Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.     

Macroscopic orientation effects in broadline NMR-spectra of model membranes at high magnetic field strength: A method preventing such effects

The partial orientation of multilamellar vesicles (MLV) in high magnetic fields has been studied and a method to prevent such effects is herewith proposed. The orientation effect was measured with ²H-, ³¹P-NMR and electron microscopy on MLVs of dipalmitoyl phosphatidylcholine with 30 mol% cholesterol. We present the first freeze—etch electron microscopy data obtained from MLV samples that were frozen directly in the NMR magnet at a field strength of 9.4 Tesla. These experiments clearly show that the MLVs adopt an ellipsoidal (but not a cylindrical) shape in the magnetic field. Best fit ³¹P-NMR lineshape calculations assuming an ellipsoidal distribution of molecular director axes to the experimentally obtained spectra provide a quantitative measure of the average semiaxis ratio of the ellipsoidal MLVs and its change with temperature. The application of so-called spherical supported vesicles (SSV) is found to prevent any partial orientation effects so that undistorted NMR powder pattern of the bilayer can be measured independently of magnetic field strength and temperature.

The usefulness of SSVs is further demonstrated by a direct comparison of spectral data such as ³¹P-and ²H-NMR lineshapes and relaxation times as well as ²H-NMR dePaked spectra obtained for both model systems. These experiments show that spectral data obtained from partially oriented MLVs are not unambiguous to interpret, in particular, if an external parameter such as temperature is varied.

     

A simple method for the preparation of small unilamellar vesicles

A simple and quick method for the preparation of small unilamellar vesicles (SUV) was developed. SUV are spontaneously formed by swelling of the specially prepared phospholipid film in water/buffer. Normally, large multilamellar vesicles (MLV) are formed when a phospholipid film is dissolved in water. To prevent the formation of multilamellar structures we used the slightly charged phospholipids which exhibit infinite swelling while the formation of large structures was prevented by the deposition of the phospholipid film on the support with small surfaces. These two requirements were met by mixing a small amount of ionic detergent into phospholipid which was deposited on microcrystals. The size and size distribution of the produced vesicles depend on the size and homogeneity of the microcrystals. When 1.5 wt% of cetyltetramethylammonium bromide (CTAB) in egg yolk phosphatidylcholine was deposited on zeolite X microcrystals with crystallite sizes of approx. 0.4 μm a homogeneous population of vesicles with average diameter 21.5 nm was obtained.     

Single bilayer vesicles prepared without sonication. Physico-chemical properties.

Single shelled lecithin vesicles of uniform size (diameter = 300 A) are prepared without sonication by solubilizing unsonicated lecithin dispersions with sodium cholate and removing the detergent from the mixed lecithin - cholate micelles by gel filtration on Sephadex G-50. A homogeneous population of pure lecithin single-bilayer vesicles free of multilamellar structures is obtained. The vesicle diameter is somewhat larger than the average diameter of sonicated vesicles. The curvature of the bilayer seems to be sufficiently large to allow for similar packing densities (areas/molecule) on the outer and inner layer of the bilayer. The morphology and some physico-chemical properties of these vesicles are described and compared with those of sonicated vesicles.     

Differences in the physical properties of lipid monolayers and bilayers on a spherical solid support.

A monolayer of 1,2-dipalmitoyl-d62-glycero-3-phosphocholine (DPPC-d62) coated onto silanized silica beads (spherical supported monolayer: SSM) is studied by 2H-NMR and DSC. The results are compared with those obtained from a single bilayer on the same solid support (spherical supported vesicles: SSV) and from multilamellar vesicles (MLV). The phase transition temperature (Tm) of the SSMs is significantly higher than that of the bilayer systems and the extent of this difference depends on the lipid density in the monolayer that is determined during its preparation. 2H-NMR reveals a gel and fluid phase coexistence in the SSM transition region. A comparison of the 2H-NMR line shapes suggests the presence of highly curved structures for the fluid phase of the SSM samples. From a comparison of SSM and SSV transverse relaxation in the fluid phase we can conclude that the lateral diffusion coefficient D1 in supported monolayers is similar to that in bilayers.     

Diffusion behavior of lipid vesicles in entangled polymer solutions.

