Verification and validation of the horseradish peroxidase (HRP)-mediated enzymatic cross-linking in collagen/PEG hydrogels for wound dressing applications.

Collagen/poly(ethylene glycol) (COL/PEG) hydrogels are attractive wound dressings but typically rely on weak hydrogen bonding at the polymer interface. Here, a catechol-enzymatic strategy is introduced in which dopamine (DA) self-polymerization reduces graphene oxide to conductive rGO while horseradish peroxidase/hydrogen peroxide (HRP/H2O2) catalyzes covalent coupling between native PEG hydroxyls and collagen phenolic residues. Spectroscopic and thermo-mechanical signatures, attenuation of -OH/amide bands in FTIR, a glass-transition shift from -9 °C to -4 °C, increased storage modulus with elastic dominance (E' ≫ E″, tan δ < 0.3), and higher thermal decomposition temperatures, are consistent with a denser network and the formation of PEG-COL ether linkages without prior PEG or collagen functionalization. Microstructural analyses (SEM/AFM) show greater fibrillar interconnectivity and roughness, while XRD indicates increased amorphous character upon enzymatic cross-linking. Functional performance relevant to wound care was demonstrated: rapid exudate uptake (equilibrium swelling 94 % within 10 min), broad-spectrum antibacterial activity (>99 % reduction of E. coli and ≥90 % of S. aureus), sustained antioxidant capacity, and cytocompatibility (≥80 % viability in human dermal fibroblasts and keratinocytes) with negligible hemolysis (<1 %). In vitro scratch assays reached 100 % closure by 48 h. In a porcine full-thickness wound model, HRP-cross-linked, rGO-reinforced COL/PEG hydrogels achieved accelerated re-epithelialization by day 21, robust angiogenesis and granulation, and no detectable irritation or systemic pathology. These results establish HRP-mediated, DA-assisted cross-linking as a minimally modified route to conductive, antioxidant COL/PEG hydrogels with tunable mechanics and clinically relevant regenerative efficacy.