透明质酸键合分枝状聚乙烯亚胺作为siRNA载体的研究

Hyaluronic acid-graft-branch polyethylenimine-a novel vector for siRNA delivery

目的构建以透明质酸为骨架材料键合分枝状聚乙烯亚胺(polyethylenimine,PEI,相对分子质量为25 000)的新型转siRNA载体,研究其对肿瘤细胞siRNA的转染。方法透明质酸(hyaluronicacid,HA)作为骨架材料,利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐[1-ethyl-3-(3-dimethyl-laminopropyl)carbodiimide hydrochloride,EDC.HCl]交联剂,与PEI嫁接,形成HA-PEI聚合载体材料。用核磁共振氢谱(1H-NMR)对载体材料进行化学表征,以确定其结构。用凝胶电泳阻滞实验观察HA-PEI与小干扰siRNA的结合能力,MTT法检测siRNA/PEI-HA复合体的细胞毒性,用原子力显微镜观察siRNA/PEI-HA复合体的大小。用B16F1细胞株进行基因沉默效率实验。结果质量分数为24%的PEI偶联到HA上,HA-PEI与siRNA在体积比为3∶1时可以完全缩合siRNA,其大小约20 nm,在相同浓度下HA-PEI的毒性低于PEI。体外转染实验显示,在含高血清条件下,siRNA/PEI-HA对B16F1细胞的基因沉默效率比PEI高。结论以透明质酸为骨架材料键合聚乙烯亚胺形成转基因载体是一种有潜在研究价值的新型靶向非病毒载体。

Objective To develop a novel vector for siRNA delivery with low molecular weight polyethylenimine（PEI）grafted to the hyaluronic acid（HA）.Methods The properties of HA-PEI were characterized by 1H-NMR.The siRNA binding ability of HA-PEI was erected by gel electrophoresis retardation assay.The particle size of siRNA/HA-PEI complex was measured by using AFM.The cytotoxicity of siRNA/PEI-HA complex was evaluated by MTT assay.The siRNA / PEI-HA complex in vitro PGL3-Luc gene silencing efficiency was evaluated by plasmid / liposome transfection method.Results The particle size of siRNA/HA-PEI complex was about 20 nm.Gel electrophoresis showed that the polymer was able to completely condense siRNA at the ratio of 3∶ 1（V∶ V）.The cytotoxicity of siRNA/PEI-HA complex to B16F1 cells was lower than that of siRNA/PEI complex.The survival rate of siRNA/PEI-HA complex cell was significantly higher,and there is a higher efficiency in gene silencing.siRNA/PEI-HA complex appeared to silence PGL3-Luc gene in the range of 50%-85% depending on the serum concentration up to 50%（φ）.Conclusions PEI-HA complex as a specific target cells siRNA carrier is shown a powerful effect.