壳聚糖复合纳米基因载体的制备

Preparation of chitosan nanocomposite gene vector

目的制备壳聚糖复合纳米基因载体,并探讨其理化性质、细胞毒性、稳定性及体外转染效率。方法采用复凝聚法制备包封聚乙烯亚胺(Polyethylenimine,PEI)/DNA复合物的壳聚糖复合纳米基因载体,用纳米粒度分析仪测定其粒径和Zeta电位;透射电镜观察其形态;MTT法检测其细胞毒性;在PBS溶液(pH 7.4)及含10%小牛血清的RPMI1640培养基中,于37℃条件下放置0、1、3、5 d,1%琼脂糖凝胶电泳检测其稳定性;体外转染CNE细胞,评价其转染活性。结果当N(PEI的氨基)/P(DNA的磷酸根)≥6时,能够形成稳定的壳聚糖复合纳米粒,平均粒径约为300 nm,表面电荷约为30 mV;壳聚糖复合纳米基因载体呈球形,圆整且分散性好;复合纳米基因载体的细胞毒性较低;1%琼脂糖凝胶电泳分析显示,DNA被完全包裹在复合纳米载体中,且5 d内无游离DNA释放;体外转染活性与壳聚糖/DNA复合物相比,提高了约1 000倍,且转染能力不受血清的干扰。结论制备的壳聚糖复合纳米基因载体是一种高效、低毒的非病毒载体,具有作为体内基因治疗载体的应用潜力。

Objective To prepare chitosan nanocomposite gene vector and investigate its physico-chemical property, cytotoxicity, stability and in vitro transfection efficiency.. Methods Chitosan nanoparticles entrapping complexes of polyethylenimine （PEI） / DNA were prepared by a complex coacervation method, and measured for size and Zeta potential by uanoparticle size analyser, observed for morphology by transmission electron microscopy, and determined for cytotoxicity by MTY method. The prepared chitosan nanocomposite gene vector was stored in PBS （pH 7. 4） and RPMI1640 containing 10% calf serum at 37 % for 0, 1, 3 and 5 d separately, and evaluated for stability by 1% agarose gel electrophoresis, CNE ceils were transfected with the prepared vector in vitro, and the transfection efficiency was evaluated. Results Stable chitosan nanoparticles were formed when the N （amino group of PEI） / P （phosphate of DNA） ratio was not less than 6. The resultant chitosan nanocomposite gene vector were in even and intact spherical shape, at mean diameter of about 300 nm, which showed a surface potential of about 30 mV and low cytotoxieity. The result of 1% agarpse gel electrophoresis showed that DNA is completely wrapped in nano-vector without free DNA released in 5 d. The transfection activity in vitro of thte nanoparticles was 1 000 times higher than that of chitosan / DNA complexes, on which no interference of serum was observed. Conclusion The prepared chitosan nanocomposite gene vector is a high effective non-viral vector with low toxicity, which may be a potential vector for gene therapy in vivo.