Targeted delivery of a triplex-forming oligonucleotide to hepatic stellate cells
Targeted delivery of a triplex-forming oligonucleotide to hepatic stellate cells
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DOI：
10.1021/bi047529j
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发表时间：
2005-03-22
期刊：
影响因子：
2.9
通讯作者：
Mahato, RI
中科院分区：
文献类型：
--
作者：
Ye, ZY;Cheng, K;Mahato, RI
肝纤维化的特征是肝星状细胞（HSC）中细胞外基质（ECM），即纤维状胶原的异常积聚。早期，我们开发了一种抗基因方法，使用α 1（I）型胶原基因启动子特异性三链体形成寡核苷酸（TFO）来抑制胶原基因表达。为了提高TFO向肝细胞、尤其是向HSC的总递送，我们通过磷酸化对硝基苯基-α-D-吡喃甘露糖苷，还原其硝基，并将其与硫光气反应以产生用于与BSA缀合的对异硫氰酸基苯基-6-磷酸-α-D-吡喃甘露糖苷（itcM 6P）来合成甘露糖6-磷酸-牛血清白蛋白（M6 P-BSA）。通过二硫键将P-33-TFO与M6 P-BSA偶联，并测定（M6 P）（20）-BSA-TFO偶联物的稳定性。在大鼠尾静脉注射后，（M6 P）（20）-BSA-P-33-TFO迅速从循环中清除，并主要蓄积在肝脏中。注射后30 min，注射的（M6 P）（20）-BSA-P-33-TFO中约有66%在肝脏中蓄积，显著高于注射33 P-TFO后的蓄积量。大部分注射的（M6 P）（20）-BSA-P-33-TFO被HSC吸收，如通过在肝脏灌注和在Nycodenz梯度上分离时测定消化的肝细胞中的放射性所证明的。因此，该TFO缀合物可用于治疗肝纤维化。
Liver fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM), namely, fibrillar collagens in the hepatic stellate cells (HSCs). Earlier, we developed an antigene approach, using a type alpha 1 (I) collagen gene promoter specific triplex-forming oligonucleotide (TFO) to inhibit collagen gene expression. In this paper, to enhance overall delivery of TFOs to the liver and more specifically to HSCs, we synthesized mannose 6-phosphate-bovine serum albumin (M6P-BSA) by phosphorylating p-nitrophenyl-alpha-D-mannopyranoside, reducing its nitro group, and reacting it with thiophosgene to produce p-isothiocyanatophenyl-6-phospho-alpha-D-mannopyranoside (itcM6P) for conjugation with BSA. P-33-TFO was conjugated with M6P-BSA via a disulfide bond, and the stability of the (M6P)(20)-BSA-TFO conjugate was determined. Following tail vein injection into rats, (M6P)(20)-BSA-P-33-TFO rapidly cleared from the circulation and accumulated mainly in the liver. Almost 66% of the injected (M6P)(20)-BSA-P-33-TFO accumulated in the liver at 30 min postinjection, which was significantly higher than that deposited after injection of 33P-TFO. A large proportion of the injected (M6P)(20)-BSA-P-33-TFO was taken up by the HSCs as evidenced by determination of radioactivity in the digested liver cells upon liver perfusion and separation on a Nycodenz gradient. Therefore, this TFO conjugate may be used for the treatment of liver fibrosis.