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Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

Gene Therapy volume 11, pages 636–644 (2004)Cite this article

Abstract

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

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Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Grants-in-Aid for Scientific Research on Priority Areas from the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan

    IA Khalil, H Kamiya & H Harashima

  2. CREST, Japan Science and Technology Corporation (JST), Japan

    IA Khalil, Y Baba, N Kaji, H Kamiya & H Harashima

  3. Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan

    S Futaki & M Niwa

  4. Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan

    Y Baba & N Kaji

  5. Single-molecule Bioanalysis Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan

    Y Baba

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  1. IA Khalil
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Khalil, I., Futaki, S., Niwa, M. et al. Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation. Gene Ther 11, 636–644 (2004). https://doi.org/10.1038/sj.gt.3302128

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  • DOI: https://doi.org/10.1038/sj.gt.3302128

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