Molecular-weight-dependent antioxidant functions of silk fibroin fragments for engineering sustained-release composite hydrogels

Chronic wounds have persistent oxidative stress impairing tissue regeneration, necessitating antioxidant strategies that are both biologically effective and durable. While silk fibroin (SF) exhibits intrinsic antioxidant activity, how its molecular weight governs antioxidant performance across chemical and cellular contexts remains unclear. We systematically elucidate SF's molecular-weight-dependent antioxidant behavior and translate these insights into designing a sustained-release composite gel. SF fragments spanning ultra-low to high molecular weights were prepared and characterized. Ultra-low-molecular-weight silk fibroin (∼10.7 kDa) exhibited the strongest chemical antioxidant activity, achieving >60% DPPH scavenging and > 95% hydroxyl radical elimination, attributed to enriched tyrosine content and reduced β-sheet crystallinity. However, this superior radical-scavenging capacity did not translate directly to cellular repair. Instead, low-molecular-weight silk fibroin (L-SF, ∼27 kDa) provided the most robust and concentration-stable cellular protection, significantly restoring viability in oxidatively damaged fibroblasts and enhancing intracellular antioxidant defenses, including catalase (CAT) and superoxide dismutase activities ( p  < 0.01). Building on this divergence, a composite high-molecular-weight silk fibroin/sodium carboxymethyl cellulose (H-SF/CMC-Na) hydrogel was engineered using H-SF for mechanical reinforcement and L-SF surface coating to impart sustained antioxidant activity. The optimized formulation (H-SF: CMC-Na = 2: 1, 40% solids) satisfied mechanical and conformability requirements for wound dressing applications and enabled sustained release of bioactive SF over 24 h. Gel extracts significantly improved cellular antioxidant indicators, increasing CAT activity 7.6-fold. This work establishes molecular-weight-guided design as a unifying principle for SF-based antioxidant biomaterials, demonstrating that effective wound dressing candidates must balance radical-scavenging capacity with cellular compatibility and sustained biological function.