Comparative gene transfer between cationic and thiourea lipoplexes
Comparative gene transfer between cationic and thiourea lipoplexes
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DOI：
10.1002/jgm.1417
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发表时间：
2010-01-01
影响因子：
3.5
通讯作者：
Mignet, Nathalie
中科院分区：
文献类型：
--
作者：
Breton, Marie;Leblond, Jeanne;Mignet, Nathalie
背景我们之前开发了脂多硫脲脂质作为中性 DNA 缩合剂，用于全身基因递送。脂多硫脲结构的优化产生了有效的转染剂。为了进一步评价这些脂质，我们研究了硫脲脂质复合物的内化过程以及它们与阳离子脂质复合物的细胞内转染机制。方法采用MTT测试来评估细胞毒性。通过荧光素酶读数确定转染效率。通过流式细胞术评估对碘化丙啶和增强型绿色荧光蛋白的渗透。通过标记 DNA 的共聚焦显微镜监测内化和 DNA 释放的动力学。使用内吞作用抑制剂来研究脂质复合物内化的机制。结果虽然硫脲/DNA复合物表现出与阳离子复合物几乎相似的转染水平，但硫脲脂质复合物的内化程度要低六倍。复合物能够使细胞质膜通透至 30 kDa 分子。最后，DNA 在细胞质中的释放时间不到 10 分钟，而阳离子脂质复合物则需要 30 分钟。 结论 尽管与阳离子脂质相比，硫脲脂质复合物的内化作用较弱，但硫脲脂质复合物能够以相似的水平转染细胞，因为它具有更强的破坏细胞质膜稳定性和释放 DNA 的能力。 版权所有 (C) 2009 John Wiley & Sons, Ltd.
Background We have previously developed lipopolythiourea lipids as neutral DNA condensing agents for systemic gene delivery. Optimization of the lipopolythiourea structure led to efficient transfecting agents. To further evaluate these lipids, we investigated the internalization process of the thiourea lipoplexes and their intracellular mechanism of transfection versus that of cationic lipoplexes.Methods The MTT test was used for cytotoxicity assessment. Transfection efficiency was determined by luciferase read-out. Permeation to propidium iodide and enhanced green fluorescent protein was evaluated by flow cytometry. Kinetics of internalization and DNA release were monitored by confocal microscopy with labelled DNA. Endocytosis inhibitors were used to study the mechanisms of lipoplex internalization.Results Although thiourea/DNA complexes exhibit an almost similar level of transfection compared to that of cationic complexes, the thiourea lipoplexes were shown to be six-fold less internalized. Complexes were able to permeabilize the cytoplasmic membrane to 30 kDa molecules. Finally, DNA was shown to be released in less than 10 min in the cellular cytoplasm versus 30 min for cationic lipoplexes.Conclusions Despite a weaker internalization compared to cationic lipids, the thiourea lipoplexes were able to transfect cells at a similar level as a result of its greater ability to destabilize the cytoplasmic membrane and release DNA Copyright (C) 2009 John Wiley & Sons, Ltd.