Beisner Julia, Dong Meng, Taetz Sebastian, Piotrowska Kamilla, Kleideiter Elke, Friedel Godehard, Schaefer Ulrich, Lehr Claus-Michael, Klotz Ulrich, Mürdter Thomas E
Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University of Tübingen, Stuttgart, Germany.
J Pharm Sci. 2009 May;98(5):1765-74. doi: 10.1002/jps.21553.
The antisense oligonucleotide 2'-O-methyl-RNA is a selective telomerase inhibitor targeting the telomerase RNA component and represents a potential candidate for anticancer therapy. The poor cellular uptake of 2'-O-methyl-RNA is a limiting factor that may contribute to the lack of functional efficacy. To improve delivery of 2'-O-methyl-RNA and consequently antitumoral efficiency in human lung cancer cells, we have investigated several transfection reagents. The transfection reagents DOTAP, MegaFectin 60, SuperFect, FuGENE 6 and MATra-A were tested for intracellular delivery. A FAM-labeled 2'-O-methyl-RNA was used to assess the intracellular distribution by confocal laser scanning microscopy in A549 human non-small cell lung cancer cells. Telomerase activity was measured using the telomeric repeat amplification protocol. Cell viability after transfection was quantified by the MTT assay. All transfection reagents enhanced 2'-O-methyl-RNA uptake in A549 cells but the cationic lipid reagents DOTAP and MegaFectin 60 were most efficient in the delivery of 2'-O-methyl-RNA resulting in telomerase inhibition. Among both DOTAP exhibited the lowest cytotoxicity. Our experiments show that DOTAP is the most suitable transfection reagent for the delivery of 2'-O-methyl-RNA in human lung cancer cells according to its relatively low cytotoxicity and its ability to promote efficient uptake leading to the inhibition of telomerase.
反义寡核苷酸2'-O-甲基核糖核酸是一种靶向端粒酶RNA成分的选择性端粒酶抑制剂，是抗癌治疗的潜在候选药物。2'-O-甲基核糖核酸较差的细胞摄取率是一个限制因素，可能导致功能疗效的缺乏。为了改善2'-O-甲基核糖核酸的递送并因此提高其在人肺癌细胞中的抗肿瘤效率，我们研究了几种转染试剂。测试了转染试剂DOTAP、MegaFectin 60、SuperFect、FuGENE 6和MATra-A的细胞内递送情况。使用FAM标记的2'-O-甲基核糖核酸通过共聚焦激光扫描显微镜评估A549人非小细胞肺癌细胞中的细胞内分布。使用端粒重复序列扩增协议测量端粒酶活性。通过MTT法对转染后的细胞活力进行定量。所有转染试剂均增强了A549细胞对2'-O-甲基核糖核酸的摄取，但阳离子脂质试剂DOTAP和MegaFectin 60在递送2'-O-甲基核糖核酸方面最有效，从而导致端粒酶抑制。在这两种试剂中，DOTAP表现出最低的细胞毒性。我们的实验表明，根据其相对较低的细胞毒性以及促进有效摄取从而抑制端粒酶的能力，DOTAP是在人肺癌细胞中递送2'-O-甲基核糖核酸最合适的转染试剂。
