Silk fibroin has been widely used in biomedical and clinical fields owing to its good biocompatibility. In the present work, self-crosslinking of fibroin molecules was carried out using the hydrogen peroxide (H2O2)-horseradish peroxidase system, followed by preparation of the fibroin membranes, aiming at improving the mechanical property of fibroin-based material and expanding its applications. P-Hydroxyphenylacetamide (PHAD), as the model compound of tyrosine residues in fibroins, was used to investigate the possibility of horseradish peroxidase (HRP)-catalyzed crosslinking. The results were characterized by means of 1H NMR and UPLC-TQD. The efficacy of enzymatic crosslinking of silk fibroins was examined by determining the changes in the relative viscosity, amino acid compositions, and SEC chromatogram. The obtained data indicated that H2O2-HRP incubation led to PHAD polymerization, and the molecular weight of fibroin proteins was also noticeably increased after the enzymatic treatment. CD and ATR-FTIR spectra revealed that H2O2-HRP treatments had an evident impact on the conformational structure of silk fibroins. The mechanical property and thermal behavior for the modified fibroin membrane were noticeably improved compared to the untreated. Meanwhile, the obtained membrane exhibited good biocompatibility according to the cell growth experiment. The present work provides a novel method for preparation of the fibroin-based materials for biomedical applications.
Keywords: Enzymatic crosslinking; H2O2; Horseradish peroxidase (HRP); P-Hydroxyphenylacetamide (PHAD); Silk fibroin.