Giant liposomes in physiological buffer using electroformation in a flow chamber
Giant liposomes in physiological buffer using electroformation in a flow chamber
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DOI：
10.1016/j.bbamem.2005.03.012
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发表时间：
2005-06-30
影响因子：
3.4
通讯作者：
Mayer, M
中科院分区：
文献类型：
--
作者：
Estes, DJ;Mayer, M
我们描述了一种在高离子强度溶液中获得巨脂质体（直径10-100 μ m）的方法，以便在生理条件下进行膜结合试验。利用ITO电极上的电形成，我们在流动室的甘油溶液中形成表面附着的巨大脂质体，然后将高离子强度（高达2 M KCl）的溶液引入该室。离子溶液与脂质体内外的等摩尔甘油溶液交换。在脂质体的内部和外部之间的折射率的初始不匹配允许观察溶液替换。离子和小极性分子在几分钟内进出表面附着的脂质体。相反，在大分子溶液中形成的脂质体保留了大于4 kDa的分子，即使交换了脂质体外的溶液，也可以将这些分子包封数小时或数天。我们认为溶质通过脂质小管进入脂质体，脂质小管将脂质体附着在ITO电极表面的脂质膜上。这里提出的方法，使其直接进行流动通过结合分析的条件下巨大的脂质体的生理离子强度。我们对膜联蛋白V进行了膜结合试验，膜联蛋白V是一种钙依赖性蛋白，与磷脂酰丝氨酸（PS）结合。膜联蛋白V的结合依赖于PS的浓度，随着离子强度增加到生理水平而降低。(c) 2005 Elsevier B.V.版权所有
We describe a method to obtain giant liposomes (diameter 10-100 mu m) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels. (c) 2005 Elsevier B.V. All rights reserved.