Construction of ATOX1 targeted nano gel based on cuproptosis mechanism and its role in reversing TACE refractoriness of hepatocellular carcinoma

INTRODUCTION: Transarterial chemoembolization (TACE) is the standard treatment for intermediate-stage hepatocellular carcinoma (HCC); however, TACE refractoriness remains a major clinical challenge. Increasing evidence suggests that post-embolization hypoxia-driven metabolic reprogramming, particularly dysregulated copper homeostasis, contributes to therapeutic resistance. OBJECTIVE: This study aimed to elucidate the role of copper metabolism in TACE-refractory HCC and to develop a mechanism-driven nanoembolic strategy targeting the copper chaperone ATOX1 to induce cuproptosis and reverse TACE refractoriness. METHODS: Clinical serum samples from HCC patients with TACE refractoriness or favorable response were analyzed for copper levels and copper transport-related proteins. A thermosensitive nanogel (Cu²⁺/DC_AC50@PNA) co-loaded with copper ions and the ATOX1 inhibitor DC_AC50 was synthesized and characterized. Its embolic performance, antitumor efficacy, and biosafety were evaluated in rabbit renal artery and VX2 orthotopic liver tumor models. In vitro assays using Huh-7 and LM3 cells under normoxic and hypoxic conditions assessed cell viability, migration, invasion, and cuproptosis-related signaling. RESULTS: Patients with TACE refractoriness exhibited significantly elevated serum copper levels and increased ATOX1 and ATP7B expression, indicating systemic copper dyshomeostasis. Cu²⁺/DC_AC50@PNA demonstrated favorable physicochemical properties, sustained embolic capability, and excellent biocompatibility. In vivo, this nanoembolization system markedly suppressed tumor growth, increased tumor necrosis, reduced metastasis, and prolonged survival compared with conventional cTACE. Mechanistically, Cu²⁺/DC_AC50@PNA disrupted intracellular copper transport, activated cuproptosis, inhibited HIF-1α/VEGF-mediated angiogenesis, and remodeled the tumor immune microenvironment by enhancing CD8⁺ T-cell infiltration. In vitro findings corroborated potent antitumor and anti-metastatic effects, particularly under hypoxic conditions. CONCLUSION: Copper metabolism dysregulation is a defining feature of TACE-refractory HCC. Targeting ATOX1-mediated copper homeostasis using Cu²⁺/DC_AC50@PNA represents a novel and effective strategy to induce cuproptosis, overcome TACE refractoriness, and improve therapeutic outcomes in HCC.