Dynamic light scattering was used to follow the tracer diffusion of phospholipid/cholesterol vesicles in aqueous polyacrylamide solutions and compared with the diffusive behavior of polystyrene (PS) latex spheres of comparable diameters. Over the range of the matrix concentration examined (Cp = 0.1-10 mg/ml), the diffusivities of the PS spheres and the large multilamellar vesicles exhibited the Stokes-Einstein (SE) relation, while the diffusivity of the unilamellar vesicles did not follow the increase of the solution's viscosity caused by the presence of the matrix molecules. The difference between the diffusion behaviors of unilamellar vesicles and hard PS spheres of similar size is possibly due to the flexibility of the lipid bilayer of the vesicles. The unilamellar vesicles are capable of changing their shape to move through the entangled polymer solution so that the hindrance to their diffusion due to the presence of the polymer chains is reduced, while the rigid PS spheres have little flexibility and they encounter greater resistance. The multilamellar vesicles are less flexible, thus their diffusion is similar to the hard PS spheres of similar diameter.     

Quantal release of serotonin

We have studied the origin of quantal variability for small synaptic vesicles (SSVs) and large dense-cored vesicles (LDCVs). As a model, we used serotonergic Retzius neurons of leech that allow for combined amperometrical and morphological analyses of quantal transmitter release. We find that the transmitter amount released by a SSV varies proportionally to the volume of the vesicle, suggesting that serotonin is stored at a constant intravesicular concentration and is completely discharged during exocytosis. Transmitter discharge from LDCVs shows a higher degree of variability than is expected from their size distribution, and bulk release from LDCVs is slower than release from SSVs. On average, differences in the transmitter amount released from SSVs and LDCVs are proportional to the size differences of the organelles, suggesting that transmitter is stored at similar concentrations in SSVs and LDCVs.     

Preparation and some properties of giant liposomes and proteoliposomes

Optimal conditions for formation of giant liposomes and proteoliposomes were investigated. A suspension of small unilamellar vesicles made of various phospholipids in a buffer of 0-3 M KCl, 0.1 mM EDTA, and 20 mM MOPS (pH 7.0) was subjected to a freeze-thaw treatment. Giant multilamellar liposomes of diameter ranging from 10 to 60 microns were found to form from phospholipid mixtures containing phosphatidylethanolamine as a major component and phosphatidylserine as a minor component. The concentration of KCl optimal for the giant vesicle formation was 30-500 mM. By applying a patch-pipette to a giant liposome, suitable conditions for obtaining a high-resistance (giga-ohm) seal were sought. It was found that use of a patch-pipette of relatively small tip diameter (less than 1 micron), the presence of divalent metal cations in the suspension medium and inflation of vesicles in a hypotonic solution facilitated giga-seal formation. In a suspension of asolectin (soybean phospholipid) vesicles which had been subjected to the freeze-thaw treatment, giant unilamellar vesicles were found. They could be held on the tip of a suction pipette and impaled with a microelectrode filled with an EGTA solution. Small unilamellar proteoliposomes were prepared by the cholate-dialysis method from asolectin and sarcoplasmic reticulum vesicles, and were subjected to a freeze-thaw cycle. When the ratio of exogenous phospholipid to protein was larger than 10, giant multilamellar vesicles were formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid mixing during freeze-thawing of liposomal membranes as monitored by fluorescence energy transfer

A new pair of fluorescence-energy-transferring probes, dansylphosphatidylethanolamine and dioctadecylindocarbocyanine, were incorporated separately into phospholipid vesicles to monitor intervesicle lipid mixing under various conditions. The transfer efficiencies of mixtures of sonicated vesicles labeled with 2 wt% donor dansylphosphatidylethanolamine (DnsPE) or with 1 wt% acceptor dioctadecylindocarbocyanine (DiI-C18) were negligible, but increased to about 25% after the vesicles had been frozen in a solid CO2/ethanol bath, thawed and diluted. The freeze-thaw-induced mixing of lipids between vesicles, signified by energy transfer, was dependent on lipid concentration and was promoted by 0.5-1.5 M KCl, 0.5 M potassium trichloroacetate and 5 mM sodium acetate (pH 4) and inhibited by 0.5 M LiCl, 0.5 M glycerol, 0.5 M sucrose, 0.15 M KCl and 0.15-1.5 M NaCl. These results support and complement previously reported measurements of the trapped volumes, turbidities and population size distributions of similarly treated liposomes. Comparison of the responses of paucilamellar vesicles with those of multilamellar vesicles suggests that lipid mixing during freeze-thawing can occur either during interaction of the outermost bilayers of vesicles or during interaction of all bilayers, possibly as a result of breakdown and reformation of bilayer structure.     

