No abstract

Gelatin-based water-insoluble nanofibers with a diameter of 160 nm were obtained from electrospinning aqueous solutions containing gelatin with phenolic hydroxyl (Ph) moieties (Gelatin-Ph) and horseradish peroxidase (HRP). The water insolubility of the nanofibers was accomplished through HRP-catalyzed cross-linking of the Ph moieties by exposing the electrospun nanofibers to air containing hydrogen peroxide. The HRP activity in the electrospun nanofibers was 65% that of native HRP. The cytocompatibility necessary for tissue engineering applications of the water-insoluble Gelatin-Ph nanofibers was confirmed by the adhesion and viability of human embryonic kidney-derived HEK293 cells.

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The delivery of nucleic acids is indispensable for tissue engineering and gene therapy. However, the current approaches involving DNA/RNA delivery by systemic and local injections face issues such as clearance, off-target distribution, and tissue damage. In this study, we report plasmid DNA (pDNA) delivery using gelatin electrospun nanofibers obtained through horseradish peroxidase (HRP)-mediated insolubilization. The nanofibers were obtained through the electrospinning of an aqueous solution containing gelatin possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph) and HRP with subsequent HRP-mediated cross-linking of the Ph moieties by exposure to air containing 16 ppm H2O2 for 30 min. Then, Lipofectamine/pDNA complexes were immobilized on the nanofibers through immersion in the solution containing the pDNA complexes, resulting in transfection and sustained delivery of pDNA. Cells cultured on the resultant nanofibers expressed genome-editing molecules including Cas9 protein and guide RNA (gRNA), resulting in targeted gene knock-in and knock-out. These results demonstrated the potential of Gelatin-Ph nanofibers obtained through electrospinning and subsequent HRP-mediated cross-linking for gene therapy and tissue regeneration by genome editing.