Spatiotemporally Engineered Protein Delivery via Integrated Covalent Coupling and Pathological Triggers for Precision Microenvironment Reprogramming in Tissue Injury Repair

Abstract Tissue injury presents a complex microenvironment with temporal pathological dynamics, necessitating precision therapeutic strategies. However, current interventions lack effective measures that precisely respond to and modulate the dynamic changes in the microenvironment. Herein, a biomaterial‐driven platform based on covalent conjugation and enzyme‐responsive release, enabling the spatiotemporally controlled delivery of regenerative and immunomodulatory factors, is developed. Following tissue injury, the early‐phase protease thrombin and the delayed‐phase matrix metalloproteinase‐2 (MMP2) serve as endogenous triggers that correspond to distinct stages of the microenvironmental evolution. By site‐specific covalent conjugation via Sortase A (SrtA)‐mediated transpeptidation to intraluminal aligned fibers, combined with outer encapsulation within T‐HA (thrombin‐responsive cleavable hyaluronic acid gel), it is discovered that sequential delivery (CNTF/FGF2 followed by IL‐4) significantly enhances spinal cord injury (SCI) recovery compared to reverse sequencing (IL‐4 followed by CNTF/FGF2). Likewise, SrtA‐mediated protein conjugation to HA demonstrates that FGF2 delivery preceding IL‐4 resulted in superior cardiac functional restoration in the myocardial infarction (MI) model. This spatiotemporally system optimizes the critical therapeutic window for inflammation and tissue remodeling after injury, offering a versatile paradigm for addressing the challenges posed by the dynamic nature of injured tissues.