Cholesterol-butyl-biguanide conjugate as a novel excipient enhances the efficacy of doxorubicin liposomes against triple-negative breast cancer by potentiating immunogenic cell death.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited therapeutic options and poor prognosis. Although doxorubicin (DOX)-loaded liposomes are clinically used, their tumor-targeting efficiency and overall therapeutic efficacy remain suboptimal. To overcome these limitations, we designed and synthesized a novel cationic lipid excipient, cholesterol-butyl-biguanide (CBB), and formulated DOX-loaded CBB liposomes (Lip CBB@DOX) via active loading. Physicochemical characterization confirmed well-defined vesicular morphology, a positive surface charge, and pH-responsive drug release. In vitro, Lip CBB@DOX markedly increased DOX uptake by 4T1 cells, leading to enhanced cytotoxicity, reduced migration and proliferation, and elevated apoptosis. Mechanistic studies showed that Lip CBB@DOX potentiated DOX-induced immunogenic cell death, as indicated by increased calreticulin exposure and HMGB1 release. In a 4T1 murine model, Lip CBB@DOX produced the greatest tumor growth inhibition without aggravating systemic toxicity; histology revealed reduced tumor-associated extramedullary hematopoiesis and extensive tumor necrosis. Collectively, these results indicate that CBB is a safe and effective liposomal excipient that enhances DOX delivery and amplifies ICD-mediated antitumor immunity, offering a promising strategy to improve TNBC therapy and informing future optimization of biguanide-modified liposomal carriers.