Despite the ubiquity of transbilayer asymmetry in natural cell membranes, the vast majority of existing research has utilized chemically well-defined symmetric liposomes, where the inner and outer ...bilayer leaflets have the same composition. Here, we review various aspects of asymmetry in nature and in model systems in anticipation for the next phase of model membrane studies.
Liposomes are widely investigated for their use as drug delivery systems, where they have to meet strict stability criteria. Hence, it is of common interest to establish appropriate storage ...conditions to improve the shelf life of liposomes. In general, long-term stability can be achieved by freezing as well as freeze-drying, and different carbohydrates or polyalcohols, such as mannitol or glycerol are considered as cryoprotective agents to inhibit liposomal fusion or degradation during freezing procedures.
Here, we determined the impact of different cryoprotectants on physicochemical parameters of sterically stabilized PEGylated liposomes, which become increasingly important for pharmaceutical applications. We investigated particle stability in terms of size, lamellarity and thickness of the lipid bilayer using photon correlation spectroscopy and small angle X-ray scattering. Besides, we evaluated the impact of cryoprotectants on the thermal lipid phase behavior of either frozen/thawn or lyophilised/rehydrated PEGylated liposome formulations by differential scanning calorimetry.
Optimal results for the preservation of the average size of the extruded unilamellar liposomes during freezing were achieved using a mixture of glycerol and carbohydrate concentrations of about 1% (w/v), irrespective of the carbohydrate used. We found no significant changes in the bilayer organisation, and the transition behavior of lipids was almost uneffected by freezing. In case of freeze-drying, similar carbohydrate concentrations as used for freezing were sufficient to maintain the size of PEGylated liposomes after reconstitution of the dried lyophilised cakes, but our small angle X-ray scattering data provide strong evidence that the lyophilisation/rehydration process affects lipid membrane reorganisation on a molecular level such that a swelling of the bilayer might occur. These internal structural changes, which are not detected by standard particle size analysis, might well influence drug release profiles and the pharmacological performance of a liposomal drug delivery system.
Freely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer ...leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet's lipid composition: the overall lipid composition is determined by gas chromatography-mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr
. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.
Accurate details on the sampled atomistic resolution structures of lipid bilayers can be experimentally obtained by measuring C–H bond order parameters, spin relaxation rates and scattering form ...factors. These parameters can be also directly calculated from the classical atomistic resolution molecular dynamics simulations (MD) and compared to the experimentally achieved results. This comparison measures the simulation model quality with respect to ‘reality’. If agreement is sufficient, the simulation model gives an atomistic structural interpretation of the acquired experimental data. Significant advance of MD models is made by jointly interpreting different experiments using the same structural model. Here we focus on phosphatidylcholine lipid bilayers, which out of all model membranes have been studied mostly by experiments and simulations, leading to the largest available dataset. From the applied comparisons we conclude that the acyl chain region structure and rotational dynamics are generally well described in simulation models. Also changes with temperature, dehydration and cholesterol concentration are qualitatively correctly reproduced. However, the quality of the underlying atomistic resolution structural changes is uncertain. Even worse, when focusing on the lipid bilayer properties at the interfacial region, e.g. glycerol backbone and choline structures, and cation binding, many simulation models produce an inaccurate description of experimental data. Thus extreme care must be applied when simulations are applied to understand phenomena where the interfacial region plays a significant role. This work is done by the NMRlipids Open Collaboration project running at https://nmrlipids.blogspot.fi and https://github.com/NMRLipids. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.
•Atomistic simulations well describe the lipid bilayer acyl chain structure and dynamics.•Atomistic resolution quality of lipid bilayer models decreases toward water phase.•Atomistic MD has potential to interpret NMR and X-ray experiments simultaneously.•Force field development is needed to fully unleash the power of atomistic MD.
