Sequential Mannose-Targeted Nanotherapy Orchestrates Tumor-Associated Macrophage Repolarization and Chemotherapeutic Response in Triple-Negative Breast Cancer

Recently, explorations are being undertaken involving chemo-immunotherapy based on targeting cancer cells and tumor-associated macrophages (TAMs). However, these strategies mainly focus on delivering TAM modulators and chemotherapy simultaneously, without considering the effect of chemotherapy on TAMs and which may compromise TAM-modulatory action. This necessitates investigations involving sequential treatment wherein pretreatment would enable efficient M2-to-M1 repolarization before administration of chemotherapy. In this study, mannose-targeted liposomes encapsulating resiquimod (M-LIPO-RES) and doxorubicin (M-LIPO-DOX) were developed to target M2-type TAMs and cancer cells, respectively. The formulations (M-LIPO-RES and M-LIPO-DOX) were characterized for particle size, zeta potential, surface chemistry, morphology, drug entrapment efficiency, release profile, and hemolysis potential. The results showed that mannose-targeted liposomes possess a marked increase in cellular uptake in M2-polarized macrophages and MDA-MB-231 cells, revealing their potential for receptor-mediated cellular internalization. M-LIPO-RES-mediated macrophage polarization from the M2 to M1 phenotype was confirmed by analysis of the cytokine profile and estimation of TAM-associated surface proteins (e.g., CD206 and CD86), both in vitro and in vivo. The biodistribution study indicated the preferential accumulation of the developed formulation at the tumor site following intravenous administration. Furthermore, the anticancer effect was studied in the immunocompetent 4T1 mouse model, which indicated improved anticancer effect after sequential treatment (M-LIPO-RES followed by M-LIPO-DOX). Additionally, cardiac markers (i.e., troponin and CK-MB) were decreased in the targeted formulation group as compared to other treated groups, indicating a favorable cardiac safety profile. The presented targeted nano strategy preserves the immune-modulatory effect and reveals great potential for immuno-chemo response in triple-negative breast cancer (TNBC) therapy.