Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation
Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation
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DOI：
10.1038/sj.gt.3302128
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发表时间：
2004-04-01
期刊：
影响因子：
5.1
通讯作者：
Harashima, H
中科院分区：
文献类型：
--
作者：
Khalil, IA;Futaki, S;Harashima, H
利用激光共聚焦显微镜和流式细胞仪分析研究了与octagrinine和stearyl-octagrinine相关的内化机制。八精氨酸能够以不完全涉及经典内吞内化途径的方式通过细胞膜转运。然而，当硬脂基部分连接到八精氨酸的N-末端时，内化主要转向内吞依赖性途径。硬脂酰八精氨酸的转染效率显著高于八精氨酸。为了了解八精氨酸N端硬脂酰化促进基因转移的机制，比较了八精氨酸和硬脂酰八精氨酸介导的基因转移过程。八精氨酸和硬脂酰八精氨酸都能够携带质粒DNA进入细胞。在硬脂酰-八精氨酸的情况下，内化的质粒DNA的量以及递送到细胞核的量更高。尽管八精氨酸和硬脂酰八精氨酸的内化机制不同，但它们与质粒DNA的复合物通过相同的途径内化，推测为网格蛋白介导的内吞途径。原子力显微镜的结果显示，硬脂酰-octagrinine，但不是octagrinine，可以完全凝聚成稳定的复合物，可以高度吸附到细胞表面，随后高度内化的DNA。因此，使用硬脂酰化-八精氨酸提供了质粒DNA进入细胞的更高内化，这是由于增强的细胞缔合以及更高的核递送。在这项研究中提出的结果提供了一个更好的理解的机制，提高转染使用硬脂酰化octagrinine。使用硬脂酰化肽的概念可能有助于开发更有效的非病毒基因载体。
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.