Preparation Of Functionalized Graphene And Its Cytotoxicity And Gene Transfection Performances

The large specific surface area and the aromatic conjugated structure,and etc of graphene provide for the loading of gene and other chemical drugs. However, the pristine graphene has disadvantages such as aqueous dispersion and easy of agglomeration, which limit its applications,especially in biomedical fields. The focus of current researches concentrate on using appropriate methods to modify graphene, which can increase its dispersion properties and biocompatibility. This paper will use mixed acid(H2SO4 and HNO3,V/V=3:1) oxidation of graphene are oxide graphene(GO),and analyze why the graphene oxide-chitosan-arginylglycylaspartic acid peptide nanoparticle(GO-CHIT-RGD NPs) produce greater toxicity on melanoma cells(A375). This research also combines the features of arginylglycylaspartic acid peptide(RGD), aminophenylboronic acid(ABA), 4-aminophenyl β-D-Galactopyranoside(GAL), polyamidoamine(PAMAM) and gluconic acid(GA) and uses 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) and N-hydroxy-succinimide(NHS) coupling method to make composite materials of GO-RGD, GO-ABA,GO-GAL and GO-PAMAM-GA. This paper evaluated the toxicity of GO-RGD, GO-ABA and GO-GAL composite materials on cell cytotoxicity, and analyse the selective cytotoxicity of different cell lines and evaluate GO-PAMAM-GA composite materials’ transfection properties on different cells as gene vectors. The following are the specific steps:1. The success of the graphene oxidation to GO was verified and characterized by x-ray photoelectron spectroscopy(XPS), Fourier transform infrared(FTIR) spectroscopy, transmission electron microscopy(TEM) and zeta potential; The GO cytotoxicity was evaluated by(3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyl tetrazoliumbromide(MTT) and water-soluble tetrazolium salts(WST-8) methods. the rate of αVβ3 on A375, He La and MCF-7 cells were evaluated by ELISA, use active oxygen detection reagent box to detect active oxygen on A375、HeLa and MCF-7 cells induced by different concentration of GO-CHIT-RGD NPs. It shows that the oxided graphene dealt by mixd acid has better biocompatibility and stability in liquor. The special toxicity of GO-CHIT-RGD NPs on A375 cells is the combing result of GO, CHIT and RGD. Besides, it cannot impact on the activity of normal cell line(endothelial cells cultured from human umbilical vein, HUVEC). This is because the integrin rate on A375 cells is high and material has special binding with it, which results in the rise of reactive oxygen insides A375 cells and as a result the cells died.2. Use EDC and NHS as coupling agent of synthesis to produce GO-RGD, GO-ABA, GO-GAL under very mild conditions and use FTIR and zeta potential to evaluate successful coupling and characterize surface potential and particle size. This paper uses WST-8 method to evaluate toxicity of GO-RGD and GO-ABA on A375, HeLa and MCF-7 cells,GO-GAL on A375, HeLa and HepG-2 cells;This is because the integrin rate on A375 cells is high and material has special binding with it, which results in the rise of reactive oxygen insides A375 cells and as a result the cells died.3. Use EDC and NHS as coupling agent of synthesis to produce GO-PAMAM-GA under mild conditions and use FTIR and zeta potential to evaluate successful coupling and characterize surface potential and particle size. GO-PAMAM-GA of toxicity on A375, HeLa and MCF-7 cells and the toxicity of GO,GO-PAMAM on human neuroblastoma cells(SKNSH) were evaluated by WST-8; use GO-PAMAM-GA complexes as gene vector to mediate pEGFP plasmid transfecting into A375 cells and MCF-7 cells. It shows that the biocompatibility of GO-PAMAM-GA is not very good. However, it can transfer pEGFP-N1 into cells and express successfully,but GO and GO-PAMAM are good biocompatibility with SK-N-SH.