免疫纳米微粒靶向胰腺癌细胞输送siRNA的方法研究

 目的：构建一种向胰腺癌细胞安全、高效输送siRNA的免疫纳米载体。方法：检测纳米载体IONP-PEI（非靶向组）及其与siRNA复合物的表征；通过琼脂糖凝胶电泳检测siRNA结合力、MTS法检测细胞活力和流式细胞术检测转染率以确定其复合siRNA的最佳N/P比值；细胞免疫荧光和普鲁士蓝染色观察scFvCD44v6偶联IONP-PEI的靶向纳米载体（靶向组）在细胞内分布；流式细胞术、荧光显微镜观察、real-time PCR和Western blotting检测靶向组和非靶向组的转染率和转染siKRAS后的干扰效果。结果：IONP和PEI的最适质量比为0.75；纳米载体复合siRNA的最佳N/P比值为20；IONP-PEI/siRNA复合物的电位为（21.73±8.07）mV，粒径为（51.3±2.2）nm。荧光显微镜显示,非靶向组和靶向组转染后均在细胞内,靶向组的转染率为（89.75±1.81）%，高于非靶向组的（59.87±4.52）%,且靶向组的荧光强度高于非靶向组。靶向组的KRAS mRNA的相对表达量为（34.02±615）%，低于非靶向组的（51.09±6.70）%；Western blotting显示靶向组的KRAS蛋白表达量低于非靶向组。结论：非靶向组和靶向组均能够将siRNA转染进细胞内，且靶向组具有更高的转染效率和更好的干扰基因表达效果。本课题构建的scFvCD44v6-IONP-PEI是一种高效、安全和靶向识别胰腺癌细胞的免疫纳米载体。

Nanoparticle for siRNA delivery and its pancreatic cancer targeting ability

AIM:  To synthesize a safe, efficient and targeted nanoparticulate carrier for siRNA delivery to pancreatic cancer cells. METHODS: Iron oxide nanocrystal with carboxylic acid group-polyethyleneimine (IONP-PEI) was synthesized and investigated as a nonviral carrier of siRNA to the pancreatic cells. The size, surface and charge using zeta potential were characterized. The perfect charge ratio between amino groups of IONP-PEI and phosphate groups of siRNA (N/P) was determined by the transfection efficiency detection, gel retardation assay and MTS assay. An antibody-directed nonviral vector, scFvCD44v6-IONP-PEI nanoparticle attaching to the cancer-associated CD44v6 single-chain variable fragment, was constructed as a cancer-targeting nanocarrier for siRNA delivery. Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFvCD44v6-IONP-PEI/siRNA complexes in the cells. The transfection efficiency, fluorescence intensity and the expression of KRAS at mRNA and protein levels in the cells transfected by IONP-PEI/siRNA and scFvCD44v6-IONP-PEI/siRNA were detected by flow cytometry, fluorescence microscopy, real-time PCR and Western blotting, respectively. RESULTS：The mass ratio of IONP to PEI was 0.75. The suitable ratio of N/P was 20. The averaged size and surface zeta potential of IONP-PEI/siRNA in deionized water were (51.3±2.2)nm (diameter) and (21.73±8.07)mV,respectively. Red fluorescence was seen in both targeting and nontargeting groups, which clearly revealed the intracellular distribution of siRNA and delivery agents. Transfection efficiencies in targeting and nontargeting groups were (89.75±1.81)% and (59.87±4.52)%, respectively. Down-regulation of the KRAS mRNA in Panc-1 cells transfected with siKRAS by scFvCD44v6-IONP-PEI and IONP-PEI was up to (34.02± 6.15)% and (51.09±6.70)%, respectively. The protein level of KRAS was lower in targeting group than that in nontargeting group. CONCLUSION：scFvCD44v6-IONP-PEI is a safe and efficient nanoparticulate carrier for gene delivery. It is more effective to transfer siRNA into the cells and mediate gene silencing effect in vitro than the nontargeting group.