中国病理生理杂志Issue(9)：1567-1573,7.DOI:10.3969/j.issn.1000-4718.2014.09.005
免疫纳米微粒靶向胰腺癌细胞输送siRNA 的方法研究
Nanoparticle for siRNA delivery and its pancreatic cancer targeting abili-ty
摘要
Abstract
AIM:To synthesize a safe , efficient and targeted nanoparticulate carrier for siRNA delivery to pan-creatic 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 frag-ment, was constructed as a cancer-targeting nanocarrier for siRNA delivery .Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFv CD44v6-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 scFv CD44v6-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 re-vealed 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)%, re-spectively .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 .关键词
胰腺肿瘤/纳米微粒/基因治疗/靶向Key words
Pancreatic neoplasms/Nanoparticles/Genetic therapy/Targeting分类
医药卫生引用本文复制引用
李佳佳,陈茵婷,曾林涓,练国达,陈少杰,李雅晴,黄开红..免疫纳米微粒靶向胰腺癌细胞输送siRNA 的方法研究[J].中国病理生理杂志,2014,(9):1567-1573,7.基金项目
国家自然科学基金资助项目（No．30670951；No．81072045）；国家自然青年科学基金资助项目（No．81302140）；中山大学医科青年培育项目 （）
