Synthesis and characterization of thiolated chitosan-pDNA nanoparticles as gene carriers

Objective: To study the preparation of thiolated chitosan and thiolated chitosan-plasmid DNA(pDNA)nanoparticles.Methods:Thiolated chitosan was prepared in the synthesis reaction of chitosan and thioglycolic acid with amide linkage forming by the catalysis of 1-thyl-3-(-dimethylaminopropyl)-carbodoomide hydrochloride(EDAC).The degree of thiol groups modification of the thiolated chitosan conjugate was determined by 5,5′-dithiobis(2-nitrobenzoiol acid)(DTNB) assay.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 shape and size of thiolated chitosan-pcDNA3.1(+)-EGFP nanoparticles were observed by transmission electron microscope(TEM),and encapsulation rate was measured by ultraviolet spectrophotometer.The nanoparticles were treated by DNase I,then the products were measured by gel electrophoresis.Results:Chitosan was thiolatized by the catalysis of EDAC.1 gram of thiolated chitosan processed(202.85±3.05)μmol thiol groups(n=6)by calculating.It was demonstrated that full binding of thiolated chitosan with the pDNA and stability of nanoparticles.Encapsulation efficiency was higher than 90%.TEM images showed that nanoparticles were approximatively spherical in shape and particle size was in the range from 300 to 350 nm.The encapsulated pDNA protection against DNase I degradation was confirmed by DNase I treatment and gel electrophoresis.Conclusion:Thiolated chitosan-pcDNA3.1(+)-EGFP nanoparticles constructed are characterized for their proper particle size,high encapsulation efficiency of pDNA and ability protect pDNA from degradation.So Thiolated chitosan may serve as an effective nonviral gene carrier.