Spermine-modified Bombyx Mori Silk Fibroin As A Gene Delivery Carrier

Promoting the formation of vasculature network in three-Dimensional scaffold is critical to stimulate dermal tissue regeneration. Vascular endothelial growth factor(VEGF) and angiopoietin-1(Ang-1) are both vascular endothelial cell-specific angiogenesis growth factors, but their half-lives are shorter. A suitable gene delivery can effectively pack and condense plasmid DNA(p DNA) containing growth factor code, mediating p DNA transfection and expression to stimulate the formation of vasculature network and dermal tissue regeneration. Bombyx mori silk fibroin(BSF) has good biocompatibility, controllable biodegradation, contains a mass of polar amino acids with ionized side chains, making silk fibroin more chemical reaction sites. Surface charge of silk fibroin after chemical modification can turn negative into positive, and silk fibroin may be a gene delivery carrier for angiogenesis growth factors to promote vasculature network regeneration in dermal regeneration scaffold.In this paper, silk fibroin was modified by spermine, making surface charge of silk fibroin turn negative into positive, creating cationic silk fibroin technology; the cationic silk fibroin(CMBSF) and polyethyeneimine(PEI) packed and condensed p DNA containing VEGF and Ang-1 codes to form positively charged complexes which protected p DNA from enzymatic degradation, creating a new VEGF165-Ang-1 coexpression gene delivery. In in vitro experiment, on the one hand the cationic silk fibroin/p DNA complexes transfected EA.hy926 cell to study the influences of the formation ways of the complexes on gene transfection efficiency and cytotoxicity, on the other hand, the cationic silk fibroin/p DNA complexes incorporated the porous silk fibroin scaffold to investigate the effects of the formation ways of the complexes on transfecting endothelial cells and promoting the formation of vasculature network by chick embryo chorioallantoic experiment. In in vivo experiment, the cationic silk fibroin/p DNA complexes incorporated the porous silk fibroin scaffold, and the scaffold was implanted in the back dermal defect of SD rat to research whether or not p DNA can transfect cell in situ wound, promoting vasculature network formation and dermal tissue regeneration.First, Bombyx mori silk fibroin was modified by spermine. EDC/NHS activated the hydroxyl of silk fibroin side chain, and positively charged spermine covalently reacted with the side chain of silk fibroin. Surface zeta potential value of spermine-modified silk fibroin turned negative into positive, and the isoelectric point(p I) of silk fibroin increased from 4.20 to 9.04. Secondly, the influences of the cationic silk fibroin/p DNA complexes on transfecting EA.hy926 cells in vitro were investigated, and the results showed that compared to the PEI/p DNA complexes group,(CMBSF+PEI)/p DNA complexes and CMBSF/BSF/PEI/p DNA complexes had higer tansfection efficiency,better cell viability and less cytotoxicity by laser confocal microscope, scanning electron microscope and cell counting kit-8 reagent. In addition, the effects of(CMBSF+PEI)/p DNA complexes and CMBSF/BSF/PEI/p DNA complexes on the vasculature formation of chick embryo chorioallantoic were studied, and the results indicated that compared to silk fibroin scaffolds without p DNA complexes group and PEI/p DNA complexes group, respectively,silk fibroin scaffolds containing( CMBSF+PEI) /p DNA complexes and CMBSF/BSF/PEI/p DNA complexes had full of blood vessels, and vascularization area ratio of chick chorioallantoic membrane significantly increased by Image-Pro Plus software analysis. Finally, the influences of the cationic silk fibroin /VEGF165-Ang-1p DNA complexes on vasculature network formation and dermal tissue regeneration in the back dermal defect of SD rat were researched, after 1week implantation, new tissue area ratio and vascular growth factors expression had no significant differences between experiment group(silk fibroin scaffold containing p DNA complexes) and control group(silk fibroin scaffold without p DNA complexes group); after 2 week implantation, new tissue area ratio, microvessel density and vascular growth factors expression had significant differences between experiment group and control group; after 4 week implantation in experiment group, new tissue area ratio obviously increased and collagen produced fibrous, but microvessel density and vascular growth factors expression significantly decreased, furthermore, silk fibroin scaffold degraded, and new tissues fully grew into the pore structures of silk fibroin scaffold. These results indicated that silk fibroin scaffold containing p DNA complexes promoted the vasculature formation and simulated the dermal defect regeneration.In this paper, Bombyx mori silk fibroin was modified by spermine to obtain the cationic silk fibroin for the first time. The influences of(CMBSF+PEI)/p DNA complexes and CMBSF/BSF/PEI/p DNA complexes on gene transfection efficiency and cytotoxicity were studied, in addition, VEGF165-Ang-1p DNA complexes incorporated the porous silk fibroin scaffold to study the effect on transfection efficiency, the vasculature formation and dermal defect regeneration. The results showed that(10 μg CMBSF+5 μg PEI)/2 μg p DNA complexes and 20 μg CMBSF/150 μg BSF /10 μg PEI/2 μg p DNA complexes were optimal parameters, and they had better transfection efficiency and less cytotoxicity, moreover, they promoted the vasculature formation and simulated the dermal defect regeneration in chick embryo chorioallantoic and the dermal defect regeneration of SD rat experiments.