Department of Pharmaceutics and Analytical Chemistry Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen O, Denmark.
Pharm Res. 2012 Mar;29(3):669-82. doi: 10.1007/s11095-011-0589-0. Epub 2011 Oct 5.
Tumor necrosis factor α (TNF-α) plays a key role in the progression of rheumatoid arthritis and is an important target for anti-rheumatic therapies. TNF-α expression can be silenced with small interfering RNA (siRNA), but efficacy is dependent on efficient and safe siRNA delivery vehicles. We aimed to identify polymeric nanocarriers for anti-TNF-α siRNA with optimal efficacy and minimal off-target effects in vitro.
TNF-α silencing with polymeric siRNA nanocarriers was compared in lipopolysaccharide-activated RAW 264.7 macrophages by real-time reverse transcription (RT)-PCR. Expression of non-target genes involved in inflammation, apoptosis, and cell cycle progression was determined by RT-PCR, toxicity evaluated by propidium iodide and annexin V staining.
PAMAM dendrimers (G4 and G7) and dextran nanogels mediated remarkably high concentration-dependent gene silencing and low toxicity; dioleoyltrimethylammoniumpropane-modified poly(DL-lactide-co-glycolide acid) nanoparticles, thiolated, trimethylated chitosan and poly[(2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol] polyplexes were less efficient transfectants. There were minor changes in the regulation of off-target genes, mainly dependent on nanocarrier and siRNA concentration.
Dextran nanogels and PAMAM dendrimers mediated high gene silencing with minor toxicity and off-target transcriptional changes and are therefore expected to be suitable siRNA delivery systems in vivo.
肿瘤坏死因子 α（TNF-α）在类风湿关节炎的进展中起着关键作用，是抗风湿治疗的重要靶点。TNF-α 的表达可以用小干扰 RNA（siRNA）沉默，但疗效取决于高效和安全的 siRNA 递药载体。我们旨在鉴定用于抗 TNF-α siRNA 的聚合物纳米载体，使其在体外具有最佳疗效和最小的脱靶效应。
通过实时逆转录（RT）-PCR 比较脂多糖激活的 RAW 264.7 巨噬细胞中聚合物 siRNA 纳米载体的 TNF-α 沉默作用。通过 RT-PCR 确定参与炎症、细胞凋亡和细胞周期进展的非靶基因的表达，通过碘化丙啶和膜联蛋白 V 染色评估毒性。
PAMAM 树突（G4 和 G7）和葡聚糖纳米凝胶介导的基因沉默浓度依赖性极高，毒性低；二油酰基三甲基铵丙烷修饰的聚（DL-乳酸-co-乙醇酸）纳米粒子、巯基化、三甲基化壳聚糖和聚[（2-羟丙基）甲基丙烯酰胺 1-甲基-2-哌啶甲醇]聚合物复合物的转染效率较低。脱靶基因的调控变化较小，主要取决于纳米载体和 siRNA 的浓度。
葡聚糖纳米凝胶和 PAMAM 树突介导的高基因沉默作用，毒性小，脱靶转录变化小，因此有望成为体内合适的 siRNA 递药系统。
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