Silk Fibroin Nanofiber–Immobilized Horseradish Peroxidase Enables Continuous Preparation of Injectable Hydrogels With Negligible Residual Enzyme for Drug Delivery

ABSTRACT Injectable hydrogels formed via horseradish peroxidase (HRP)‐catalyzed crosslinking in the presence of hydrogen peroxide (H 2 O 2 ) are promising materials for drug delivery and tissue engineering because of their tunable drug release profiles and mechanical properties. However, the potential immunogenicity of residual HRP poses a major challenge for clinical use. In this study, an HRP‐immobilized mechanically ground silk fibroin nanofiber (SFNF‐HRP) membrane flow‐through reactor was developed to prepare injectable hyaluronic acid (HA) hydrogels with negligible HRP contamination. The reactor enables continuous crosslinking of phenol‐modified HA (HA‐Ph) under physiological conditions. Enzyme‐linked immunosorbent assay (ELISA) signals from the resulting hydrogels were close to the assay's limit of detection (2.01 ng/mL), precluding reliable quantification of residual HRP. The mechanical properties of the hydrogel were readily tuned by varying the linear velocity of the precursor solution, yielding Young's moduli of 0.96–1.7 kPa. The uniform crosslinking density of the hydrogels was confirmed by the consistent Young's modulus observed throughout the 1.5 h continuous preparation. The system also enabled the sustained release of macromolecular model drugs. Release rates could be tuned by controlling the linear velocity during hydrogel preparation, supporting the potential utility of this continuous platform for advanced drug delivery systems.