Formulation, Optimization, And Pharmacological Evaluation Of Macrophage Membrane-Camouflaged Redox-Sensitive Liposomes For Breast Cancer Theranostics

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive malignancy associated with limited targeted therapeutic options, high relapse rates, and considerable toxicity from conventional chemotherapy. Doxorubicin (DOX) remains an important therapeutic agent, but its clinical utility is limited by systemic toxicity and nonspecific distribution. The present study aimed to develop and optimize a macrophage membrane-camouflaged, redox-sensitive liposomal nanocarrier co-encapsulating doxorubicin and indocyanine green (MM-RSL/DOX/ICG) for combined chemotherapy, photothermal therapy, and fluorescence imaging of breast cancer. Methods: MM-RSL/DOX/ICG was prepared by incorporating DOX and ICG into disulfide-containing liposomes followed by macrophage membrane coating. A 3² full factorial design was employed to optimize the lipid-to-drug molar ratio and DSPE-PEG-SS content using particle size, polydispersity index (PDI), and DOX and ICG encapsulation efficiencies as responses. The optimized formulation was characterized for physicochemical properties, morphology, encapsulation efficiency, colloidal stability, glutathione (GSH)-responsive drug release, in-vitro cytotoxicity, and chemo-photothermal activity. Its therapeutic performance was further evaluated in a 4T1 breast cancer model, including survival and safety assessments. Results: The factorial design identified a lipid-to-drug molar ratio of 10:1 and DSPE-PEG-SS content of 12.5 mol% as the optimum formulation conditions. The optimized MM-RSL/DOX/ICG exhibited a particle size of 158.4 ± 6.1 nm, PDI of 0.262 ± 0.022, and zeta potential of −21.3 ± 1.1 mV, with DOX and ICG encapsulation efficiencies of 80.4 ± 2.4% and 77.6 ± 2.3%, respectively. The formulation maintained satisfactory colloidal stability for 30 days. GSH markedly accelerated drug release, with cumulative release at 48 h reaching 88.0% for DOX and 84.0% for ICG under GSH-rich conditions, compared with substantially lower release under physiological conditions. In 4T1 cells, MM-RSL/DOX/ICG combined with 808-nm laser irradiation showed enhanced cytotoxicity, with an IC₅₀ of 0.18 µg/mL and combination-index values below 1, indicating synergistic chemo-photothermal activity. In vivo, the combined formulation with laser irradiation increased median survival to 58 days, compared with 24 days for saline controls, while maintaining body weight and showing comparatively favorable biochemical, hematological, and histopathological safety profiles. Conclusion: The optimized macrophage membrane-camouflaged redox-sensitive liposome demonstrated efficient co-delivery of DOX and ICG, GSH-responsive release, enhanced in-vitro chemo-photothermal cytotoxicity, prolonged survival, and an improved safety profile in the experimental breast cancer model. The findings support MM-RSL/DOX/ICG as a promising multifunctional theranostic platform for TNBC, although further validation in advanced tumor models, comprehensive toxicological studies, and translational investigations are required before clinical application.