A membrane filtering method for the purification of giant unilamellar vesicles

The use of giant unilamellar vesicles (GUVs) for investigating the properties of biomembranes is advantageous compared to the use of small-sized vesicles such as large unilamellar vesicles (LUVs). Experimental methods using GUVs, such as the single GUV method, would benefit if there was a methodology for obtaining a large population of similar-sized GUVs composed of oil-free membranes. We here describe a new membrane filtering method for purifying GUVs prepared by the natural swelling method and demonstrate that, following purification of GUVs composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes suspended in a buffer, similar-sized GUVs with diameters of 10–30 μm are obtained. Moreover, this method enabled GUVs to be separated from water-soluble fluorescent probes and LUVs. These results suggest that the membrane filtering method can be applied to GUVs prepared by other methods to purify larger-sized GUVs from smaller GUVs, LUVs, and various water-soluble substances such as proteins and fluorescent probes. This method can also be used for concentration of dilute GUV suspensions.     

Stabilization of small unilamellar phospholipid vesicles during spray-drying

In the presence of 10% (0.3 M) sucrose in the aqueous medium, small unilamellar phospholipid vesicles are preserved during freeze-drying and spray-drying. Moreover, the bilayer integrity and permeability barrier are maintained during these processes.     

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine     

A new method for the reconstitution of membrane proteins into giant unilamellar vesicles

In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.     

Cholesterol transfer from small and large unilamellar vesicles

The rates of transfer of [14C]cholesterol from small and large unilamellar cholesterol/egg yolk phosphatidylcholine vesicles to a common vesicle acceptor were compared at 37 degrees C. The rate of exchange of cholesterol between vesicles of identical cholesterol concentrations (20 mol%) did not differ from the rate of transfer from donor vesicles containing 20 mol% cholesterol to egg yolk PC vesicles. Further, the rate of transfer of [14C]cholesterol from vesicles containing 15 mol% dicetyl phosphate (to confer a negative charge) was not different from the rate of transfer from neutral vesicles. However, the half-time for transfer of [14C]cholesterol from large unilamellar donor vesicles was about 5-times greater (10.2 h, 80 nm diameter) than from small unilamellar vesicles (2.3 h, 23 nm diameter). These data suggest that increased curvature in small unilamellar vesicles reduces cholesterol-nearest neighbor interactions to allow a more rapid transfer of cholesterol into the aqueous phase.     

Large vesicle contamination in small,unilamellar vesicles

Small, unilamellar phospholipid vesicles have been prepared using a new, high-powdered cup sonifier that avoids contact of the sample with a titanium probe. These vesicles have been characterized by gel filtration chromatography both before and after fractionation by high-speed centrifugation. Plots of the turbidity of centrifuged vesicles between 300 and 650 nm against the reciprocal fourth power of the scattering wavelength were linear with zero intercepts (extrapolated to infinite wavelength). In the presence of minute quantities of large, multilamellar vesicles, these plots remained linear but had intercepts quantitatively proportional to the amount of contaminating large vesicles. Since this measurement requires only a standard spectrophotometer and very small quantities of lipid, this method is suggested as a useful assay for determining contamination of small vesicle preparations by large vesicles. Two applications of this method as well as a practical limitation are discussed.     

Interaction of bilirubin with small unilamellar vesicles of dipalmitoylphosphatidylcholine

Interaction of bilirubin with phospholipid bilayers was studied at physiological pH above and below the gel-liquid crystalline phase transition of small unilamellar vesicles of dipalmitoylphosphatidylcholine. Chromatographic, calorimetric and 1H-NMR evidences strongly suggest that dianion form of bilirubin binds to the polar heads of the phosphatidylcholines protruding from the outer leaflet of the vesicles, whilst acid bilirubin, which is insoluble in water, is hydrophobically inserted into the lipophilic region of the bilayers. The surface-bound bilirubin is promptly removed from vesicles, whilst the acid form hydrophobically inserted into the vesicles is firmly bound to the membrane in the gel state. This pool of bilirubin could perturb the chemico-physical properties of the membrane (i.e., fluidity, phase transition, etc. ...) thus contributing to perturbation of the biological properties of living cells.     

