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目的分析一种siRNA药物载体薄膜在溶液中的稳定性,并评价其基因沉默效应。方法利用层层自组装(layer-by-layer self-assembly,LBL)技术制备壳聚糖(chitosan,CHS)-透明质酸(hyaluronic acid,HA)-siRNA多层薄膜。通过UV-vis吸光度法对siRNA在薄膜上的生长过程以及在不同缓释液中的稳定性分别进行验证。结合荧光图像和流式细胞术,采用直接与细胞接触的方式检测该siRNA载体薄膜的HEK293T细胞转染效应。结果 CHS-HA-siRNA多层薄膜在260 nm处有很强的吸收峰,且随着含siRNA的组装层数的增加,在260 nm的吸光度值越大。该siRNA载体薄膜在1 mol/L NaCl溶液中释放效果最好,在pH 7.4的PBS缓冲液中次之,在纯水中稳定性最好。过低或过高的盐离子浓度对促进薄膜的崩解无优势。阳性对照组7-eGFP-siRNA和2-eGFP-siRNA具有较好的沉默绿色荧光蛋白基因的效应;在细胞培养2 d后的转染试验中,阳性对照组7-eGFP-siRNA较2-eGFP-siRNA组显示出更强的基因沉默效应。结论成功构建了一种非病毒siRNA递送载体系统,为未来实现siRNA局部给药开辟了一条可行的途径。
Objective To analyze the stability of a siRNA drug carrier film in solution and evaluate its gene silencing effect. Methods Chitosan (CHS) -hyaluronic acid (HA) -siRNA multilayers were prepared by layer-by-layer self-assembly (LBL). The growth of siRNA on the membrane and the stability in different sustained-release solutions were verified by UV-vis absorbance. Combined with fluorescence imaging and flow cytometry, direct transfection with HEK293T cells was detected by direct contact with cells. Results The CHS-HA-siRNA multilayer film had a strong absorption peak at 260 nm, and the absorbance at 260 nm increased with the increase of the number of layers containing siRNA. The siRNA carrier membrane released best in 1 mol / L NaCl solution, followed by PBS solution at pH 7.4, and its stability in pure water was the best. Too low or too high salt ion concentration has no advantage in promoting the disintegration of the film. The positive control group 7-eGFP-siRNA and 2-eGFP-siRNA had a better effect of silencing the green fluorescent protein gene. In the transfection experiment after 2 days of cell culture, the positive control group 7-eGFP-siRNA was more effective than 2-eGFP -siRNA group showed stronger gene silencing effect. Conclusion A non-viral siRNA delivery vector system has been successfully constructed, which opens up a viable approach for the local administration of siRNA in the future.