsequent apoptosis in HCC cells. In vivo antitumor experiments, immunohistochemistry, H&E staining, and serum biochemical assays collectively showed that the dual-ligand formulation had good safety and the mostpotent antitumor activity. Collectively, these findings demonstrated that GER@GC-FA-LIP is a safe and effective targeted drug delivery system with promising application prospects in HCC therapy.