Micro- and nanoparticles of chitosan and carboxymethyl chitosan were synthesized, both with and without ascorbic acid. Methods were developed to form complexes between these micro- and nanoparticles and plant proteases—ficin, papain, and bromelain. It was demonstrated that the activity of cysteine protease complexes with carboxymethyl chitosan micro- and nanoparticles was higher compared to those with chitosan micro- and nanoparticles. Additionally, the complexes of ficin, papain, and bromelain with chitosan and carboxymethyl chitosan micro- and nanoparticles synthesized in the presence of ascorbic acid exhibited greater proteolytic activity than those formed with particles prepared without ascorbic acid. Molecular docking studies revealed that the amino acid residues of ficin, papain, and bromelain primarily interact with chitosan and carboxymethyl chitosan through hydrogen bonding and hydrophobic interactions. The amino acid residues in the active sites of these enzymes participate in a complex formation, which likely contributes to the increased activity and stability of cysteine proteases in complexes with chitosan and carboxymethyl chitosan micro- and nanoparticles.

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.

Bleeding remains a leading cause of death in trauma and surgery, which drives the search for effective local hemostatic materials. Chitosan is a promising candidate because of its biocompatibility, biodegradability, and intrinsic hemostatic activity, yet chitosan hydrogels remain poorly characterized in terms of hemocompatibility. This study assessed the hemocompatibility and hemostatic activity of chitosan hydrogels with varying degrees of crosslinking and their complexes with ficin. Hydrogels were synthesized using 1,4-butanediol diglycidyl ether and characterized by ζ-potential measurements. Hemocompatibility was evaluated through hemolysis, hemoglobin spectroscopy, erythrocyte cytoarchitecture, and lymphocyte viability assays. Ficin was immobilized by adsorption and covalent binding, and hemostatic properties were assessed via swelling and kinetic blood coagulation. The hydrogels showed a statistically significant inverse dose-dependent hemolytic effect, attributed to their low colloidal stability. In contrast, hemoglobin spectra, erythrocyte cytoarchitecture, and lymphocyte viability showed no statistically significant changes. Adsorption immobilization provided higher protein loading and proteolytic activity than covalent binding. Ficin-containing compositions significantly accelerated blood coagulation compared with pristine hydrogels. These findings confirm the potential of the developed hydrogels and their ficin complexes as hemocompatible hemostatic materials.