Study on peptides passing through cell membranes by the electrostatic interaction.

血脑屏障(BBB)是一种高度选择性的膜屏障，调节血液中物质进入脑实质的运输。最近，我们成功地通过吸附介导的跨细胞作用(AMT)将合成肽输送到大脑中，AMT是基于阳离子配体和细胞膜上的阴离子位置之间的静电相互作用。例如，合成的阳离子多肽MeTyr-Arg-MeArg-D-Leu-NH(CH2)8称为OO1-C8，它通过AMT机制高度通过BBB转运。在本研究项目中，我们制备了一种荧光标记的多肽001-C8-NBD(NBD：5-硝基苯并-3-氧杂-2，4-二唑)，通过测量荧光OO1-C8-NBD的跨上皮转运，以及通过对活的非固定细胞的三维可视化分析，成功地应用于阐明和可视化AMT在Caco-2细胞中的过程。接下来，我们合成了促酪氨酸激素释放激素(TRH)和止痛肽Kyotorphin(Kyo)类似物，其中引入了辛二胺(Oda=C8)和NBD残基；这些多肽被设计成具有适当的阳离子和亲脂性，需要通过AMT机制进行BBB转运。到目前为止，我们研究了TRH-C8-NBD通过BBB的被动转运机制，而不是AMT机制，而Kyo-C8-NBD没有通过BBB。这一事实表明，我们需要重新考虑通过BBB的多肽的设计。此外，我们制备了三种含对硼苯丙氨酸的二肽，将用于阐明肿瘤细胞的中子俘获治疗。

The blood-brain barrier (BBB) is a highly selective membranous barrier regulating the transport of substances in blood into the brain parenchyma. Recently we succeeded to open away to deliver synthetic peptides into the brain by the adsorptive-mediated transcytosis (AMT) which is based on the electrostatic interaction between the cationic ligands and the anionic sites on the cell membranes. For example, a synthetic cationic peptide MeTyr-Arg-MeArg-D-Leu-NH(CH_2)_8 termed OO1-C8 was highly transported through the BBB via the AMTmechanism. In the present research project, we prepared a fluorescence-labeled peptide 001-C8-NBD (NBD : 5-nitrobenzo-3-oxa-2, 4-diazole) which was successfully applied to elucidate and visualize the process of AMT in Caco-2 cells by measuring the transepithelial transport of fluorescent 0O1-C8-NBD, as well as by visual three-dimentional analysis of living, nonfixed cells by confocal laser microscopy.We next synthesized both the tyrotropic hormone releasing hormone (TRH) and an analgesic peptide kyotorphin (KYO) analogs in which the octanediamine (Oda=C8) and NBD residues were introduced ; these peptides were designed as possessing suitable cationic and lipophilic character requiring for the BBB transport by the AMT mechanism. So far we examined, TRH-C8-NBD was transported through the BBB by the passive transport mechanism but not the AMT mechanism, while KYO-C8-NBD did not pass through the BBB.This fact suggests that we need to reconsider for the design of the peptides passing through the BBB.Furthermore, we prepraed three kinds of p-boronophenylalanine containing dipeptides which will be applied to the elucidation of neutron capture therapy of cancer cells.