Department of Pharmaceutics and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Int J Pharm. 2013 May 1;448(1):231-8. doi: 10.1016/j.ijpharm.2013.03.035. Epub 2013 Mar 25.
Nowadays a large number of clinical trials suffer from lacking an efficient method for drug delivery into target cells with minimal side effects. Due to the great significance of this issue in novel and effective therapies, more attempts are required in order to distinguish better conditions for biomedical drug delivery. Since embryonic stem cells (ESCs) are under scrutiny of many new studies, development of novel methods for their genetical and functional modifications is of great value. On the other hand, the application of short nucleic acids in new therapeutic approaches is increasing. In this study the efficiency of small interfering RNA (siRNA) uptake with two transfection reagents generation five of polyamidoamine dendrimer (PAMAM G5) as a cationic dendrimer and N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl-sulfate (DOTAP) as a cationic lipid and one commercially available reagent were evaluated in mouse ESCs using flow cytometry. Prior to the cellular investigations; atomic force microscopy; gel electrophoresis; siRNA binding and release assays; and size and zeta potential measurements were utilized to characterize the physicochemical properties of reagent-siRNA nano-complexes. The safety of the nano-complexes was subsequently assessed by MTT assay. Functional effects of siRNA (complementary strand for OCT4 transcript) transfection in ESCs with the mentioned reagents were analyzed using a quantitative real-time polymerase chain reaction (qPCR). Surprisingly DOTAP at higher molar ratios and PAMAM at lower molar ratios could successfully knock down the OCT4 transcription relatively better than commercial reagent. Our findings supported the appropriate efficiency of the mentioned transfection reagents for short nucleic acid transfection. From a clinical point of view, this research helps allocation of short nucleic acids into stem cells therapies.
如今，许多临床试验都面临着缺乏将药物有效递送至靶细胞而副作用最小的方法的问题。由于这个问题在新的有效治疗方法中有重要意义，因此需要更多的尝试来区分生物医学药物输送的更好条件。由于胚胎干细胞 (ESC) 受到许多新研究的关注，因此开发用于其基因和功能修饰的新方法具有重要价值。另一方面，短核酸在新治疗方法中的应用正在增加。在这项研究中，使用两种转染试剂——第五代聚酰胺胺树枝状大分子 (PAMAM G5) 作为阳离子树枝状大分子和 N-[1-(2,3-二油酰氧基)]-N,N,N-三甲基丙铵甲基硫酸盐 (DOTAP) 作为阳离子脂质以及一种市售试剂，通过流式细胞术评估了小干扰 RNA (siRNA) 在小鼠 ESC 中的摄取效率。在进行细胞研究之前，利用原子力显微镜、凝胶电泳、siRNA 结合和释放测定以及粒径和 zeta 电位测量来表征试剂-siRNA 纳米复合物的物理化学性质。随后通过 MTT 测定评估纳米复合物的安全性。使用定量实时聚合酶链反应 (qPCR) 分析了用所述试剂转染 siRNA（OCT4 转录物的互补链）对 ESC 的功能影响。令人惊讶的是，较高摩尔比的 DOTAP 和较低摩尔比的 PAMAM 可以相对更好地成功敲低 OCT4 转录，优于商业试剂。我们的研究结果支持了所述转染试剂对短核酸转染的适当效率。从临床角度来看，这项研究有助于将短核酸分配到干细胞治疗中。
