Nanodelivery of Y-27632 by RGD-modified liposome enhances radioimmunotherapy of hepatocellular carcinoma via tumor microenvironment matrix stiffness reprogramming

Background: Hepatocellular carcinoma (HCC) causes a significant mortality burden worldwide. Radiotherapy (RT) is the primary locoregional treatment modality for HCC. However, the efficacy of RT in HCC is limited by tumor microenvironment (TME) hypoxia, immunosuppression, and extracellular matrix (ECM) stiffness. Methods: We developed a novel RGD-modified liposomal platform (RGD@LP-Y) that encapsulates the ROCK inhibitor Y-27632 through thin-film hydration. We characterized the RGD@LP-Y by the transmission electron microscope (TEM), UV-Vis spectrophotometer, and dynamic light scattering instrument (DLS). A high-stiffness hydrogel co-culture system mimicking mechanical TME was established to explore the role of RGD@LP-Y on matrix stiffness remodeling. In vitro evaluations included cytotoxicity, reactive oxygen species (ROS) generation, mitochondrial function, immunogenic cell death (ICD) markers, and immune cell activation. Mechanistic investigations encompassed matrix stiffness regulation analysis, flow cytometry profiling of pro-inflammatory macrophages, dendritic cell (DC) maturation, transcriptome sequencing, and western blotting. In vivo validation used xenograft models treated with intravenous RGD@LP-Y and localized RT. Biosafety was confirmed through organ histology, serum biochemistry analysis, and hemolysis assay. Results: RGD@LP-Y downregulated matrix stiffness markers (YAP/COL1) and activated PI3K/AKT/NF-κB signaling to drive pro-inflammatory macrophage polarization and DC maturation. The synergistic effects were observed in combination with RT. The treatment of RGD@LP-Y and RT inhibited HCC proliferation, induced apoptosis, suppressed mitochondrial respiration, elevated intracellular ROS, and thus enhanced ICD. In vivo, RGD@LP-Y+RT demonstrated potent tumor suppression and immune activation without systemic toxicity. Conclusion: RGD@LP-Y enhances RT sensitivity by remodeling ECM stiffness, modulating the hypoxia and immunosuppressive conditions within TME, and enhancing the ICD. The study provides a safe combinatorial approach for HCC therapy.