Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Department of Biostatistics, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Mol Ther. 2018 Dec 5;26(12):2798-2811. doi: 10.1016/j.ymthe.2018.08.022. Epub 2018 Sep 1.
Trans-differentiation of quiescent hepatic stellate cells (HSCs) into active myofibroblasts secretes excess amounts of extracellular matrix (ECM) proteins. miR-29b1 has the potential to treat liver fibrosis, because it targets several profibrotic genes. We previously demonstrated that miR-29b1 and the hedgehog (Hh) pathway inhibitor GDC-0449 could, together, inhibit the activation of HSCs and ECM production in common bile-duct-ligated (CBDL) mice. Herein, we determined the effect of chemical modifications of miR-29b1 on its stability, immunogenicity, and Argonaute-2 (Ago2) loading in vitro, after modifying its antisense strand with phosphorothioate (PS-miR-29b1), 2'-O-methyl-phosphorothioate (OMe-miR-29b1), locked nucleic acid (LNA-miR-29b1), and N,N'-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN-miR-29b1). Chemical modifications significantly improved stability of miR-29b1 in 50% FBS. Among all the modified miRNAs tested, OMe-PS-miR-29b1 showed the highest stability with low immunogenicity, without the loss of efficacy in vitro. Therefore, OMe-PS-miR-29b1 was complexed with poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-tetraethylenepentamine (mPEG-b-PCC-g-DC-g-TEPA) cationic micelles, and anti-fibrotic efficacy was evaluated in CBDL mice. There was a significant improvement in liver histology and decrease in the levels of injury markers. Further, mRNA/protein levels of collagen, α-SMA, and TIMP-1 were significantly lower for the OMe-PS-miR-29b1-loaded micelles compared to miR-29b1-loaded micelles. In conclusion, micellar delivery of OMe-PS-miR-29b1 is a promising strategy to treat liver fibrosis.
静止的肝星状细胞（HSCs）向活性肌成纤维细胞的转分化会分泌大量细胞外基质（ECM）蛋白。miR-29b1 具有治疗肝纤维化的潜力，因为它靶向几个促纤维化基因。我们之前的研究表明，miR-29b1 和 hedgehog（Hh）途径抑制剂 GDC-0449 可以共同抑制胆总管结扎（CBDL）小鼠中 HSCs 的激活和 ECM 的产生。在此，我们确定了用硫代磷酸酯（PS-miR-29b1）、2'-O-甲基硫代磷酸酯（OMe-miR-29b1）、锁核酸（LNA-miR-29b1）和 N,N'-二乙基-4-（4-硝基萘-1-基偶氮）-苯基胺（ZEN-miR-29b1）修饰其反义链后，miR-29b1 在体外的稳定性、免疫原性和 Argonaute-2（Ago2）加载的影响。化学修饰显著提高了 miR-29b1 在 50%胎牛血清中的稳定性。在所测试的所有修饰的 miRNA 中，OMe-PS-miR-29b1 表现出最高的稳定性和低免疫原性，而不会降低体外疗效。因此，将 OMe-PS-miR-29b1 与聚乙二醇-嵌段-聚（2-甲基-2-羧基丙稀碳酸酯-接枝-十二醇-接枝-四乙撑五胺）阳离子胶束复合，并在 CBDL 小鼠中评估其抗纤维化效果。肝组织学有显著改善，损伤标志物水平降低。此外，与 miR-29b1 载药胶束相比，载 OMe-PS-miR-29b1 的胶束的胶原、α-SMA 和 TIMP-1 的 mRNA/蛋白水平显著降低。综上所述，载 OMe-PS-miR-29b1 的胶束递药是治疗肝纤维化的一种很有前途的策略。
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