Cardoso A L C, Simões S, de Almeida L P, Pelisek J, Culmsee C, Wagner E, Pedroso de Lima M C
Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
J Gene Med. 2007 Mar;9(3):170-83. doi: 10.1002/jgm.1006.
RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra- and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)-associated liposomes for siRNA complexation and gene silencing.
Cationic liposomes composed of DOTAP : Cholesterol associated with or without transferrin (Tf) were complexed with siRNA at different lipid/siRNA charge ratios. Complexation and protection of siRNA from enzymatic degradation was assessed with the PicoGreen intercalation assay and gel electrophoresis. Cellular internalization of these siRNA Tf-lipoplexes was detected by confocal microscopy. Luciferase assay, immunoblot and fluorescence-activated cell sorting (FACS) analysis were used to evaluate reporter gene silencing in Huh-7 hepatocarcinoma and U-373 glioma cells. c-Jun knockdown in HT-22 cells was evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Cytotoxicity of the siRNA complexes was assessed by Alamar blue, lactate dehydrogenase and MTT assays.
Complexation of siRNA with the cationic liposomes in the presence of Tf results in the formation of stable particles and prevents serum-mediated degradation. Confocal microscopy showed fast cellular internalization of the Tf-lipoplexes via endocytosis. In the GFP glioma cells Tf-lipoplexes showed enhanced gene silencing at minimum toxicity in comparison to Tf-free lipoplexes. Targeting luciferase in the hepatocarcinoma cell line resulted in more than 70% reduction of luciferase activity, while in HT-22 cells 50% knockdown of endogenous c-Jun resulted in a significant protection from glutamate-mediated toxicity.
Cationic liposomes associated with Tf form stable siRNA lipoplexes with reduced toxicity and enhanced specific gene knockdown activity compared to conventional lipoplexes. Thus, such formulations may constitute efficient delivery systems for therapeutic siRNA applications.
RNA干扰为特定基因沉默提供了一种强大的技术。然而，小干扰RNA（siRNA）的治疗应用需要有效的载体，用于这些核酸的稳定络合、保护以及细胞外和细胞内递送。在此，我们评估了转铁蛋白（Tf）相关脂质体用于siRNA络合和基因沉默的潜力。
由DOTAP：胆固醇组成的阳离子脂质体，与或不与转铁蛋白（Tf）结合，在不同脂质/siRNA电荷比下与siRNA络合。用PicoGreen嵌入分析法和凝胶电泳评估siRNA的络合和免受酶降解的保护作用。通过共聚焦显微镜检测这些siRNA-Tf脂质复合物的细胞内化。荧光素酶测定、免疫印迹和荧光激活细胞分选（FACS）分析用于评估Huh-7肝癌细胞和U-373胶质瘤细胞中报告基因的沉默。通过定量实时聚合酶链反应（RT-PCR）评估HT-22细胞中c-Jun的敲低情况。通过Alamar蓝、乳酸脱氢酶和MTT测定评估siRNA复合物的细胞毒性。
在Tf存在下，siRNA与阳离子脂质体的络合导致形成稳定颗粒，并防止血清介导的降解。共聚焦显微镜显示Tf脂质复合物通过内吞作用快速细胞内化。在GFP胶质瘤细胞中，与不含Tf的脂质复合物相比，Tf脂质复合物在最低毒性下显示出增强的基因沉默。在肝癌细胞系中靶向荧光素酶导致荧光素酶活性降低70%以上，而在HT-22细胞中，内源性c-Jun的50%敲低导致对谷氨酸介导的毒性有显著保护作用。
与传统脂质复合物相比，与Tf相关的阳离子脂质体形成稳定的siRNA脂质复合物，毒性降低且特异性基因敲低活性增强。因此，此类制剂可能构成用于治疗性siRNA应用的有效递送系统。
