Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection
Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection
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DOI：
10.1021/bi9602019
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发表时间：
1996-05-07
期刊：
影响因子：
2.9
通讯作者：
Szoka, FC
中科院分区：
文献类型：
--
作者：
Xu, YH;Szoka, FC
为了了解 DNA 如何从细胞中的阳离子脂质体/DNA 复合物中释放，我们研究了哪些生物分子介导 DNA 从阳离子脂质体复合物中释放。通过溴化乙锭 (EtBr) 荧光的增加来定量单价[1,2-二油酰基-3-（三甲基铵）丙烷]或多价（双十八烷基酰胺甘氨酰精胺）脂质的释放。使用琼脂糖凝胶电泳上质粒迁移的变化来检查质粒对 DNAse I 降解的敏感性。 DNA 与阳离子脂质的物理分离得到证实，并在蔗糖密度梯度上进行了定量。含有模拟质膜面向细胞质的单层的组合物（例如磷脂酰丝氨酸）的阴离子脂质体从复合物中快速释放DNA。释放发生在接近 1/1 的电荷比 (-/+) 处，并且不受离子强度或离子类型的影响。具有高负线性电荷密度的水溶性分子（例如硫酸葡聚糖或肝素）也会释放 DNA。然而，离子水溶性分子，如 ATP、tRNA、DNA、聚（谷氨酸）、亚精胺、精胺或组蛋白，即使在 100 倍电荷过量 (-/+) 下也不会发生这种情况。基于这些结果，我们提出，在阳离子脂质/DNA复合物通过内吞作用内化到细胞中后，它使内体不稳定，内体横向扩散到复合物中并与阳离子脂质形成电荷中性离子对。这导致 DNA 从阳离子脂质中移位并将 DNA 释放到细胞质中。这种机制解释了阳离子脂质/DNA 复合物与细胞相互作用的各种观察结果。
TO understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3-(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at a 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.