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

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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.