Avoiding artifacts in liposome leakage measurements via cuvette- and liposome-surface modifications

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.