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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 nanoparticles and study their physico-chemical properties and in vitro activity. Methods Firstly, a good dispersible carbon nano-powder was prepared by sol method and then chitosan-carbon nanoparticles were prepared by mutual attraction of positive and negative charges. Secondly, plasmid DNA of green fluorescent protein (PEGFP-C1) Electrophoretic adsorption of PEGFP-C1 plasmid DNA and chitosan - carbon nanoparticles combine to form carrier gene nanoparticles; Third, observe the morphological features by scanning electron microscopy, laser particle size analyzer to determine the particle size distribution and surface potential (Zeta potential ). MTT assay was used to detect the cytotoxicity of chitosan-carbon nanocarrier on HepG2 cells and COS7 cells. Gel-retardation assay was used to determine the DNA carrier rate of the vector. DNaseⅠprotein was used to study the protective effect of the chitosan- Nanoparticle Introduction Experiment Qualitatively assess the activity of nanoparticles entering into cells in vitro and observe the effect of introduction by fluorescence microscopy. Results The surface of chitosan-CNTs possessed a positive charge of <0.3. The binding efficiency to DNA was high, and DNA was protected from degradation by DNase I. The FITC-labeled nanoparticles were able to successfully enter COS7 cells and HepG2 cells . Conclusion Chitosan-carbon nanoparticles can enter the cell interior with high efficiency of introduction and can be used as gene delivery non-viral vector system, which deserves further study.