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.

A closer look at calcium-induced interactions between phosphatidylserine-(PS) doped liposomes and the structural effects caused by inclusion of gangliosides or polyethylene glycol- (PEG) modified lipids.

The effects of polyethylene glycol- (PEG) modified lipids and gangliosides on the Ca2+ induced interaction between liposomes composed of palmitoyl-oleoyl phosphatidylethanolamine (POPE) and palmitoyl-oleoyl phosphatidylserine (POPS) was investigated at physiological ionic strength. Förster resonance energy transfer (FRET) studies complemented with dynamic light scattering (DLS) and cryo-transmission electron microscopy (Cryo-EM) show that naked liposomes tend to adhere, rupture, and collapse on each other's surfaces upon addition of Ca2+, eventually resulting in the formation of large multilamellar aggregates and bilayer sheets. Noteworthy, the presence of gangliosides or PEGylated lipids does not prevent the adhesion-rupture process, but leads to the formation of small, long-lived bilayer fragments/disks. PEGylated lipids seem to be more effective than gangliosides at stabilizing these structures. Attractive interactions arising from ion correlation are proposed to be a driving force for the liposome-liposome adhesion and rupture processes. The results suggest that, in contrast with the conclusions drawn from previous solely FRET-based studies, direct liposome-liposome fusion is not the dominating process triggered by Ca2+ in the systems studied.

Improved accuracy and reproducibility of spontaneous liposome leakage measurements by the use of supported lipid bilayer-modified quartz cuvettes.

Recent studies have revealed avid interactions between liposomes and several solid materials, such as quartz, polystyrene (PS) and poly(methyl methacrylate) (PMMA), commonly found in cuvettes used for spectroscopic measurements. These interactions risk leading to detrimental changes in liposome structure and integrity that, if overlooked, may compromise the measurements. In case of leakage experiments based on probing the spontaneous release of encapsulated hydrophilic markers, the liposome-cuvette interactions may result in the recording of erroneously high degrees of leakage. In the present study we investigate the possibilities of preventing unwanted liposome-cuvette interactions through the use of quartz cuvettes passivated with supported lipid bilayers (SLBs). The results show that this strategy leads to higher reproducibility and significantly improved accuracy of the leakage measurements. The usefulness of the method is validated in comparative experiments focused on how changes in temperature and lipid phase state, as well as inclusion of poly(ethylene glycol)-conjugated lipids (PEG-lipids), affect the release of liposome encapsulated carboxyfluorescein (CF).

Effect of gangliosides on structure and integrity of polyethylene glycol (PEG)-stabilized liposomes.

The in vivo efficacy and tolerance of polyethylene glycol (PEG)-decorated drug nanocarriers, such as liposomes, is compromised bytheir tendency to induce the generation of PEG-specific immunoglobulin M (IgM) antibodies. Recently, a number of independent studies have reported on an attenuated anti-PEG immune response upon incorporation of gangliosides in the membrane of PEGylated liposomes.In the present study we investigate the effect of gangliosides on the self-assembled structures found in lipid dispersions based on hydrogenated egg phosphatidylcholine (HEPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG(2000)). Results from cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) investigations show that gangliosides promote structural transitions from liposomes to bilayer disks. In case of samples comprising 5 mol% PEG-conjugated lipids (PEG-lipid), inclusion of 2.5 mol% ganglioside (porcine ganglioside extract) results in the presence of a small but significant amount of disks. With increasing ganglioside content the population of disks grows at the expense of the liposomes. Comparative investigations using isolated ganglioside components reveal that disialoganglioside GD1a is more potent than monosialoganglioside GM1 in promoting disk formation. Experiments involving liposome encapsulated carboxyfluorescein confirm that the ganglioside-induced structural transformations have a detrimental effect on the total entrapped aqueous volume of the samples. The reported coexistence of liposomes and bilayer disks may if overlooked have important implications for the therapeutic efficacy and immunogenicity of ganglioside-supplemented liposomal formulations.

Osmoprotective effect of ubiquinone in lipid vesicles modelling the E. coli plasma membrane.

Bacteria need to be able to adapt to sudden changes in their environment, including drastic changes in the surrounding osmolarity. As part of this adaptation, the cells adjust the composition of their cytoplasmic membrane. Recent studies have shown that ubiquinones, lipid soluble molecules involved in cell respiration, are overproduced by bacteria grown in hyperosmotic conditions and it is thus believed that these molecules can provide with osmoprotection. Hereby we explore the mechanisms behind these observations. Liposomes with a lipid headgroup composition mimicking that of the cytoplasmic membrane of E. coli are used as suitable models. The effect of ubiquinone-10 (Q10) on water transport across the membranes is characterized using a custom developed fluorescence-based experimental approach to simultaneously determine the membrane permeability coefficient and estimate the elastic resistance of the membrane towards deformation. It is shown that both parameters are affected by the presence of ubiquinone-10. Solanesol, a molecule similar to Q10 but lacking the quinone headgroup, also provides with osmoprotection although it only improves the resistance of the membrane against deformation. The fluorescence experiments are complemented by cryogenic transmission electron microscopy studies showing that the E. coli membrane mimics tend to flatten into spheroid oblate structures when osmotically stressed, suggesting the possibility of lipid segregation. In agreement with its proposed osmoprotective role, the flattening process is hindered by the presence of Q10.