载血管紧张素转换酶短发卡RNA-聚乙二醇-壳聚糖纳米粒制备以及生物学特性

The preparation and biological characteristics of PEG-chitosan nanoparticles containing ACEsh RNA

目的制备载血管紧张素转换酶(ACE)短发卡RNA(sh RNA)壳聚糖纳米粒,并且给予聚乙二醇(PEG)表面修饰,分析其相关物理学、生物学特性,以期获得一种缓释的非病毒载体介导系统。方法应用本实验室前期实验中筛选出的能显著下调ACE基因的靶序列,采用菌液用碱裂解法大量抽提和纯化重组质粒,随机酶切测序,并检测质粒浓度与纯度,采用离子交联法制备壳聚糖纳米粒,PEG修饰壳聚糖纳米粒,复凝聚法制备不同p H值、体积比以及PEG化的载ACEsh RNA壳聚糖纳米。喷金电镜下对各种壳聚糖纳米粒悬液扫描并照相,观察纳米粒与形态。应用凝胶阻滞分析验证载ACEsh RNA壳聚糖纳米多聚复合物的形成及电荷性质。结果1壳聚糖纳米粒的粒径随着溶液p H值的升高而增大,当p H值为5.5时的PEG壳聚糖纳米粒平均粒径(125.8±5.6)nm左右,大小均匀,多分散度最小,分布比较集中,zeta电位为正,有利于与带负电荷的质粒结合;PEG化后也对粒径以及zeta电位无明显影响,但是与壳聚糖相同条件相比,分散度均明显减小,可见更适合制备均匀的纳米粒,利于和质粒结合;壳聚糖与质粒体积比(质量比)为1∶1制备的壳聚糖纳米粒平均粒径较小,故通过细胞膜的通透性较好,多分散度较小,分布比较集中,zeta电位也为正值,有利于与带负电荷的质粒结合。2壳聚糖纳米以及PEG化壳聚糖纳米质粒复合物均有效结合质粒,由于中和了质粒所带的负电荷,凝胶电泳时,质粒不出孔,而裸质粒则出孔;p H<7时壳聚糖分子中大部分的氨基带正电荷能与质粒DNA有效地结合,质粒不出孔;在体积比为1∶1、1∶2、1∶3时,壳聚糖纳米粒能有效地结合质粒。结论所制备的载ACE sh RNA-PEG壳聚糖纳米粒大小均匀,粒径分布范围较窄,并且筛选出在p H=5.5时,与质粒体积比为1∶1时,以PEG壳聚糖纳米粒作为载体有良好的结合力,为后期体外转染培养细胞以及体内转染提供了实验基础。

Objective To prepare angiotensin converting enzyme（ACE） short hairpin RNA（sh RNA） chitosan nanoparticles which are subsequently surface-modified with polyethylene glycol（PEG）, and to analyze the relevant physical and biological characteristics of this complex in order to obtain a sustained release non-viral vector-mediated system. Methods The significantly down-regulated ACE gene target sequence screened out from our pre-experiment was applied in the study. The substantial extraction and purification of recombinant plasmid were completed by alka-line lysis of bacteria. The sequencing was detected by random enzyme digestion, and the concentration and purity of plasmid were measured. The chitosan nanoparticles were prepared by ionic crosslinking method and modified by PEG.Then, PEG-chitosan nanoparticles containing ACEsh RNA of different p H values and the volume ratios were prepared by complex coacervation. Different suspensions of chitosan nanoparticles were studied and photographed under scan-ning electron microscopy with gold-coating to observe their morphology. The formation and charge characteristics of ACE-sh RNA-containing chitosan nanoparticles complex were analyzed and verified by gel retardation assay. Results1 The size of chitosan nanoparticles increased with the rise of solution p H value. At the p H value of 5.5, the PEG chitosan nanoparticles were even in size [mean（125.8 ±5.6）nm], with minimum polydispersity, concentrated distribution and positive zeta potential which facilitated binding to the negatively charged plasmid. After PEG modification,there were no significant change in particle size and zeta potential. However, comparing with non-PEG chitosan under the same conditions, the dispersity of PEG modified chitosan nanoparticles was even lowered, suggesting that PEG modification may be more suitably used to prepare uniform-sized nanoparticles which readily binds to with plasmids.A smaller mean size of chitosan nanoparticles obtained with 1 ∶1 volume ratio（mass ratio） of chitosan nanoparticles to plasmid, presented with better membrane permeability, smaller dispersity, more concentrated distribution and positive zeta potential, which favored their binding to the negatively charged plasmid. 2Both chitosan nano- and PEG-chitosan nano-plasmid complexes may effectively bound to plasmid. As the negatively charged plasmid was neutralized, the plasmid retained inside the spores in contrast to the opposite as did the naked plasmid during gel electrophoresis. At volume ratio of 1∶1, 1∶2 and 1∶3, the chitosan nanoparticles can effectively combine with plasmid. Conclusion The prepared PEG-chitosan nanoparticles containing ACE-sh RNA has uniform size and narrower particle size distribution.At a PH of 5.5 and the volume ratio to plasmid of 1∶1, PEG chitosan nanoparticles as a vector have good binding capacity. This study provide a foundation for the future in vitro transfection of cultured cells and in-vivo transfection experiments.