Discovery of Peptide ligands for hepatic stellate cells using phage display.
Discovery of Peptide ligands for hepatic stellate cells using phage display.
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DOI：
10.1021/acs.molpharmaceut.5b00177
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发表时间：
2015-06-01
影响因子：
4.9
通讯作者：
Cheng K
中科院分区：
文献类型：
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作者：
Chen Z;Jin W;Liu H;Zhao Z;Cheng K
无论其原因如何，肝纤维化的特点是肝脏中细胞外基质（ECM）的过度积累。肝星状细胞 (HSC) 是纤维化肝脏中 ECM 和促纤维化细胞因子过量产生的主要产生者。因此，开发 HSC 特异性递送系统对于抗纤维化药物的成功至关重要。本研究的目的是鉴定针对胰岛素样生长因子 2 受体 (IGF2R) 的肽配体，该受体在 HSC 上过度表达。我们期望将肽配体用于未来HSC靶向药物递送系统的开发。进行基于蛋白质和全细胞的噬菌体展示生物淘选来鉴定噬菌体/肽候选物。采用噬菌体 ELISA、细胞摄取和细胞活力测定来评估这些肽配体与重组人 IGF2R 和 HSC 的结合亲和力和特异性。 IGF2R siRNA 用于沉默人肝星状细胞 (LX-2) 中的 IGF2R 蛋白表达，以确认所鉴定的肽配体的特异性。在已鉴定的候选肽中，肽 431 对重组人 IGF2R 蛋白和 HSC 表现出最高的结合亲和力和特异性。肽-431 的平衡解离常数 (Kd) 对于 LX-2 细胞为 6.19 μM，对于大鼠肝星状细胞 HSC-T6 为 12.35 μM。用 siRNA 沉默 IGF2R 后，LX-2 细胞对肽 431 的细胞摄取显着减少。 Peptide-431 还能增强 LX-2 和 HSC-T6 细胞对促凋亡肽（KLA 肽）的摄取，这表明 Peptide-431 可以用作靶向配体，不仅可以将抗纤维化药物递送到大鼠 HSC 中，还可以将抗纤维化药物递送到人 HSC 中。肽 431 的二聚化进一步将其与 LX-2 细胞的结合亲和力增加了约九倍。
Regardless of its cause, liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) in the liver. Hepatic stellate cells (HSCs) are the main producers responsible for the excessive production of ECM and profibrogenic cytokines in fibrotic liver. Therefore, development of HSC-specific delivery systems is essential for the success of antifibrotic agents. The objective of this study is to identify peptide ligands targeting the insulin like growth factor 2 receptor (IGF2R), which is overexpressed on HSCs. We expect to use the peptide ligands for the future development of HSC-targeted drug delivery system. Protein- and whole cell-based phage display biopannings were conducted to identify phage/peptide candidates. Phage ELISA, cellular uptake and cell viability assay were employed to evaluate the binding affinity and specificity of these peptide ligands to recombinant human IGF2R and HSCs. IGF2R siRNA was used to silence the IGF2R protein expression in human hepatic stellate cells (LX-2) to confirm the specificity of the identified peptide ligands. Among the identified peptide candidates, the peptide-431 shows the highest binding affinity and specificity to recombinant human IGF2R protein and HSCs. The equilibrium dissociation constant (Kd) of the peptide-431 is 6.19 μM for LX-2 cells and 12.35 μM for rat hepatic stellate cells HSC-T6. Cellular uptake of the peptide-431 in LX-2 cells is significantly reduced after silencing IGF2R with siRNA. The peptide-431 also enhances the uptake of a proapoptotic peptide (KLA peptide) in LX-2 and HSC-T6 cells, indicating that the peptide-431 can be used as a targeting ligand to deliver antifibrotic agents into not only rat but also human HSCs. Dimerization of the peptide-431 further increase its binding affinity to LX-2 cells by approximately nine-fold.