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

利用膜通透性多肽载体进行细胞内递送是近年来发展起来的一种方法，它已成功地用于将各种生物活性分子运送到细胞内以改变细胞功能。本研究的目的是利用荧光显微镜实时观察内在化的多肽载体，并分析细胞内靶向信号(包括细胞核和线粒体的靶向信号)附着到多肽载体上对其细胞定位的影响。细胞的共聚焦显微镜观察表明，大部分多肽载体是通过内吞作用被细胞摄取的，但由于多肽在胞浆中的高度积累，很难分析扩散到胞浆中的多肽载体。然而，在无血清培养或高浓度载体存在的情况下，可观察到有效的胞内扩散。将SV40核定位信号肽成功地连接到载体上，促进了结合物在细胞核中的聚集，但对线粒体定位信号的连接没有明显的影响。因此，我们得出结论，线粒体定位信号肽可能只对细胞内新产生的蛋白有效，而不是对细胞内传递的蛋白有效。这些发现将有助于细胞内靶向载体的研究。

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.