基因壳聚糖纳米粒表面修饰和转染研究

目的：研究递送基因纳米粒表面修饰对体外基因转染的影响。方法：利用末端活化的聚乙二醇(PEG)制备PEG化基因壳聚糖纳米粒;通过两端活化的PEG将糖蛋白配基连接到纳米粒表面,完成肝靶向纳米粒的制备;用透射电镜观察表面修饰对纳米粒粒径大小、粒子形态的影响;使用蛋白质测定试剂盒测算纳米粒表面蛋白连接量;利用体外转染实验考察表面修饰对纳米粒转染活性的影响;用倒置荧光显微镜观察并用流式细胞仪测定转染结果。结果：纳米粒PEG化使转染效率大幅度升高,半乳糖基牛血清白蛋白(Galn—BSA)使体系的转染效率比PEG化纳米粒略有下降,但比不经修饰的纳米粒转染活性高。壳聚糖纳米粒的表面PEG化能提高纳米粒的体外稳定性,从而提高体外转染效率,并适合于进行冷冻干燥。结论：长循环壳聚糖基因递送纳米粒在基因治疗研究中可能会发挥重要作用。

Study on Surface Modification and Transfection of Gene-chitosan Nanoparticles

Objective: To investigate the effect of surface chemical modification to gene-delivering chitosan nanoparticles on efficiency in vitro of gene transfection. Methods:Polyethylene glycol (PEG)-modified gene-chitosan nanoparticles were prepared with bipoilar-activiated PEG. And through the activiated PEG, galactosyated bovine serum albumin (Galn-BSA) was linked to the surface of the nanoparticles. The hepar-targeted gene delivery nanoparticle were made. Transmission electron microscopy was used to observe the diameters and surface morphology of the particles after the modification. Protein-determination Kit was applied to determine the amount of Galn-BSA linked to the particles. Transfection in vitro was conducted to investigate the transfection efficiency. And inverted microscopy and flow cytometry were carried out to evaluate transfective outcome. Results:The PEG modified nanoparticles resulted in a great increase of transfection efficiency. Galn-BSA could lead to a slight decrease in transfection efficiency than PEG-only modified nanoparticles, but its was still higher than non-modified particles. The chemical modification to chitosan nanoparticles with PEG could improve the stability of the particles in vitro so that transfection efficiency in vitro was enhanced. And these nanoparticles could stand freeze-drying. Conclusions:Chitosan nanoparticles gene-delivery system could have a good perspective in the field of gene therapy.