SS-PEI/核酸纳米复合物的构建分析

目的：构建一种可生物降解的基因载体,并观察其体外转染DNA/siRNA的效率和细胞毒性。方法：采用低分子质量PEI与3,3′-二硫代二丙酸在催化剂作用下合成可降解的PEI衍生物SS-PEI,用红外波谱仪和核磁波谱仪分析其化学结构;动态光散射分析动态光散射（dynamic light scattering,DLS）SS-PEI/DNA和SS-PEI/siRNA的粒径;采用流式细胞技术（FCM）和荧光激活细胞分类术（FACS）分别分析SS-PEI对GFP-DNA和FAM-dsRNA的转染率;通过GFP表达水平分析SS-PEI对GFP质粒和GFP-siRNA的转染效率;采用考马斯蓝法分析SS-PEI/DNA和SS-PEI/siRNA的细胞毒性。结果：红外波谱和核磁波谱与SS-PEI理论波谱一致;DLS结果显示,SS-PEI/DNA复合物粒径为95~175nm,SS-PEI/siRNA平均粒径约200nm。FCM结果显示,SS-PEI对DNA的最大转染率为（25.2±2.3）%,稍低于HWPEI组的（33.8±3.1）%;SS-PEI/GFP-DNA转染后GFP的表达水平与HWPEI转染组接近〔（150±7）FI（A.U.）/mg蛋白vs（168±18）FI（A.U.）/mg蛋白〕,差异无统计学意义,t=1.62,P=0.18;细胞毒性分析显示,SS-PEI/DNA组细胞活力（89±5.2）%,显著高于HWPEI/DNA组的（70±4.9）%,差异有统计学意义,t=4.61,P=0.001。FACS结果显示,SS-PEI对FAM-dsRNA的转染率为（86.5±2.5）%;SS-PEI转染GFP-siRNA,靶基因GFP被显著敲除,相对表达水平为（42±3.5）FI（A.U.）/mg蛋白,与对照组的（79±10.8）FI（A.U.）/mg蛋白相比差异有统计学意义,t=5.64,P=0.004。结论：SS-PEI的合成可明显提高装载核酸的效率,具有低毒和高效等特点。

Preparation and analysise of SS-PEI/nucleic acid complexes

OBJECTIVE：To synthesize a bioreducible gene vector SS-PEI and to analyze its cytotoxicity and capacity of binding DNA and siRNA.METHODS：SS-PEI was synthesized by chemical coupling of the 3′-dithiobispropanoic acid with the low molecular weight PEI（800Da）via an EDC/NHS activation reaction.The 1HNMR spectra and Fourier transform infrared（FTIR）spectra of the SS-PEI polymer were detected,respctrively.The size of SS-PEI/DNA and SS-PEI/siRNA were measured by bynamic light scattering（DLS）.The transfection rates of SS-PEI of transfecting DNA and siRNA were tested by flow chtometry（FCM）and fluorescence-activated cell sorting（FACS）,respectively.Expression level of GFP protein was measured to evaluate the transfection capacity of SS-PEI mediating DNA and siRNA delivery.The cytotoxicity of SS-PEI was determined using coomassie blue staining.RESULTS：1H NMR and FTIR spectra of the polymer were in full accordance with the expected structure.The sizes of SS-PEI/DNA were in the range from 95to 175nm and the average diameter of SS-PEI/siRNA was 200nm.FCM indicated that the highest transfection rate of SS-PEI/DNA was（25.2±2.3）% which was slightly lower than that of the HWPEI/DNA（33.8±3.1）%.The expression level of GFP protein in the group SS-PEI/GFP-DNA was similar to that of the group HWPEI/GFP-DNA（150±7）FI（A.U.）/mg protein vs（168±18）FI（A.U.）/mg protein;t=1.62,P=0.18].However,the cell viability of the group SS-PEI/DNA was significantly higher than that of the group HWPEI/DNA（89±5.2）%vs（70±4.9）%,t=4.61,P=0.001].FACS indicated that transfection rate of SS-PEI/siRNA was（86.5±2.5）%,and the expression level of GFP protein after transfecting SS-PEI/GFP-siRNA was notably descended than that of the control group,which were（42±3.5）and（79±10.8）FI（A.U.）/mg protein,（t=5.64,P=0.004）.CONCLUSION：SS-PEI can enhance the efficacy of loading DNA and siRNA and it is safe and in high performance.