A method for quick measurement of protein binding to unilamellar vesicles.

A general method for measuring interaction of liposome-protein (or potentially small molecules) was developed. This method utilizes biotinylated liposomes to incubate with interactants. Streptavidin-coated paramagnetic resins were then added and the liposomes (along with bound materials) can be quickly separated under a magnetic field or by low speed centrifugation. Subsequently, concentration of unbound materials (in the supernatants) can be directly determined. The described method is particularly useful for proteins or compounds that are not very soluble under certain assay conditions.     

Kinetics of melittin-induced fusion of dipalmitoylphosphatidylcholine small unilamellar vesicles

We have studied the kinetics of fusion of dipalmitoylphosphatidylcholine small unilamellar vesicles at 51 degrees C which is induced by bee venom melittin at a protein-to-lipid molar ratio of 1/60. This was done by following with a stopped-flow fluorometer the reduction in the ratio of the excimer to monomer fluorescence intensities of 1-palmitoyl-2-(10-pyrenyldecanoyl)-sn-glycero-3-phosphorylcholine that accompanies fusion. At a low melittin concentration and low ionic strength, for which case the protein is monomeric, the value of the rate constant for fusion is 0.006 s-1. This is much smaller than that of 0.06 s-1 obtained for a high melittin concentration at low ionic strength, i.e. for the protein in the tetrameric form which is not induced by a high salt concentration. The value of the rate constant for fusion for a low melittin concentration in the presence of 2 M NaCl, i.e. for the protein in the tetrameric form which is induced by a high salt concentration, is 0.12 s-1. This is twice as large as that for fusion induced by the tetramer in a low ionic strength solution. These findings show that the state of aggregation of the protein in solution and, to a lesser extent, electrostatic interactions play an important role in the kinetics of melittin-induced fusion of vesicles.     

Hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by porcine pancreatic phospholipase A2

The hydrolysis of small unilamellar vesicles made of dipalmitoylphosphatidylcoline by pancreatic phospholipase A2 has been studied under various conditions of temperature and enzyme and substrate concentration using the following three different experimental protocols. When the enzyme was added to the substrate vesicles after being separately adjusted to the temperature of the experiments hydrolysis occurred instantaneously only in the temperature range where the lipid is known to exist in its gel phase, while above the transition range no hydrolysis occurred. Within the transition range, the time course of hydrolysis was characterized by initial very slow rate of hydrolysis (latency phase) followed by an abrupt increase in the rate after a time tau, which is a complex function of temperature and enzyme to substrate ratio. When an enzyme-substrate mixture was first preincubated below Tm and then temperature jumped to a temperature above or within the transition range, the latency phase was markedly shortened. When the temperature jump was to the transition range, this effect is observed even if Ca2+ is absent in the preincubation mixture. However, instantaneous hydrolysis was observed upon temperature jumping the mixture to a temperature high above Tm only if Ca2+ was present in the preincubation medium. In temperature-scanning experiments, hydrolysis was followed while changing the temperature of the enzyme-substrate mixture continuously. Heating an enzyme-substrate mixture from room temperature resulted in an abrupt onset of hydrolysis when the transition range was approached. These results lead us to conclude that two distinctly different steps precede rapid hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by pancreatic phospholipase A2: a Ca2+-independent binding of the enzyme to the substrate vesicles, which for chemically pure bilayers occurs best in the gel phase. This step is followed by a Ca2+-dependent activation of the initially formed enzyme-substrate complex. The latter step only occurs under conditions where the bilayer possesses packing irregularities and probably involves a reorganization of the enzyme-substrate complex. At least one of these two steps appears to involve enzyme-enzyme interaction.     

