MMP2-Responsive Minicells Co-Delivering Melittin and Doxorubicin Mitigate Stromal Barriers to Boost Solid Tumor Chemotherapy

Abstract The extracellular matrix (ECM) secreted by cancer-associated fibroblasts (CAFs) impedes deep intratumoral infiltration of chemotherapeutic agents and immune cells, resulting in insufficient intratumoral drug accumulation and compromised antitumor efficacy. In this study, we engineered genetically bacterial minicell as a tumor microenvironment-responsive delivery platform to remodel CAFs and facilitate the deep intratumoral penetration of chemotherapeutics. Specifically, the carrier system was designed by encapsulating doxorubicin (DOX) within minicells functionalized with a matrix metalloproteinase 2 (MMP2)-cleavable melittin precursor fusion peptide, designated DOX@MCm. Upon exposure to elevated MMP2 levels in tumor tissue, active melittin is locally released, promoting DOX release while simultaneously enhancing its deep-tissue penetration by inhibiting CAF activation. In both standard and stroma-rich subcutaneous 4T1 tumor models, systemic DOX@MCm treatment significantly restrained primary tumor progression, ameliorated intratumoral fibrosis, elevated intratumoral cytotoxic CD8+ T cell populations, and exhibited minimal systemic toxicity, with no detectable organ injury or body weight loss. Furthermore, DOX@MCm markedly suppressed lung metastasis in stroma-rich subcutaneous 4T1 tumor models. Collectively, this MMP2-activatable melittin-engineered minicell delivery system achieves enhanced deep intratumoral penetration, stromal reprogramming, protease-triggered drug liberation and profoundly augmented antitumor immunity. The DOX@MCm establishes a biocompatible nanoplatform designed for synergistic therapy for stroma-rich solid tumors.