Lamellar dispersion and phase separation of chloroplast membrane lipids by negative staining electron microscopy

Aqueous dispersions of lipids isolated from spinach chloroplast membranes were studied by electron microscopy after negative staining with phosphotungstic acid. Influence of low temperature (5°C for 24 h) was also investigated. It was observed that when contacted with water, these lipids, as such, formed multilamellar structures. Upon sonication, these multilamellar structures gave rise to a clear suspension of unilamellar vesicles varying in size (diameter) between 250 and 750 Å. When samples of sonicated unilamellar vesicles were stored at 5°C for 24 h or more, they revealed a variety of lipid aggregates including liposomes, cylindrical rods (about 100 Å wide and up to 3600 Å long), and spherical micellar structures (100–200 Å in diameter)—thus indicating phase separation of lipids.     

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.     

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 μm membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 μm while maintaining an acceptable level of encapsulation of the aqueous phase.     

Cross-correlation laser scattering.

The dependence of phospholipid vesicle size on lipid composition is investigated by photon correlation spectroscopy. For each lipid composition prolonged ultracentrifugation was used to isolate a nearly uniform population of minimum-sized vesicles. The residual size variations in the samples were sufficient to cause polydispersity that made comparisons between samples difficult. Analyses of the data by the method of cumulants and by a method for approximating the particle size distributions directly are presented. The latter method made possible unambiguous comparisons that revealed small but systematic dependences of vesicle size on composition in vesicles containing mixtures of egg phosphatidylcholine and phosphatidylethanolamine, egg phosphatidylcholine and beef brain sphingomyelin, and in single lipid vesicles of egg phosphatidylcholine, dioleylphosphatidylcholine, and beef brain sphingomyelin. These size dependences are quantified within the resolution limits of the technique and their implications are discussed.     

Quantization of membrane potential generation by cytochrome c oxidase in small vesicles

Proteoliposomes incorporating cytochrome c oxidase have been prepared by the cholate dialysis method and by sonication. Sonication produces multilamellar vesicles heterogeneous in size in contrast to a more uniform preparation of unilamellar vesicles produced by the dialysis procedure. Respiratory control in both preparations ranges between 4 and 8. From an electron microscopic analysis of proteoliposome size, the average electrical capacitance/vesicle for the dialyzed and sonicated preparations is calculated as 15 X 10(-18) F and 130 X 10(-18) F, respectively. These capacitance values would lead to a quantization of membrane potential generation by the enzyme at 77 mV/turnover for the dialyzed preparation and 9 mV/turnover for sonicated vesicles. It is argued that these differences can explain the dependence of H+ translocation on the number of turnovers of cytochrome c oxidase in dialyzed preparations in contrast to the lack of dependence on number of turnovers in sonicated preparations.     

Changes of the physical properties of the liquid-ordered phase with temperature in binary mixtures of DPPC with cholesterol: A 2H-NMR, FT-IR, DSC, and neutron scattering study

The structure of the so-called liquid-ordered (lo) phase of binary mixtures of DPPC-d₆₂ with cholesterol was studied between 20-50 mol% cholesterol using ²H-NMR, FT-IR, DSC, and neutron specular reflection. Different model systems such as multilamellar vesicles, spherical supported vesicles, and oriented multilayers were used. We observed significant changes of the lo phase structure in the physiological relevant temperature region between 30-45°C. ²H-NMR in combination with lineshape simulations provides evidence for a drastic effect of cholesterol on the shape of multilamellar vesicles due to magnetic field orientation. Moreover, the data indicates a significant change of the extent of this partial orientation for DPPC-d₆₂ multilamellar vesicles containing 25 mol% cholesterol between 36-42°C. The semiaxes ratio of the (due to magnetic field orientation) ellipsoidal multilamellar vesicles changes over this temperature range by ≈25%. ²H-NMR and FT-IR measurements indicate changes of the average orientational order at the bilayer center in the same temperature range and a significant increase of the number of end-gauche conformers while the majority of the methylene groups remain in an all-trans conformation. Additionally, specular reflection of neutrons shows a concomitant reduction of the bilayer thickness by 4 Å. Based on a model of the arrangement of DPPC and cholesterol in the lo phase, a molecular mechanism is proposed in which increasing the temperature between 30 and 45°C has the effect of driving cholesterol from the bilayer center towards the head group region.     

