Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity
Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity
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DOI：
10.1016/j.bbamem.2012.05.023
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发表时间：
2012-11-01
影响因子：
3.4
通讯作者：
Huang, Juyang
中科院分区：
文献类型：
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作者：
Baykal-Caglar, Eda;Hassan-Zadeh, Ebrahim;Huang, Juyang
含有胆固醇的巨单层囊泡（GUV）通常在脂质组成中具有广泛的分布。在这项研究中，使用标准电形成方法以及湿脂质膜的改进方法从干脂质膜制备1，2-二油酰-sn-甘油-3-磷酸胆碱（DOPC）/1，2-二硬脂酰-sn-甘油-3-磷酸胆碱（DSPC）/胆固醇和1，2-二植烷酰-sn-甘油-3-磷酸胆碱（diPhyPC）/1，2-二棕榈酰-sn-甘油-3-磷酸胆碱（DPPC）/胆固醇的GUV，其由使用快速溶剂交换（RSE）方法制备的组成均匀的脂质体制成。我们通过使用荧光显微镜测量GUV的可溶解性转变温度来量化GUV的脂质组成分布，因为转变温度的较窄分布应对应于GIN脂质组成的更均匀分布。胆固醇分子可以在干燥状态下与其他脂质分层并形成胆固醇晶体。使用光学显微镜，在一些干脂质膜中观察到微米级晶体。因此，GUV脂质组成异质性的主要原因是干膜状态下脂质的分层。通过避免干膜状态，由湿脂质膜制备的GUV在脂质组成上具有更好的均匀性，并且增溶转变温度的标准偏差比由干脂质膜制备的GUV小约2.5倍。比较两个三元体系，diPhyPC/DPPC/胆固醇GUV具有较大的胆固醇组成异质性，这与通过光散射测量的diPhyPC脂质双层中胆固醇的最大溶解度（40.2 +/-0.5 mol%）低直接相关。我们的数据表明，胆固醇与diPhyPC的相互作用远不如与其他PC。湿脂质膜方法还具有从含有天然蛋白质的细胞膜制备GUV而不经历干燥状态的潜力。(C)2012爱思唯尔有限公司版权所有。
Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GIN lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2 +/- 0.5 mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state. (C) 2012 Elsevier B.V. All rights reserved.