摘要
目的:通过巯基化修饰将壳聚糖改性成巯基化壳聚糖,以巯基化壳聚糖与质粒DNA(pDNA)结合,制备巯基化壳聚糖-pDNA纳米粒。方法:在1-(3-二甲胺基丙基)-3-乙基碳二亚胺盐酸盐(EDAC)的催化作用下,巯基乙酸与壳聚糖通过酰胺键结合,形成巯基化壳聚糖。以5-5′-二硫代-双-硝基苯甲酸(DTNB)法检测巯基化壳聚糖的巯基含量;通过复凝聚法使巯基化壳聚糖和报告基因质粒pcDNA3.1(+)-EGFP结合,制备巯基化壳聚糖-pcDNA3.1(+)-EGFP纳米粒,透射电镜检测纳米粒形态和粒径,紫外分光光度法检测纳米粒对pcDNA3.1(+)-EGFP的包封率,DNase I处理该纳米粒,并用琼脂糖凝胶电泳检测其产物。结果:EDAC能有效地使壳聚糖巯基化,1g巯基化壳聚糖的巯基含量为(202.85±3.05)μmol(n=6);巯基化壳聚糖能有效地与质粒pcDNA3.1(+)-EGFP结合形成稳定的纳米粒,包封率大于90%;电镜下该纳米粒的粒径在300～350 nm之间,为不规则球型,经DNaseⅠ处理和电泳分析表明该纳米粒能保护pcDNA3.1(+)-EGFP免受DNaseⅠ的降解。结论:巯基化壳聚糖pcDNA3.1(+)-EGFP纳米粒有较好的包封效果、适度的粒径和良好的保护内部质粒抗核酸酶水解的能力,巯基化壳聚糖可能成为基因递送的非病毒载体。
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 thioglycolie 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）txmol 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 ehitosan may serve as an effective nonviral gene carrier.
出处
《天津医科大学学报》
2008年第4期466-469,共4页
Journal of Tianjin Medical University
关键词
巯基化壳聚糖
纳米粒
基因载体
转染
Thiolated chitosan
Nanoparticles
Gene carrier
Transfection