Kinetics of melittin binding to phospholipid small unilamellar vesicles

We have used the decrease in the fluorescence intensity of the single tryptophan residue of bee venom melittin at long emission wavelengths that accompanies binding of the peptide to phospholipid small unilamellar vesicles to determine the rate of binding through the use of stopped-flow fluorometry in the millisecond range. We have found the rate to depend on the degree of saturation of the lipid acyl chains as well as on the physical state of the bilayer, the net electric charge of the polar headgroups, and the lipid-to-melittin molar ratio R. For zwitterionic lipids (i) the binding process is found to exhibit negative cooperativity, and (ii) the rate-limiting step appears to be penetration of the protein into the hydrophobic region of the bilayer. For negatively charged lipids the results show that binding is a very fast process that seems to be electrostatic in nature.     

A novel method for the efficient entrapment of calcium in large unilamellar phospholipid vesicles

A technique for the efficient entrapment of high concentrations of Ca2+ in large unilamellar phospholipid vesicles (LUVs), using the carboxylic acid antibiotic ionophore A23187 (calcimycin) is demonstrated. It is shown that rapid A23187-mediated entrapment of Ca2+, corresponding to essentially 100% sequestration of the extravesicular cation may be achieved for egg yolk phosphatidylcholine LUVs (100 nm) in the presence of a transmembrane proton gradient (acidic interior). Interior-exterior concentration cation gradients of over 400-fold may be readily achieved, with interior Ca2+ concentrations in excess of 250 mM. It is shown that the extent and efficiency of the A23187-mediated uptake process is affected by the intravesicular buffering capacity and the extravesicular Ca2+ concentration in a manner that is consistent with a Ca2(+)-H+ exchange process. In the absence of a pH gradient, or the presence of a reversed gradient (basic interior), only background levels of cation uptake are detected. The driving force for A23187-mediated uptake of Ca2+ is shown to depend on the intravesicular proton pool rather than on a chelation process. This protocol provides a novel method for the efficient entrapment of high concentrations of Ca2+ and other cations in phospholipid vesicles.     

Spontaneous fusion of phosphatidylcholine small unilamellar vesicles in the fluid phase

Using a high-sensitivity differential scanning microcalorimeter capable of performing cooling scans, we have examined the phase behavior of small unilamellar vesicles (SUV) as a function of time of storage above their order-disorder phase transition. Vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were examined. Cooling scans on fresh (5-7-h postsonication) samples revealed broad, relatively simple heat capacity peaks (peak temperatures: 19.9 degrees C for DMPC, 37.8 degrees C for DPPC) free of high-temperature spikes or shoulders. Subsequent heating scans displayed a sharp peak characteristic of previously described fusion products formed below the phase transition. SUV samples stored for 1 or more days above their phase transition displayed a moderately broad, high-temperature shoulder (23.8 degrees C for DMPC and 40.2 degrees C for DPPC) in the cooling profile. For DMPC, the enthalpy associated with this peak increased in a first-order fashion with time. Hydrolysis products were not detected until 12-20 days of storage. Both the rate and extent of shoulder appearance increased with temperature (k = 0.0017 h-1, fraction of total enthalpy = 0.1 at 36 degrees C; k = 0.0037 h-1, fraction = 0.2 at 42 degrees C). Freeze-fracture electron micrographs confirmed that an intermediate-sized vesicle population (diameters 400-500 A) appeared in SUV samples stored above their phase transition. Also, the trapped volume of DMPC SUV increased from 0.26 microL/mumol after 17 h of storage to 0.54 microL/mumol after storage for 16 days at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume of distribution and transcapillary passage of small unilamellar vesicles

This study investigated the biodistribution of bovine brain sphingomyelin (SM)/cholesterol (CH) (2/1; M/M) small unilamellar vesicles (SUV) in mice, addressing specifically the volume of distribution and transcapillary passage of the SUV. The complex of nitrilotriacetic acid with In-111 or Ga-67 ions was encapsulated in the SUV as the radioactive marker for various studies. The structural integrity of liposomes in vitro and in vivo was monitored by the technique of gamma ray perturbed angular correlation. Our data suggested that initially the SM/CH SUV remained within the vascular system and occupied a volume of distribution approximately 1.28 times larger than that of erythrocytes in the vascular system of mice. However, our data also indicated that with time the SM/CH SUV could get out of the vascular system of mice and were taken up by surrounding tissues over a period of 24 hours.     

