摘要
目的制备壳聚糖-碳纳米颗粒并研究其理化性质和体外活性。方法首先,用溶胶法制备出分散性良好的碳纳米粉末,再通过正负电荷相互吸引制备出壳聚糖-碳纳米颗粒;其次,用绿色荧光蛋白(PEGFP-C1)质粒DNA作报告基因,以静电吸附的方式将PEGFP-C1质粒DNA与壳聚糖-碳纳米颗粒结合形成载基因纳米粒;再次,用扫描电镜观察其形态特征,激光粒度分析仪测定其粒度分布及表面电位(Zeta电位),MTT试验检测壳聚糖-碳纳米载体对HepG2细胞和COS7细胞的毒性作用,凝胶阻滞实验确定该基因载体的DNA携带率,DNaseⅠ保护实验研究其对所携带基因的保护作用,体外纳米粒导入实验定性评价纳米粒进入在体外进入细胞的活性,并用荧光显微镜观察导入效果。结果壳聚糖-碳纳米粒表面携带正电荷,聚合指数<0.3;与DNA结合后效率较高,且可保护DNA免受DNaseⅠ的降解;经FITC标记后能够成功地进入到COS7细胞和HepG2细胞。结论壳聚糖-碳纳米颗粒能进入到细胞内部,且导入效率较高,可以用作基因递送的非病毒载体系统,值得进一步研究。
[Objective] To prepare chitosan-carbon nanoparticle and investigate its physico-chemical properities and activity in vitro. [Methods] At first, carbon nanoparticles with good dispersibility were prepared by solation ,nethod and chitosan which was delivered with positive charges was adsorbed on the surface of them to form chitosan-ca,bon nanoparticles using the mechanism of electrostatic adsorption. Then, plasmid DNA of PEGFP-C1 was absorbed on the surface of chitosan-carbon nanoparticles, the fomer acting as reporter gene. After that, the appearances of the nanoparticles were surveyed by the scanning electron microscope; the grain distribution and zeta potentials of the nanoparticles were determined with the laser grain analyzer; the cytotoxities of the nanoparticles to HepG2 cell and L-02 cell lines were examined by the MTT method. Later on, DNA loading efficiency of nanoparticles was examined by get retardation assasy and the gene protection effect of the nanoparticles was invested by DNase Ⅰ assay. Furthermore, the introduction activities in vivo of the chitosan-carbon nanoparticles were qualitatively assessed by an importation test with HepG2 ceils and Cos7 ceils and the results was determined by fluorescencemicroscope. [Results] Chitosan-carbon nanoparticles with positive charges can protect the DNA from degradation by DNase Ⅰ . The polyindex of them was less than 0.3 and the DNA loading efficiency was high certificated by gel retardation assasy. The nanoparticles could enter the inner of COS7 ceils and HepG2 cells. [Conclusion] The chitosan-carbon nanoparticles can delivery the gene into cells effectively, so the chitosan-carbon nanoparticles are worth studying as gene medicine carriers.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2006年第6期801-806,共6页
China Journal of Modern Medicine
