In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

ABSTRACT Diabetic Foot Ulcer (DFU), a severe chronic diabetes complication with low healing and high recurrence rates, is a major global health challenge. Overexpression of matrix metalloproteinase‐9 (MMP‐9) and the consequent MMP‐9/TIMP1 (tissue inhibitor of metalloproteinase 1) imbalance delays healing by degrading the extracellular matrix, impairing granulation tissue formation, and exacerbating inflammation. Conventional therapies do not dynamically respond to fluctuating protease levels in chronic wounds. This study introduces an MMP‐9‐responsive protein release system (M9RR), where the therapeutic protein (i.e., TIMP1) is linked to a membrane‐anchoring domain at its C‐terminus via an MMP‐9‐cleavable peptide, thereby exposing the fusion protein to the extracellular side of cell membranes. The system enables targeted release of TIMP1 in high‐MMP‐9 microenvironments, thereby neutralizing excessive MMP‐9 activity. In vitro, M9RR demonstrates MMP‐9 specificity, broad mammalian cell applicability, and protection of HaCaT keratinocytes and BJ fibroblasts from MMP‐9‐induced damage. In db/db diabetic mice, the MMP‐9‐responsive TIMP1 release system (TIMP1 M9RR ) significantly improves wound contraction, granulation tissue formation, epithelial regeneration, and collagen remodeling. Additionally, a cryomicroneedle (CryoMNs)‐based co‐delivery system for basic fibroblast growth factor (bFGF) and TIMP1 M9RR shows effective diabetic wound healing. This modular M9RR system offers a precise, adaptive therapeutic strategy for DFU and holds promise for other MMP‐related diseases.