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

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.