Department of Pharmaceutics and Biopharmacy, Philipps Universität Marburg, Ketzerbach 63, 35032 Marburg, Germany.
Biomaterials. 2011 Mar;32(9):2388-98. doi: 10.1016/j.biomaterials.2010.11.081. Epub 2010 Dec 22.
Since off-target effects in non-viral siRNA delivery are quite common but not well understood, in this study various polymer-related effects observed in transfection studies were described and their mechanisms of toxicity were investigated. A variety of stably luciferase-expressing cell lines was compared concerning polymer-mediated effects after transfection with polyplexes of siRNA and poly(ethylene imine) (PEI) or poly(ethylene glycol)-grafted PEI (PEG-PEI). Cell viability, LDH release, gene expression profiles of apoptosis-related genes and promoter activation were investigated. Interestingly, PEG-PEI, which is generally better tolerated than PEI, was found to activate apoptosis in a cell line- and concentration-dependent manner. While both polymers showed sigmoidal dose-response of cell viability in L929 cells (IC(50)(PEI) = 6 μg/ml, IC(50)(PEG-PEI) = 11 μg/ml), H1299/Luc cells exhibited biphasic dose-response for PEG-PEI and stronger apoptosis at 2 μg/ml than at 20 μg/ml PEG-PEI, as shown in TUNEL assays. Gene expression profiling confirmed that H1299/Luc cells underwent apoptosis via thousand-fold activation of TNF receptor-associated factors. Additionally, it was demonstrated that NFkB-mediated CMV promoter activation in stably transfected cells can lead to increased target gene levels after transfection instead of siRNA-mediated knockdown. With these results, polymeric vectors were shown not to be inert substances. Therefore, alterations in gene expression caused by the delivery agent must be known to correctly interpret gene-silencing experiments, to understand the mechanisms of off-target effects, and most of all to further develop vectors with reduced side effects. Taking these observations into account, one established cell line was eventually identified to be suitable for RNAi experiments. As shown by these experiments, materials that have been used for many years can elicit unexpected off-target effects. Therefore, non-viral vectors must be screened for several levels of toxicity to make them promising candidates.
由于非病毒 siRNA 递送中的脱靶效应相当常见，但尚未得到很好的理解，因此在本研究中，描述了转染研究中观察到的各种与聚合物相关的效应，并研究了其毒性机制。比较了各种稳定表达荧光素酶的细胞系，研究了用 siRNA 和聚（亚乙基亚胺）（PEI）或聚乙二醇接枝的 PEI（PEG-PEI）的多聚物转染后，与聚合物相关的效应。研究了细胞活力、LDH 释放、凋亡相关基因的基因表达谱和启动子激活。有趣的是，通常比 PEI 更耐受的 PEG-PEI 被发现以细胞系和浓度依赖的方式激活凋亡。虽然两种聚合物在 L929 细胞中均表现出细胞活力的 S 形剂量反应（PEI 的 IC（50）= 6 μg/ml，PEG-PEI 的 IC（50）= 11 μg/ml），但 H1299/Luc 细胞对 PEG-PEI 表现出双相剂量反应，与 20 μg/ml 的 PEG-PEI 相比，2 μg/ml 的 PEG-PEI 表现出更强的凋亡，如 TUNEL 测定所示。基因表达谱分析证实，H1299/Luc 细胞通过 TNF 受体相关因子的千倍激活而发生凋亡。此外，还证明了稳定转染细胞中 NFkB 介导的 CMV 启动子激活可导致转染后靶基因水平增加，而不是 siRNA 介导的下调。有了这些结果，聚合物载体就不是惰性物质了。因此，必须了解递送剂引起的基因表达改变，以便正确解释基因沉默实验，了解脱靶效应的机制，最重要的是，进一步开发副作用较小的载体。考虑到这些观察结果，最终确定了一种已建立的细胞系适合进行 RNAi 实验。正如这些实验所示，多年来一直使用的材料可能会产生意想不到的脱靶效应。因此，必须对非病毒载体进行多种毒性水平的筛选，以使其成为有前途的候选物。
