Ficin-Loaded Quaternized Chitosan Hydrogels: A Functional Biobased Platform for Enhanced Hemostasis

Biopolymer-based hydrogels are promising platforms for hemostatic applications, yet most chitosan-based materials rely on passive plug formation rather than activation of the natural coagulation cascade. This study aimed to develop hemostatic hydrogels based on quaternized chitosan that activate the coagulation cascade through immobilized ficin. N-(2-Hydroxypropyl)-3-trimethylammonium chitosan (HTCC) with varying degrees of quaternization was synthesized and crosslinked with 1,4-butanediol diglycidyl ether to obtain Cross-HTCC hydrogels. The hydrogels were characterized by FTIR spectroscopy, ζ-potential measurements, and swelling tests. Ficin was immobilized by adsorption, and the effects of pH, contact time, and protein content on immobilization efficiency and proteolytic activity were examined. Ficin release and whole-blood coagulation were also assessed. The hydrogels exhibited ζ-potential values above 30 mV and swelling ratios of up to 28.9-fold. Ficin immobilization was optimal at pH 7.4, with maximum sorption reached after ~180 min. Ficin interacted with heterogeneous sorption sites through hydrogen bonding, electrostatic, and hydrophobic interactions, as confirmed by isotherm fitting, FTIR analysis, and molecular dynamics simulations. The immobilized enzyme was released within 24 h and significantly accelerated blood clotting compared with ficin-free hydrogels. These findings demonstrate that ficin-loaded Cross-HTCC hydrogels combine efficient enzyme loading with enhanced hemostatic performance and hold promise as multifunctional hemostatic materials.