Multifunctional Lipid Nanoparticles Remodeling Tumor Immune Microenvironment for Breast Cancer Chemo-Immunotherapy

Background: Breast cancer treatment is often hampered by the immunosuppressive tumor microenvironment (TME). To improve therapeutic efficacy, this study developed a folic acid-chitosan (FA-CS)-modified liposomal formulation co-delivering doxorubicin (DOX) and resiquimod (R848) for combined chemotherapy and immune modulation. Methods: The FA-CS-R848/DOX@Lip liposomes were prepared by rotary evaporation and characterized for morphology, particle size, zeta potential, drug encapsulation efficiency (EE), drug loading (DL) capacity, and drug release profiles. Cellular uptake and cytotoxicity were determined to assess the biological effects of the formulation. Antitumor efficacy and biosafety were assessed in an EO771 tumor-bearing mouse model. The macrophage phenotype, TME composition, and CD8⁺ T cell cytotoxic function were evaluated by flow cytometry. Results: The FA-CS-R848/DOX@Lip exhibited a relatively uniform spherical morphology, with an average size of 221.53 nm and a zeta potential of 5.06 mV. The EE (%) of R848 and DOX were over 70% and 80%, respectively, while the DL (%) capacities were 4.6% and 3.1%. Drug release studies showed a slow release profile. In vitro, FA-CS-R848/DOX@Lip showed greater cytotoxicity than non-targeted liposomes (p < 0.01) and higher uptake in EO771 cells and RAW264.7 macrophages. In vivo, treatment with FA-CS-R848/DOX@Lip significantly inhibited tumor growth compared with saline (p < 0.0001) without causing significant hematological or biochemical toxicity (p > 0.05). In addition, FA-CS-R848/DOX@Lip increased the MHC II/CD206 ratio of tumor-associated macrophages (TAMs), elevated the proportions of CD8⁺ and CD4⁺ T cells, reduced the proportion of myeloid-derived suppressor cells (MDSCs), and enhanced the percentages of IFNγ⁺ and Ki67⁺ CD8⁺ T cells in vivo. Conclusions: FA-CS-R848/DOX@Lip exerts potent antitumor activity with favorable biosafety in vivo. Its therapeutic effect may be associated with improved immune activation and remodeling of the TME, supporting its potential as a chemo-immunotherapeutic strategy for breast cancer.