Abstract

The barrier properties of lipid membranes are often determined by investigating their solute permeability with the help of spectroscopic methods and the use of liposome-encapsulated self-quenching fluorescent dyes, for example, Carboxyfluorescein (CF). It was shown previously that liposome–surface interactions, and thus the choice of cuvette material, influence the result of such spectroscopic permeability/leakage experiments. In this work, we explore different methods to minimize the artifacts observed in spontaneous leakage measurements performed with cholesterol-containing liposomes. The spontaneous leakage of CF from liposomes with different composition and surface properties is monitored in cuvettes composed of quartz, polystyrene (PS), and Poly(methyl methacrylate) (PMMA). Our results show that significantly different leakage profiles are recorded for the exact same liposome batch depending on the cuvette material used. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) experiments indicate that these discrepancies likely arise from side processes occurring at the solution-cuvette interface, mainly, the attaching and spreading of liposomes. Further, we show that in some cases it is possible to minimize liposome–cuvette interactions, and reduce the experimental artifacts, by supplementing the liposomes with polyethylene glycol (PEG)-grafted lipids or gangliosides, and/or by pre-adsorbing free PEG to the cuvette walls. The collected data suggest that quartz cuvettes modified by adsorption of PEG8000 are suitable for spontaneous leakage experiments with POPC:cholesterol-based liposomes, while other cuvette materials perform poorly in the same experiments.

摘要

脂质膜的阻隔特性通常通过借助光谱方法和使用脂质体包裹的自猝灭荧光染料（例如羧基荧光素 (CF)）研究其溶质渗透性来确定。先前已经表明，脂质体 - 表面相互作用以及比色皿材料的选择会影响这种光谱渗透/泄漏实验的结果。在这项工作中，我们探索了不同的方法，以尽量减少在使用含胆固醇脂质体进行的自发泄漏测量中观察到的伪影。在由石英、聚苯乙烯 (PS) 和聚甲基丙烯酸甲酯 (PMMA) 组成的比色皿中监测 CF 从具有不同成分和表面特性的脂质体的自发泄漏。我们的结果表明，根据所使用的比色皿材料，对于完全相同的脂质体批次记录的泄漏曲线显着不同。具有耗散监测的石英晶体微天平 (QCM-D) 实验表明，这些差异可能源于溶液-比色皿界面处发生的副过程，主要是脂质体的附着和扩散。此外，我们表明，在某些情况下，通过向脂质体补充聚乙二醇 (PEG) 接枝的脂质或神经节苷脂，和/或通过预吸附游离 PEG比色皿壁。收集的数据表明，通过吸附 PEG8000 改性的石英比色皿适用于 POPC：胆固醇基脂质体的自发泄漏实验，