Mechanism of oligonucleotide release from cationic liposomes
Mechanism of oligonucleotide release from cationic liposomes
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DOI：
10.1073/pnas.93.21.11493
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发表时间：
1996-10-15
影响因子：
11.1
通讯作者：
Szoka, FC
中科院分区：
文献类型：
--
作者：
Zelphati, O;Szoka, FC
我们提出了一种机制，寡核苷酸（ODN）释放阳离子脂质复合物在细胞中，占阳离子脂质-核酸-细胞相互作用的各种观察。用罗丹明标记的阳离子脂质体/荧光素标记的ODN（F-ODN）复合物处理的细胞的荧光共聚焦显微镜显示F-ODN在内化后与脂质分离并进入细胞核，将荧光脂质留在细胞质结构中。通过荧光共振能量转移研究了ODN从复合物中的置换。阴离子脂质体组合物（例如，磷脂酰丝氨酸）以约1：1（-/+）的电荷比从复合物中释放ODN。释放是独立的离子强度和pH值。单价和多价阳离子脂质的F-ODN的物理分离，通过凝胶电泳确认。需要流体而不是固相阴离子脂质体，而阳离子脂质的物理状态不影响释放。具有高负线性电荷密度的水溶性分子、硫酸葡聚糖或肝素也释放ODN。然而，ATP，亚精胺，精胺，tRNA，DNA，聚谷氨酸，聚赖氨酸，牛血清白蛋白，或组蛋白不释放ODN，即使在100倍的电荷过量（-/+）。基于这些结果，我们提出，复杂的，通过内吞作用内化后，诱导触发器的阴离子脂质从细胞质面对单层。阴离子脂质横向扩散到复合物中，并与阳离子脂质形成带电的中和离子对。这导致ODN从阳离子脂质中置换并释放到细胞质中。
We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome Compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.