The electrospun chitosan nanofibers provide excellent material for immobilized proteolytic enzymes, and are
biocompatible, nontoxic and hydrophilic matrices with large specific area. This paper deals with an application
of electrospun chitosan nanofibers and optimizing conditions for their biofunctionalization by model proteolytic
enzyme trypsin. Nanofibers fromchitosan were prepared using Nanospider™ technology and covalent immobilization
of trypsin followed. Three immobilization techniques preserving biocompatibility and utilizing amine
and/or hydroxyl groups of chitosan were optimized and compared to simple adsorption to achieve maximum
proteolytic activity per cm2 of the functionalized chitosan nanofibers (Tryp-NF). Significant differences were observed.
Trypsin immobilized by the carbodiimide one-step protocol demonstrated the highest activity of the
three procedures, ranging from 132 to 210 IU/cm2 (i.e., 548–874 IU/mg of nanofibers), depending on the initial
amount of trypsin used. Long-term storage stability together with high reusability of Tryp-NF confirmed advantages
of the immobilized enzyme. Tryp-NF showed no cytotoxicity toward growth of HeLa cells. The in vivo tests
for irritation and skin sensitization demonstrated no undesirable skin reactions.