Silk Fibroin As A Non-viral Vector For Angiogenesis Gene

Revascularization is the key challenge in tissue engineering and in situ tissueregeneration. Transgenic technology can import angiogenesis growth factor genes intotarget cells to express certain growth factor protein and contribute to angiogenesis.Vascular endothelial growth factor (VEGF165) and angiopoietin-1(Ang-1) have beenidentified as two key angiogenesis growth factors. VEGF promotes the formation ofprimitive vascular network in the early formation of blood vessels; Ang-1plays a role inthe vascular reconstruction and shaping, as well as promotes the formation of a matureand spatial structure of vascular network. Combination of VEGF165and Ang-1can notonly enhance angiogenesis but also obtain stable blood vessels. Gene delivery vector isneeded for importing gene into target cells. The branched polyethylenimine (PEI,25kDa)has been the most successful non-viral gene vector. Due to its high positive charge, PEIhas high transfection efficiency, but it has high cytotoxicity. Silk fibroin (SF) is a naturalprotein and presents excellent cell biocompatibility and biodegradability. As an anionicpolymer, SF can not combine with DNA directly. It can bind to the cationic PEI/DNAcomplex electrostatically to modify the complex surface and lower cytotoxicity. At thesame time, ASF has tripeptide sequence RGD which is known to exhibit specificinteractions with cells. It can interact specifically with receptors of vascular endothelialcells and fibroblast to replace the non-specific electrostatic interaction between PEI andcells.This paper develops SF and PEI as VEGF165and Ang-1gene delivery vector. Theshape, structure, physical characteristics, cytotoxicity and transfection efficiency wasstudied compared with PEI/DNA. Its mechanism was also discussed.First, PEI/DNA complex was prepared (DNA2μg/ml). When N/P is above3, PEIcan entrap DNA completely. MTT assay demonstrate that survival rate of cells is morethan90%when PEI is1-5μg/ml. When PEI is3μg/ml (DNA2μg/ml), GFP-positivepercentage is highest. Here PEI/DNA is5μg/ml (3μg PEIå'Œ2μg DNA), which canboth obtain highest transfection efficiency and lowest cell cytotoxicity. ASF and BSF were used to encapsulate PEI/DNA to obtain ASF/PEI/DNA andBSF/PEI/DNA complexes. EDS can test the wt%of different elements on the surfaceof complexes and demonstrated ASF can bind to the cationic PEI/DNA complex. Theresults of AFM and DLS indicated ASF and BSF can both entrap PEI/DNA. PEI/DNAshowed274.3±3.4nm particle size and ASF/PEI/DNA showed337.6±7.4nm particlesize. The addition of silk fibroin to the PEI/DNA complex decreased its ξ-potentials.ASF/PEI/DNA complex can avoid hydrolysis.L929cells were added ASF/PEI/DNA and BSF/PEI/DNA, transfection wasobserved by LSCM and GFP-positive percentage was detected in the flow cytometer.Results showed that transfection efficiency of ASF/PEI/DNA was higher than that ofPEI/DNA. The results of CHL cells were the same.1d and2d after transfection,GFP-positive percentage of ASF/PEI/DNA (60:3:2) was higher than PEI/DNA. Theresults shows ASF and PEI can raise transfection efficiency of VEGF-Ang-1DNA.Cell cytotoxicity of ASF/PEI/DNA, PEI/DNA and BSF/PEI/DNA was examined.Survival rate of cells with ASF/PEI/DNA was higher than that of PEI/DNA. Theaddition of VEGF165and Ang-1genes raised VEGF more greatly than the addition ofVEGF165. ASF/PEI/DNA group showed the most VEGF than any other group. Thisindicates ASF and PEI can not only raise transfection efficiency, but also can makeVEGF gene express and secrete VEGF.This paper uses Antheraea pernyi silk fibroin and PEI for the first time to serve asdelivery vector for VEGF165and Ang-1genes. The results showed improvedtransfection efficiency and low cytotoxicity, providing a new non-viral gene vector fortissue engineering and in situ tissue regeneration.