Membrane proteins usually need to be extracted from their native environment and separated from other membrane components for in-depth in vitro characterization. The use of styrene/maleic acid (SMA) ...copolymers to solubilize membrane proteins and their surrounding lipids into bilayer nanodiscs is an attractive approach toward this goal. We have recently shown that a diisobutylene/maleic acid (DIBMA) copolymer similarly solubilizes model and cellular membranes but, unlike SMA(3:1), has a mild impact on lipid acyl-chain order and thermotropic phase behavior. Here, we used fluorescence spectroscopy, small-angle X-ray scattering, size-exclusion chromatography, dynamic light scattering, and
P nuclear magnetic resonance spectroscopy to examine the self-association of DIBMA and its membrane-solubilization properties against lipids differing in acyl-chain length and saturation. Although DIBMA is less hydrophobic than commonly used SMA(3:1) and SMA(2:1) copolymers, it efficiently formed lipid-bilayer nanodiscs that decreased in size with increasing polymer/lipid ratio while maintaining the overall thickness of the membrane. DIBMA fractions of different molar masses were similarly efficient in solubilizing a saturated lipid. Coulomb screening at elevated ionic strength or reduced charge density on the polymer at low pH enhanced the solubilization efficiency of DIBMA. The free-energy penalty for transferring phospholipids from vesicular bilayers into nanodiscs became more unfavorable with increasing acyl-chain length and unsaturation. Altogether, these findings provide a rational framework for using DIBMA in membrane-protein research by shedding light on the effects of polymer and lipid properties as well as experimental conditions on membrane solubilization.
Liposomes, Lipid Bilayers and Model Membranes Georg Pabst, Norbert Kucerka, Mu-Ping Nieh, John Katsaras / Georg Pabst, Norbert Kucerka, Mu-Ping Nieh, John Katsaras
2014, 2014-03-04
eBook
As a result of their unique physical properties, biological membrane mimetics, such as liposomes, are used in a broad range of scientific and technological applications. Liposomes, Lipid Bilayers and ...Model Membranes: From Basic Research to Application describes state-of-the-art research and future directions in the field of membranes, which has evo
Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two ...leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.
We studied the influence of sodium and calcium chloride on the global and local membrane properties of fluid palmitoyl-oleoyl phosphatidylcholine bilayers, applying synchrotron small-angle x-ray ...diffraction, spin-labeling electron paramagnetic resonance spectroscopy, and differential scanning calorimetry, as well as simultaneous density and acoustic measurements. The salt concentration was varied over a wide range from 0 to 5
M. We found that NaCl leads to a continuous swelling of the bilayers, whereas the behavior of the bilayer separation
d
W in the presence of CaCl
2 is more complex, showing an initial large
d
W value, which decreased upon further addition of salt and finally increased again in the high concentration regime. This can be understood by a change of balance between electrostatic and van der Waals interactions. We were further able to show that both salts lead to a significant increase of order within the lipid bilayer, leading to a decrease of bilayer elasticity and shift of main phase transition temperature. This effect is more pronounced for Ca
2+, and occurs mainly in the high salt-concentration regime. Thus, we were able to reconcile previous controversies between molecular dynamics simulations and x-ray diffraction experiments regarding the effect of salts on neutral lipid bilayers.
We have studied the concentration and temperature dependent influence of cholesterol, stigmasterol, and sitosterol on the global structure and the bending fluctuations of fluid dimyristoyl ...phosphatidylcholine and palmitoyl oleoyl phosphatidylcholine bilayers applying small-angle x-ray scattering, as well as dilatometry and ultrasound velocimetry. Independent of the lipid matrix, cholesterol was found to be most efficient in modulating bilayer thickness and elasticity, followed by sitosterol and stigmasterol. This can be attributed to the additional ethyl groups and double bond at the C17 alkyl side-chain of the two plant sterols. Hence, it seems that some flexibility of the sterol hydrocarbon chain is needed to accommodate within the lipid bilayer. In addition, we did not observe two populations of membranes within the putative liquid-ordered/liquid-disordered phase coexistence regime of binary sterol/lipid mixtures. Instead, the diffraction patterns could be interpreted in terms of a uniform phase. This lends further support to the idea of compositional fluctuations of unstable sterol rich domains recently brought up by fluorescence microscopy experiments, which contrasts the formation of stable domains within the miscibility gap of binary lipid/sterol mixtures.
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