In Vivo Antitumor Apoptosis, in Vitro Cytotoxicity and ROS Assay Studies of Macrophage Membrane-Camouflaged Redox-Sensitive Liposomes for Breast Cancer Therapy

Abstract

The present study investigated the in vitro cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, and in vivo antitumor efficacy of biomimetic redox-sensitive liposomes co-loaded with doxorubicin (DOX) and indocyanine green (ICG) and camouflaged with macrophage membrane. The developed macrophage membrane-camouflaged redox-sensitive liposomes (MM-RSL/DOX/ICG) were designed to provide glutathione-responsive drug release and combined chemo-photothermal therapy. Cytotoxicity was evaluated in 4T1 breast cancer cells using the MTT assay, while apoptosis and intracellular ROS generation were assessed using Annexin V-FITC/PI staining and DCFH-DA, respectively. The antitumor activity was further investigated in 4T1 tumor-bearing BALB/c mice following intravenous administration of the formulations, with or without 808 nm laser irradiation. The MM-RSL/DOX/ICG formulation exhibited enhanced cytotoxicity compared with free DOX and non-camouflaged liposomes, with an IC₅₀ of 0.62 µg/mL, which was further reduced to 0.18 µg/mL following 808 nm laser irradiation. Combination treatment demonstrated synergistic cytotoxic activity, with a combination index ranging from 0.28 to 0.62. Annexin V-FITC/PI analysis showed substantial induction of apoptosis, with the laser-treated MM-RSL/DOX/ICG group producing 48.5% early apoptotic and 33.2% late apoptotic cells. ROS generation was also markedly increased, reaching 158 ± 12 a.u. compared with 28 ± 4 a.u. in the saline group. In vivo, the laser-assisted MM-RSL/DOX/ICG formulation produced the highest tumor growth inhibition (94.1 ± 1.4%) at day 21, with near-complete tumor regression observed in 4 of 8 animals. Furthermore, the biomimetic formulations showed an improved safety profile compared with free DOX/ICG, with comparatively normal biochemical parameters and major-organ histology. Overall, the findings demonstrate that macrophage membrane-camouflaged redox-sensitive DOX/ICG liposomes can enhance cytotoxicity, apoptosis, ROS-mediated activity, and antitumor efficacy, particularly when combined with 808 nm laser irradiation, supporting their potential as a biomimetic chemo-photothermal therapeutic platform for breast cancer.