Preparation Of Thiolated Chitosan-pcDNA3.1(+)-EGFP Nanoparticles And Study On Its Transfection Activity To HeLa Cells In Vitro

Objective:To study the preparation of thiolated chitosan.The thiolated chitosan-plasmid DNA(pDNA) nanoparticles were made by thiolated chitosan solution reacted with the pDNA solution.To study the expression of GFP gene transfer in HeLa cell line by thiolated chitosan as carrier in vivo.To investigate cytotoxicity of thiolated chitosan-based nanoparticles to HeLa cells.Methods.Thiolated chitosan was prepared in the synthesis reaction of chitosan and thioglycolic acid with amide linkage forming by the action of coupling agents 1-thyl-3-(-dimethylaminopropyl)- carbodoomide hydrochloride(EDAC ).Infrared spectrum of product was measured by Fourier transform infrared detector,and the degree of thiol groups modification of thiolated chitosan was determined by 5,5'-dithiobis(2-nitrobenzoiol acid )(DTNB) assay,and sulfur content was determined by elemental analyser.When the plasmid pcDNA3.1(+)-EGFP was used as the reporter gene,thiolated chitosan- pcDNA3.1(+)-EGFP nanoparticles were prepared using a complex coacervation process by mixing chitosan and pDNA.The encapsulation rate was measured by ultraviolet spectrophotometer.The diameter and surface charge of the nanoparticles were determined by Zetasize and grainsize analyzer.The morphologies and particle size of thiolated chitosan-pcDNA3.1 (+)-EGFP nanoparticles was measured by transmission electron microscope(TEM).The nanoparticles were treated by DNase I,then depolyrnerized by heparin,and integrity of plasmid gene was measured by gel electrophoresis.The cytotoxity of the nanoparticlesto to HeLa cells was examined with the MTT assay,when chitosan-based nanoparticles were used as a control.The transfection activities of the thiolated chitosan-pcDNA3.1(+)-EGFP nanoparticles in HeLa cells were assessed by approximatively measuring the ratio of fluore-positive cells in an in vitro gene transfection test,when lipofectin were used as a control.Results:Infrared spectrum showed that chitosan is thiolatized by the catalysis of EDAC.DTNB assay exhibit that 1 gram of thiolated chitosan processed (202.85±3.05)μmol thiol groups(n=6).It is demonstrated that full binding of thiolated chitosan and the pDNA with N/P value being 9.4:1 and stability of nanoparticles.Encapsulation efficiency was higher than 90%.Grainsize and Zetasize analyzer showed thiolated chitosan-pcDNA3.1(+)-EGFP nanoparticles were 288.7nm in average size and(+25.2±2.1)Mv of zeta electric potential.TEM images showed that nanoparticles were approximatively spherical in shape and particle size was in the range from 300 to 350 nm.The ability that protectedencapsulated pDNA against Dnase I degradation was confirmed by the pDNA protection test.The nanoparticles showed no cytotoxicity to HeLa cells.No significant difference in viabilities of the cells between unmodified chitosan based nanoparticles and thiolated chitosan based nanoparticles(P＞0.05).The GFP Genes were effectively expressed by the HeLa cells when transfected by thiolated chitosan-pcDNA3.1(+)-EGFP nonaparticles.Thiolated chitosan as gene carrier was observed a lower transfection efficiency than lipofectin(P＜0.01).Conclusions:Yhiolated chitosan- pcDNA3.1(+)-EGFP nanoparticles constructed were characterized for their proper particle size,high encapsulation efficiency of pDNA and ability protect pDNA from degradation.The ability that protected pDNA from degradation and stability of thiolated chitosan- pcDNA3.1(+)-EGFP nanoparticles were confirmed.The pDNA were ransferred into the cells by thiolated chitosan-pcDNA3.1(+)-EGFP nonaparticles constructed by the technology.Reporter genes effectively expressed by the HeLa cells.The nanoparticles were shown no cytotoxicity to the HeLa cells.So Thiolated chitosan may serve as a promising nonviral gene vector for gene transfer and gene therapy.