Development of intracellular targeting peptide vectors and the real-time observation in cells.

利用膜渗透性肽载体的细胞内递送是最近发展起来的一种方法，已成功地将各种生物活性分子转运到细胞中以改变细胞功能。本研究的目的是利用荧光显微镜实时观察内化肽载体，分析细胞核和线粒体等细胞内靶向信号附着在肽载体上对其细胞定位的影响。细胞共聚焦显微镜观察发现，大部分多肽载体通过胞吞作用被细胞吸收，但由于多肽在细胞质中大量积聚，因此很难对扩散到细胞质中的多肽载体进行分析。然而，在不含血清或存在高浓度载体的培养基中，观察到肽载体的有效细胞质扩散。将SV40核定位信号肽附着在载体上成功地促进了偶联物在细胞核中的积累，而线粒体定位信号附着在载体上没有明显的效果。因此，我们得出结论，线粒体定位信号肽可能仅对细胞内新生成的蛋白质有效，而对细胞内传递的蛋白质无效。这些发现应该对细胞内靶向载体有用。

Intracellular delivery using membrane-permeable peptide vectors is a recently developed methodology that has been employed successfully to transport various bioactive molecules into cells to modify cell functions. The purpose of this study is to observe internalized peptide vectors in real time using fluorescence microscopy and to analyze the effect of the attachment of intracellular targeting signals including those for nucleus and mitochondria to the peptide vectors on their cellular localization. Confocal microscopic observation of the cells revealed that most of the peptide vectors were taken up by the cells by endocytosis, but it was difficult to analyze the peptide vectors diffusing into cytosol due to the high accumulation of the peptides in cytosol. However, in culture medium without containing serum or in the presence of high concentration of the vectors, effective cytosolic diffusion of the peptide vectors was observed. Attachment of the SV40 nuclear localization signal peptide to the vectors successfully enhanced the accumulation of the conjugates into nucleus, but no significant effect was observed by the attachment of mitochondrial localization signals. We thus concluded that mitochondria localization signal peptides may be effective only for newly in-cell generated proteins but not for intracellularly delivered proteins. These findings should be useful for the intracellular targeting vectors.