Abstract
Despite the transformative clinical impact of programmed cell death ligand 1/programmed death 1 (PD-1/PD-L1) blockade in hepatocellular carcinoma (HCC), therapeutic efficacy remains limited by the tumor immunosuppressive microenvironment (TIME), with objective response rates persistently below 20 %. To address this critical clinical challenge, we engineered ultrasound (US)-responsive lipid nanobubbles (NBs) co-encapsulating microRNA (miR)-195-5p and shikonin (SK) (designated miR-195-5p/SK-NBs), a dual-functional platform designed to synergize PD-L1 suppression with immunogenic cell death (ICD). The NBs exhibited tumor-selective accumulation through passive and active targeting mechanisms while maintaining biosafety. US-triggered microbubble destruction enabled localized miR-195-5p delivery, achieving PD-L1 downregulation and functionally recapitulating PD-1/PD-L1 blockade. Concurrently, SK-a naphthoquinone compound repurposed as a potent ICD inducer-overcame the systemic toxicity and solubility limitations through NBs-mediated delivery, triggering robust release of damage-associated molecular patterns, including adenosine triphosphate, surface-exposed calreticulin, and secreted high-mobility group box 1 protein. In subcutaneous HCC models, miR-195-5p/SK-NBs reprogrammed the TIME by enhancing splenic lymphocyte proliferation, increasing cytotoxic CD8+ T cell infiltration and IFN-γ production, and increasing proportion and cytotoxic activity of cytotoxic T lymphocyte. The reversed TIME ultimately improved the antitumor efficacy of anti-PD-1 antibody immunotherapy. The therapeutic superiority stems from coordination between PD-1/PD-L1 blockade and ICD-mediated immune activation, presenting a promising strategy for hepatocellular carcinoma treatment.