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Gelatin (GE) is a renewable biopolymer with abundant active groups that are beneficial for manufacturing functional biomaterials via GE modification. An antibacterial fibrous GE film was prepared by electrospinning the modified GE in an aqueous solution. The original GE was modified by reacting it with N,N-dimethyl epoxypropyl octadecyl ammonium chloride (QAS), and then it was cross-linked with transglutaminase (TGase). FTIR analysis illustrated that QAS was grafted onto GE through the epoxy ring-opening reaction, and the modification did not influence the main GE skeleton structure. The investigation of the solution properties showed that the grafted cationic QAS group was the main factor that decreased the surface tension of the solution, increased the electrical conductivity of the solution, and endowed GE with antibacterial activity. TGase cross-linking clearly influenced the rheological properties such that the flow pattern of the spinning solution varied from Newton-type to shear thinning, and the aqueous solution of GE-QAS-TGs transformed from liquid-like to solid-like and even induced gelatinization with increasing TGase content. A satisfactory fibrous morphology of 200–500 nm diameter was obtained using a homemade instrument under the optimized electrospinning conditions of a temperature of 35 °C, a distance between electrodes of 12 cm, and a voltage of 15 kV. The study of film properties showed that the antibacterial activity of the fibrous GE film depended only on the grafted quaternary ammonium, whereas the thermostability, wettability, and permeability were greatly influenced by both the TGase cross-linking and film-forming methods. Cytotoxicity was tested using the CCK-8 and live/dead kit staining methods in vitro, which showed that the modified GE had good biocompatibility.

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