Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression
Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression
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DOI：
10.1016/s0168-3659(99)00280-1
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发表时间：
2000-05-15
影响因子：
10.8
通讯作者：
Hashida, M
中科院分区：
文献类型：
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作者：
Sakurai, F;Inoue, R;Hashida, M
为了确定参与阳离子脂质体介导的基因转移的重要因素，在体外转染效率由质粒DNA复合DOTMA/DOPE脂质体在不同的DNA/脂质体混合比例进行了评估，使用四种类型的培养细胞的物理化学性质。观察到显着的变化，在粒径和zeta电位的配合物，以及在它们的结构，通过原子力显微镜，这取决于混合比进行评估。在转染实验中，除了RAW 264.7细胞（小鼠巨噬细胞）之外，在MET-2细胞（小鼠膀胱肿瘤）、NLH 3 T3细胞（小鼠成纤维细胞）和HUVEC（人脐静脉内皮细胞）中分别以1：5、1：7.5或1：5的最佳比例获得有效的基因表达。另一方面，细胞摄取的[P-32]DNA/脂质体复合物增加在所有类型的细胞中的混合比的增加，这是没有反映的转染效率。MTT法测定的细胞损伤是最小的，即使在最高的DNA/脂质体比例（1：10），表明在较高的比例较低的基因表达水平不是由于复合物诱导的细胞毒性。溴化乙锭嵌入试验表明，质粒DNA从复合物中的释放，以下添加带负电荷的脂质体，限制作为混合比的增加。此外，使用HUVEC的共聚焦显微镜研究表明，1：5复合物在细胞质中表现出分散的分布，而观察到1：10复合物的点状细胞内分布。这表明，在这些混合比之间，质粒DNA/阳离子脂质体复合物的细胞内运输（可能从内体或溶酶体释放）存在显著差异。两者合计，这些发现表明，DNA/脂质体混合比显着影响与阳离子脂质体复合的质粒DNA的细胞内运输，这是阳离子脂质体介导的转染中的最佳混合比的重要决定因素。(C)2000 Elsevier Science B. V.保留所有权利。
In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MET-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [P-32]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection. (C) 2000 Elsevier Science B.V. All rights reserved.