Preparation of thiolated chitosan-pcDNA 3.1(+)-EGFP nanoparticles and study on its transfection activity to Hela cells in vitro

【Objectives】To study the preparation of thiolated chitosan and thiolated chitosan-plasmid DNA (pDNA) nanoparticles, and to investigate transfection activities of thiolated chitosan-based nanoparticles to Hela cells in vitro.【Methods】Thiolated chitosan was prepared in the synthesis reaction of chitosan and thioglycolic acid by the catalysis of 1-thyl-3-(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. Nanoparticles were synthesized through complex coacervation thiolated chitosan vector with pcDNA 3.1 (+)-EGFP, the Latter acting as the reporter gene. The diameter and surface charge of the nanoparticles were determined by Zetasize and grainsize analyzer. The nanoparticles were treated by DNase I, then depolymerized by heparin, and integrity of plasmid gene was measured by gel electrophoresis. The transfection activities of the thiolated chitosan-pcDNA 3.1 (+)-EGFP nanoparticles to HeLa cells were assessed in vitro. The cytotoxity of the nanoparticles to HeLa cells was examined with the MTT assay. 【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). Grainsize and Zetasize analyzer showed thiolated chitosan-pcDNA 3.1(+)-EGFP nanoparticles were 288.7 nm in average size and + (25.2±2.1) mV of zeta electric potential. The encapsulated pDNA protection against DNase I degradation was confirmed by DNase I protection test. The nanoparticles were shown to transfect HeLa cells effectively in transfection experiment in vitro and showed no cytotoxicity to HeLa cells. 【Conclusions】Reporter genes were effectively expressed by the HeLa cells when transfected by thiolated chitosan-pcDNA 3.1 (+)-EGFP nonaparticles constructed by the technology. So thiolated chitosan may serve as a promising nonviral gene vector for gene transfer and gene therapy.