Antheraea Pernyi Silk Fibroin For Targeted Gene Delivery Of VEGF165-Ang-1

Vascularization is the crucial challenge in tissue engineering. One solution for thisproblem is to implant scaffolds that contain functional genes that promote vascularizationby providing angiogenic growth factors. Vascular endothelial growth factor (VEGF,primarily VEGF165) and angiopoietin (Ang, primarily Ang-1) are the most importantgrowth factors that promote angiogenesis and exclusively target vascular endothelial cells;both play a key role in maintaining vascular homeostasis. Growth factors are vulnerable tothe environment, the temperature and various enzymes in vivo because of their reducedhalf-lives and poor stability. The combined transfection of exogenous genetic material witha scaffold material that promotes vascularization by providing VEGF and Ang-1couldovercome the limitations associated with directly loading the growth factors. Geneexpression vectors and delivery vehicles are highly efficient at gene delivery. PEI has arelatively high transfection efficiency based on the proton sponge effect as a gene carrierfor non-viral vectors. PEI is relatively cytotoxic because of the high density of cationicgroups and non-biodegradable. ASF has favorable cytocompatibility, is biodegradable, andpossesses RGD sequences and a negative charge. So we chose ASF and PEI as the deliveryvectors to simultaneously carry VEGF165and Ang-1. We hypothesized that this techniquecould improve the cytotoxicity and improve the efficiency of gene transfection.This paper studied the transfection and cytotoxicity that ASF combined pDNAencoding VEGF165and Ang-1(GFP as a report gene) and subsequently in conjunctionwith PEI to transfect EA.hy926cells to find the best preparation of complexes. And westudied the transfection efficiency of ASF/PEI/DNA complexes to transfect EA.hy926cellsin vitro and the function of the ASF/PEI/DNA complexes on promoting the formation ofblood vessels by choosing chicken embryo villus allantois membrane (CAM) as a model.To preliminary discuss the ASF/PEI/DNA transfection efficiency, we used theASF/PEI/DNA complexes transfection EA.hy926cell and transfection was observed byLSCM and GFP-positive percentage was detected in the flow cytometer. Results showed that transfection efficiency of ASF/PEI/DNA(60/3/2) was higher than that ofASF/PEI/DNA (30/3/2) and ASF/PEI/DNA (90/3/2).Then we studied the influence of the proportion of ASF/PEI/DNA on cell transfectionand cytotoxicity to find the best preparation of complexes. The result showed that theincrease of ASF will not seriously affect the quality of the compound of the transfectionefficiency when the quality ratio of PEI/DNA is10μg/2μg. And the result measured bythe MTT test showed that cell survival rate decreased with the increase of absolute qualityratio. The transfection and cell viability of ASF/PEI/DNA(60/3/2) was higher thanPEI/DNA.Finally, we studied the transfection efficiency by implanting scaffolds that containedASF/PEI/DNA(60/10/2) complexes to transfect cells. The result of2D which was observedby CM-DiL and SEM showed that a large number of cells could be seen in the interior andthe surface of the material, and cells could be seen completely spreading state in bracket.The quantity of cells of the3d were less than that of the1d, but the cells significantlyincreased during the following days. The ASF/PEI/DNA(60/10/2) complexes promoted theformation of blood vessels on chicken embryo villus allantois membrane (CAM) bysecreting corresponding growth factor while the DNAencoding VEGF165andAng-1.We developed a novel method that studied the influence of the ASF/PEI/DNA ratioon transfection efficiency and cytotoxicity, and we also studied the transfection efficiencyand promotion on the formation of blood vessels of ASF combined pDNA encodingVEGF165and Ang-1and subsequently in conjunction with PEI to transfect EA.hy926cells implanted in the scaffold. The flow cytometry and confocal microscopy data revealedthat the transfection efficiency was significantly improved compared with PEI/DNA. Andwe found that the best preparation of complexes was ASF/PEI/DNA(60/10/2). Thecomplexes could also promote the formation of blood vessels.