Enhanced hepatic uptake and bioactivity of type α1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol
Enhanced hepatic uptake and bioactivity of type α1(I) collagen gene promoter-specific triplex-forming oligonucleotides after conjugation with cholesterol
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DOI：
10.1124/jpet.105.100347
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发表时间：
2006-05-01
影响因子：
3.5
通讯作者：
Mahato, RI
中科院分区：
文献类型：
--
作者：
Cheng, K;Ye, ZY;Mahato, RI
α1(I) 型胶原蛋白启动子特异性的三链体形成寡核苷酸 (TFO) 是治疗肝纤维化的有希望的候选者。早些时候，我们确定了正常大鼠和纤维化大鼠全身给药后 TFO 的药代动力学和生物分布。在这项研究中，我们通过二硫键将胆固醇缀合到 TFO 的 3' 端，并使用 HSC-T6 细胞系在体外测定其细胞和核摄取和生物活性，然后在全身、器官（肝脏）和亚细胞水平上进行生物分布。与胆固醇的缀合对TFO与靶双链体DNA的三链体形成能力几乎没有影响，并且P-33-TFO-胆固醇(Chol)的细胞摄取增加了2倍至约4倍。 TFO-Chol 或 TFO 转染 HSC-T6 细胞后的实时逆转录聚合酶链反应分析表明，TFO-Chol 在低浓度 (200 nM) 下对 α1(I) 型胶原初级转录物具有比裸 TFO 更高的抑制作用，但在较高浓度（500 和 1000 nM）下表现出相似的抑制作用。随着转染时间的延长，对初级转录物的抑制增加。全身给药后，P-33-TFOChol 的肝脏摄取量为剂量的 72.22%，而 P-33-TFO 的摄取量为 45.8%。肝星状细胞和肝细胞对 P-33-TFO-Chol 的摄取显着增加。更重要的是，在细胞培养系统和体内研究中，TFO-Chol的核摄取高于TFO。总之，TFO-Chol 是一种潜在的抗纤维化药物。
A triplex-forming oligonucleotide (TFO) specific for type alpha 1(I) collagen promoter is a promising candidate for treating liver fibrosis. Earlier, we determined the pharmacokinetics and biodistribution of TFO after systemic administration into normal and fibrotic rats. In this study, we conjugated cholesterol to the 3' end of the TFO via a disulfide bond and determined its cellular and nuclear uptake and bioactivity using HSC-T6 cell lines in vitro, followed by biodistribution at whole-body, organ (liver), and subcellular levels. Conjugation with cholesterol had little effect on the triplex-forming ability of the TFO with target duplex DNA, and the cellular uptake of P-33-TFO-cholesterol (Chol) increased by 2- to approximately 4-fold. Real-time reverse transcriptase-polymerase chain reaction analysis after transfection of HSC-T6 cells with TFO-Chol or TFO indicated that TFO-Chol had higher inhibition on type alpha 1(I) collagen primary transcript than naked TFO at low concentration (200 nM) but showed similar inhibition at higher concentration (500 and 1000 nM). There was increase in the inhibition on primary transcript with transfection time. The hepatic uptake of P-33-TFOChol after systemic administration was 72.22% of the dose compared with 45.8% of P-33-TFO. There was significant increase in the uptake of P-33-TFO-Chol by hepatic stellate cells and hepatocytes. More importantly, the nuclear uptake of TFO-Chol was higher than TFO in cell culture system and in vivo studies. In conclusion, TFO-Chol is a potential antifibrotic agent.