Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes
Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes
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DOI：
10.1002/jgm.796
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发表时间：
2005-11-01
影响因子：
3.5
通讯作者：
Harashima, H
中科院分区：
文献类型：
--
作者：
Futaki, S;Masui, Y;Harashima, H
背景 使用阳离子脂质体进行细胞内基因递送的关键步骤之一是质粒/脂质体复合物从内体逃逸至胞质溶胶。添加 GALA（一种 pH 敏感的融合肽）是加速这一步骤以增强所需蛋白质表达的有前途的方法。对增强方法的详细研究将拓宽其应用视野。方法采用代表性的市售阳离子脂质体(Lipofectin、Lipofectamine和Lipofectamine 2000)，研究GALA对转染的影响。通过荧光素酶测定和共聚焦显微镜观察来研究效率。结果观察到 GALA 的转染效率呈浓度依赖性增加。向质粒/脂质体复合物中添加0.1μM GALA显着提高了转染效率，特别是在Lipofectin的情况下，但较高浓度的GALA降低了转染效率。通过使用 GALA 成功减少了脂质体剂量，同时保持了高转染效率。有趣的是，虽然在 GALA 存在的情况下转染效率较高，但细胞吸收的质粒 DNA 量较低。罗丹明标记质粒的共聚焦显微镜观察未显示在存在或不存在 GALA 的情况下孵育的细胞之间的细胞定位存在显着差异，这表明 GALA 诱导的质粒释放到胞质溶胶中的轻微增加可能会导致转染效率的显着变化。 结论 确定了 GALA 介导提高转染效率的独特功能。版权所有 (C) 2005 John Wiley & Sons, Ltd.
Background One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application.Methods Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection. efficiency were studied by luciferase assay and confocal microscopic observations.Results A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 mu M GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency.Conclusion The unique features of GALA to mediate improved transfection efficiencies were identified. Copyright (C) 2005 John Wiley & Sons, Ltd.