A 2H-NMR study on the interactions of the local anesthetic tetracaine with membranes containing phosphatidylserine

The interaction of the local anesthetic tetracaine with phosphatidylserine-containing model membranes has been studied by 2H-NMR. Charged tetracaine exhibited an unusually large partition coefficient into multilamellar dispersions of phosphatidylserine. The 2H-NMR spectra consisted of a Pake doublet and a narrow line, with the former corresponding to tetracaine in the bilayer and the latter to tetracaine free in solution. A strong pH dependence of the quadrupole splittings indicated different membrane locations for charged and uncharged tetracaine. In equimolar mixtures of phosphatidylserine and phosphatidylcholine the partition coefficients and 2H-NMR spectra were much more like those observed in neat phosphatidylcholine than in neat phosphatidylserine. Dilution studies at pH 5.5 indicated that in phosphatidylserine/phosphatidylcholine mixtures tetracaine experiences a three-site exchange similar to that found earlier for tetracaine in phosphatidylcholine. Tetracaine is in fast exchange between sites weakly bound to membrane and free in solution, and in slow exchange with a strongly bound site in the membrane.     

Interleukin-2-induced small unilamellar vesicle coalescence

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (相似文献   

Acyl chain orientational order in large unilamellar vesicles: comparison with multilamellar liposomes: a 2H and 31P nuclear magnetic resonance study.

Large unilamellar vesicles (LUVs) composed of 1-[2H31]palmitoyl-2-oleoyl phosphatidylcholine (POPC-d31), with diameters of approximately 117 +/- 31 and 180 +/- 44 nm, were prepared by extrusion through polycarbonate filters with pore sizes of 0.1 and 0.2 microns, respectively. The 2H nuclear magnetic resonance (NMR) spectra obtained at 21 degrees C contain two components: a broad component (approximately 17 kHz linewidth) corresponding to the methylene groups and a narrower component originating from the methyl groups. Spectra with increasing powder pattern characteristics were obtained by reducing the rate of phospholipid reorientations by addition of glycerol (to increase the solvent viscosity) and by lowering the temperature. Full powder spectra, characteristic of liquid-crystalline bilayers, were obtained for both LUV samples at 0 degrees C in the presence of 50 wt% glycerol. Individual quadrupolar splittings were not resolved in these spectra, due to broader linewidths in the LUVs, which have significantly shorter values for spin-spin relaxation time T2 measured from the decay of the quadrupolar echo (90 microseconds) than the multilmellar vesicles (MLVs; 540 microseconds). Smoothed order parameter profiles (OPPs) were obtained for these samples by integration of the dePaked spectra. The OPPs were very similar to the OPP of POPC-d31 MLVs in 50 wt% glycerol at the same temperature, indicating that orientational order in MLVs and LUVs with a diameter of > or = 100 nm is essentially the same. The presence of 80 wt% glycerol was found to have a disordering effect on the vesicles.     

Multilamellarity, structure and hydration of extruded POPC vesicles by SANS

The small-angle neutron scattering (SANS) data of 12 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dispersions at low lipid concentration (1 mg per 100-mg heavy water) prepared by 5, 9 and 29 extrusions through filters of pores with 50, 100, 200 and 400 nm diameter are presented. They were analyzed within a theory that permits the determination of both structural and hydration parameters of the bilayers as well as the portions of multilamellar vesicles in dispersions with negligible long-range order between the vesicles. The scattering length density profile across the bilayers is approximated by assuming a central hydrocarbon core surrounded by a water-accessible coat. It is modeled by two different forms of functions. In the boat model, the scattering length density of the coat changes linearly from core to water, whereas in the strip model it is constant across the water-accessible coat. It was found that the boat model reflects the reality better than the strip model. The decrease of the multilamellar vesicle portions, either with increasing the number of extrusions at same filter size and with decreasing the filter size, was characterized quantitatively.