壳聚糖纳米粒用作基因递送载体的初步研究

PRELIMINARY STUDY OF CHITOSAN NANOPARTICLES USED AS GENE DELIVERY CARRIERS

目的 初步研究基因壳聚糖纳米粒的性质和转染活性。方法 用复凝聚法制备纳米粒 ;用透射电镜观察形态 ;用纳米粒度分析仪测定粒径、多分散度和zeta电位 ;用荧光分光光度法测定基因包封率 ;用凝胶阻滞分析和荧光扫描测定基因在纳米粒中的位置 ;用体外基因转染实验定性评价纳米粒的转染活性。结果 纳米粒形态多呈球形 ,平均粒径为 2 18 9nm ,多分散度为 0 2 76 ,zeta电位为 +2 1 2mV ;基因包封率为 99 6 %;凝胶阻滞分析和荧光扫描表明基因几乎全部被包裹在纳米粒内部 ,表面吸附很少 ;体外基因转染实验表明基因壳聚糖纳米粒能够转染人胚胎肾细胞 (HEK2 93)和肝癌细胞 (HepG2 ) ,基因能够在这两种细胞中表达。结论 壳聚糖纳米粒能将基因递送到细胞内并且基因能够表达 ,因此可以用作基因药物载体。

AIM To study characteristics and transfection activity of pDNA-chitosan nanoparticles. METHODS The pDNA-chitosan nanoparticles were prepared by complex coacervation, and its morphology was observed by transmission electronic microscopy (TEM). Particle size, polydispersity and zeta potential were determined by nanoparticle size analyser. Encapsulating efficiency of pDNA was determined by fluorescence spectrometer. The molecular localization of pDNA in the chitosan nanoparticles was determined by fluorescence scanning and gel retardation assay. Transfection activity of pDNA-chitosan nanoparticles was determined by gene transfection experiment in vitro. RESULTS The morphology of the nanoparticles are mostly spherical. The average particle size is 218.9 nm, and the polydispersity of particle size is 0.276. The zeta potential of the nanoprticles is +21.2 mV. Encapsulating efficiency of pDNA is 99.6%. Fluorescence scanning and gel retardation assay showed that pDNA was mostly encapsulated within the nanoparticles and adsorbed pDNA on the surface are very little. The gene transfection experiment in vitro suggested that the pDNA-chitosan nanoparticles can transfect HEK293 and HepG2 celles, and the gene can express in these celles. CONCLUSION The chitosan nanoparticles can delivery the gene into celles and the gene can express, so these chitosan nanoparticles may be used as gene medicine carriers.