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主被动双靶向纳米凝胶递送RRM2-siRNA的体外抗肿瘤效应
张莉,王颖,金崇,董霞,李威*
0
(第二军医大学国际合作肿瘤研究所, 上海 200433
*通信作者)
摘要:
目的 制备靶向输送针对核糖核苷酸还原酶RR小亚基M2 (RRM2)的siRNA (RRM2-siRNA)的聚N-异丙基丙烯酰胺/聚乙烯亚胺(PNIPAM/PEI)纳米凝胶,通过研究其体外的抗肿瘤效应,以期构建新型靶向纳米凝胶递送基因系统。方法 采用自由基接枝共聚合(radical graft copolymerization)反应制备具有“核-壳”结构的PNIPAM/PEI聚阳离子温度敏感型纳米凝胶,评价其粒径、zeta电位等理化性质;根据电荷相互作用原理,并通过偶联抗人表皮生长因子受体2 (Her2)抗体,形成包裹RRM2-siRNA的靶向性PNIPAM/PEI-siRNA纳米凝胶复合体。利用凝胶电泳确定PNIPAM/PEI-siRNA纳米凝胶复合体包裹siRNA效果,通过荧光显微镜定性观察和流式细胞术定量检测人胃癌细胞NCI-N87对该纳米凝胶复合体的摄取情况,利用qPCR法检测该纳米凝胶递送siRNA后靶基因RRM2的表达情况,用Transwell 法检测该纳米凝胶复合体对NCI-N87细胞迁移的影响。结果 本研究合成具有“核-壳”结构的温度敏感型PNIPAM/PEI纳米凝胶,颗粒均匀,粒径为359.8 nm,zeta电位为21.4 mV。在此基础上,我们制备出靶向凝胶包裹RRM2-siRNA的纳米凝胶复合体,确定最佳包裹比例为N/P=60 (N/P:聚阳离子纳米凝胶中氨基与质粒DNA中磷酸基的摩尔比)。不同温度下(37 ℃、42 ℃)细胞内吞实验证实,该纳米凝胶复合体具有良好的靶向性和温度响应性,且可以下调靶基因RRM2的表达,抑制NCI-N87细胞的迁移。结论 成功制备抗体靶向的PNIPAM/PEI聚阳离子温度敏感型纳米凝胶,其可以结合并投递siRNA进入靶细胞,该纳米复合体可以作为新型抗肿瘤基因治疗的给药系统。
关键词:  纳米凝胶  抗体靶向  温度响应性  药物递送系统  小分子干扰RNA
DOI:10.16781/j.0258-879x.2017.06.0720
投稿时间:2017-05-10修订日期:2017-06-02
基金项目:国家自然科学基金(31470964,81171450)
In vitro anti-tumor effect of RRM2-siRNA based on active and passive dual targeted nanogel
ZHANG Li,WANG Ying,JIN Chong,DONG Xia,LI Wei*
(International Joint Cancer Institute, Second Military Medical University, Shanghai 200433, China
*Corresponding author)
Abstract:
Objective To prepare antibody-targeting temperature-sensitive polylated N-isopropylacrylamide/polyethyleneimine (PNIPAM/PEI) nanogel for delivering siRNA against ribonucleotide reductase (RR) subunit M2 (RRM2, RRM2-siRNA) and to establish a new targeted nano-gel delivery system for anti-tumor therapy by studying its anti-tumor ability in vitro. Methods We synthesized the core-shell temperature-sensitive PNIPAM/PEI nanogel by radical graft copolymerization and evaluated its chemo-physical properties (such as, size and zeta potential) using transmission electron microscopy (TEM). According to the principle of charge interaction, the PNIPAM/PEI-siRNA nanogel encapsulated RRM2-siRNA was prepared by conjugating anti-human epidermal growth factor receptor 2 (Her2) antibody. The effect of PNIPAM/PEI-siRNA nanogel complex encapsulated siRNA was determined by agarose gel electrophoresis. The in vitro uptake of NCI-N87 cells by nanogel complex was quantitatively observed by fluorescence microscopy and flow cytometry (FCM). The expression of RRM2 after RRM2 interference using nanogel-siRNA complex in NCI-N87 cells was tested by realtime PCR. The tumor migration suppressing effect of the nanogel complex on Her2-positive tumor cells was determined by Transwell assay. Results The core-shell temperature-responsive PNIPAM/PEI nanogel was synthesized by radical graft copolymerization, with homogeneous size of 359.8 nm, and zeta potential of 21.4 mV. Furthermore, the nanogel complexes encapsulated RRM2-siRNA with different ratios of N/P (N/P ratio: the molar ratio of nitrogen-from-polyethylenimine to phosphate-from-RNA) were prepared and the electrophoresis results showed that the optimal N/P ratio was 60. The cellular uptake experiment showed that the nanogel had good temperature sensitivity and tumor targeting ability at different temperatures (37 ℃, 42 ℃); and the nanogel complex down-regulated the expression of RRM2 and inhibited the migration of NCI-N87 cells. Conclusion The antibody-targeting temperature-sensitive PNIPAM/PEI nanogel is successfully prepared, and it can bind and deliver siRNA into target cells and can be used as a new drug delivery system for anti-tumor therapy.
Key words:  nanogel  antibody-targeting  temperature-response  drug delivery system  small interfering RNA