NMR of molecules interacting with lipids in small unilamellar vesicles

Detailed characterization of protein, peptide or drug interactions with natural membrane is still a challenge. This review focuses on the use of nuclear magnetic resonance (NMR) for the analysis of interaction of molecules with small unilamellar vesicles (SUV). These phospholipid vesicles are often used as model membranes for fluorescence or circular dichroism experiments. The various NMR approaches for studying molecule-lipid association are reviewed. After a brief survey of the SUV characterization, the use of heteronuclear NMR (phosphorous, carbon, fluorine) is described. Applications of proton NMR through transferred nuclear Overhauser effect to perform structural determination of peptide are presented. Special care is finally given to the influence of the kinetic of the interactions for the proton NMR of bound molecules in SUV, which can constitute a good model for the study of dynamical processes at the membrane surface. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

A simple method for preparation of D-rhamnose

A rapid procedure for the preparation of D-rhamnose from bacterial lipopolysaccharide (LPS) has been developed. It involves purification of LPS from Pseudomonas syringae pv. phaseolicola by phenol extraction and hydrophobic interaction chromatography (HIC), followed by mild hydrolysis and cleavage of the O-antigen into D-fucose and D-rhamnose. The monosaccharides were separated by column chromatography, and D-rhamnose recovered after filtration over Sephadex-LH 20.     

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.     

Kinetic and thermodynamic studies of the fusion of small unilamellar phospholipid vesicles

Small phospholipid vesicles, prepared so as to minimize impurities, fuse relatively slowly resulting in the time-dependent development of a characteristic endotherm in differential scanning calorimetry and corresponding changes in the Raman spectrum. The stability of small vesicles towards fusion increases with increasing acyl chain length for the series C-14 through 18. Within the protocols of these experiments, the fusion rate remains unchanged whether the vesicles are held at 10°C below T_m or at T_m itself. We have determined enthalpies of transition for small vesicles and fusion product for C-14 through C-18. In each case ΔH for small vesicles is lower than that of the corresponding multilamellar vesicles, while the fusion product ΔH is intermediate between small and multilamellar vesicles. The apparent lack of concensus in the literature as to the nature of the fusion process is ascribed to the variety of protocols used as well as the presence or absence of fusion-inducing impurities.     

The interaction of apolipoprotein A-I with small unilamellar vesicles of L-alpha-dipalmitoylphosphatidylcholine

The interaction between apolipoprotein A-I and small unilamellar vesicles of dipalmitoylphosphatidylcholine at the lipid phase transition resulted in complete release of vesicle contents at molar ratios of lipid to protein from 4000:1 down to 50:1. This indicated the existence of two types of stable complexes: a vesicular apo-A-I complex with a maximum of two to three apo-A-Is/vesicle, and a micellar complex (disc) with a stoichiometry of about 50 phosphatidylcholines/apo-A-I (mol/mol). We characterized the complexes by density gradient centrifugation, by gel filtration, and by immunoprecipitation using an anti-apo-A-I antibody. The morphology of the discs was similar to that of previously reported discs. Apo-A-I-induced release of vesicle contents was monitored by the relief of self-quenching of vesicle-encapsulated carboxyfluorescein. Using this assay we characterized the nature of the interaction between apo-A-I and phospholipid vesicles. The formation of complexes between vesicles and apo-A-I followed a two-step process; below or above the lipid phase transition temperature (Tc), apo-A-I bound to phosphatidylcholine vesicles but caused little leakage of contents. Kinetic analysis of the interaction between apo-A-I and dipalmitoylphosphatidylcholine vesicles below Tc indicated that about 1 in 500 collisions leads to a stable apo-A-I-vesicle complex. The second step involved passage of those complexes through Tc, which resulted in a very rapid transition into discs or vesicular complexes. Vesicular complexes contain apo-A-I which was no longer capable of interacting with pure lipid. Discs, on the other hand, interacted with vesicles at their phase transition.