Phase behaviour and morphology of binary mixtures of DPPC with stearonitrile, stearic acid, and octadecanol at the air-water interface

The behaviour of dipalmitoylphosphatidylcholine (DPPC), mixed with stearonitrile (SN), was investigated at the air-water interface by surface pressure-area (pi-A) measurements and by direct visualisation of monolayers by Brewster angle microscopy (BAM). The pi-A-X diagram of system DPPC/SN was compared with the corresponding diagrams of systems DPPC/stearic acid (SA) and DPPC/octadecanol (OD) at 20 degrees C. Monolayers of the three systems reach the closest packing of alkyl chains in the 0.4-0.6 range of XDPPC. Thermodynamic analysis indicates miscibility in the three binary systems with negative deviations from the ideal behaviour. Morphological features of system DPPC/SN change significantly with XDPPC and temperature in the range 10-30 degrees C. At 10 and 20 degrees C mixed monolayers form condensed states from low pi all over the composition range. At 30 degrees C, the liquid-expanded (LE)--liquid-condensed (LC) phase transition occurs at increasing pi with XDPPC. The shape and size of condensed domains change with XDPPC and pi. Contrarily to the behaviour of pure components, mixed monolayers of DPPC/SN exhibit orientational order in the 0.2-0.6 mol fraction range of DPPC. BAM observation confirmed the partial miscibility indicated by GE data in a limited range of compositions at 30 degrees C.     

Phase behaviour of stearic acid-stearonitrile mixtures. A thermodynamic study in bulk and at the air-water interface

The solid-liquid phase behaviour of stearic acid (SA) and stearonitrile (SN) in binary mixtures was investigated by differential scanning calorimetry (DSC), and the formation of SA-SN mixed monolayers at the air-water interface was followed by surface pressure-area (pi-A) measurements and by Brewster angle microscope (BAM) observation. The solid-liquid phase diagram is a eutectic type phase diagram, with the eutectic composition 0.90相似文献   

Incorporation of beta-lactoglobulin in a lipid/porphyrin monolayer at the air--water interface

A catanionic lipid/porphyrin monolayer was formed at the air-water interface by the tetra-anionic porphyrin, tetra-sodium-meso-tetra(4-sulfonatophenyl)porphine (TSPP), mixed with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) in a 1:4 molar ratio. This binary mixture (TSPP/4DODAB) was used as the incorporation matrix of beta-lactoglobulin (betaLG). Binary and ternary systems (TSPP/4DODAB/zbetaLG, where z stands for the number of protein residues per TSPP) were characterized by surface pressure versus area (pi-A) measurements and by Brewster angle microscopy (BAM) observation at the air-water interface. Pi-A measurements and BAM images show that protein is incorporated in the expanded regime of the monolayer and is gradually expelled upon compression at high surface pressures. The successive compression-expansion cycles indicate that the protein under adsorbed to the floating film is reincorporated after the expansion of the monolayer. At low subphase pH, TSPP tends to aggregate decreasing the interaction with DODAB molecules. Electrostatic and hydrophobic interactions are responsible for the presence of betaLG at the interfacial film.     

Phase behaviour of binary mixtures involving tristearin, stearyl stearate and stearic acid: thermodynamic study and BAM observation at the air-water interface and AFM analysis of LB films

The binary mixtures involving tristearin (TS), stearyl stearate (SS) and stearic acid (SA) were studied by surface pressure-area (pi-A) measurements and by Brewster angle microscopy (BAM), at the air-water interface, and the Langmuir-Blodgett (LB) monolayers, transferred onto mica substrates, were analysed by AFM. The thermodynamic analysis indicated miscibility in the whole composition range for the system SA/TS, and partial miscibility for systems SA/SS and TS/SS. This behaviour was further confirmed by BAM observation and AFM analysis of LB films. The AFM imaging of collapsed monolayers revealed domains with a multilayered structure varying with system and composition. The layers thickness determined by cross section analysis are consistent with estimated molecular lengths and conformations proposed for the molecules, assuming nearly perpendicular or tilted orientations of the hydrocarbon chains to the interface.     

Phase behaviour of stearic acid-stearonitrile mixtures: A thermodynamic study in bulk and at the air-water interface

The solid-liquid phase behaviour of stearic acid (SA) and stearonitrile (SN) in binary mixtures was investigated by differential scanning calorimetry (DSC), and the formation of SA-SN mixed monolayers at the air-water interface was followed by surface pressure-area (π-A) measurements and by Brewster angle microscope (BAM) observation. The solid-liquid phase diagram is a eutectic type phase diagram, with the eutectic composition 0.90 < X_SN < 0.95 and T^eut = 40.9 °C. The DSC results also suggest that the two components are immiscible in the solid phase but form a liquid mixture with positive deviations to the ideal behaviour. At the air-water interface, the two components form liquid condensed monolayers in the entire range of compositions, at low surface pressures, while solid mixed monolayers only form at high surface pressures for X_SN < 0.8. Thermodynamic analysis indicates that SA and SN are miscible in the liquid condensed phase, with negative deviations from the ideal behaviour. The variation of the collapse surface pressure of mixed monolayers also indicates miscibility at the air-water interface.     

Microdomains in mixed monolayers of oleanolic and stearic acids: thermodynamic study and BAM observation at the air-water interface and AFM and FTIR analysis of LB monolayers

Monolayers of oleanolic acid (OLA) mixed with stearic acid (SA) were studied at the air-water interface. The surface pressure-area (pi-A) isotherms, measured over the whole composition range, and BAM observations were used to investigate the phase behaviour and self-organization of these components in a two-dimensional structure. Pure OLA forms a very compressible monolayer, and BAM observation revealed the coexistence of large and irregular solid domains of different thickness dispersed in a gas matrix, compatible with the two most probable orientations of the OLA molecule at the interface. Mixtures of OLA/SA form condensed monolayers from low surface pressures and the thermodynamic analysis indicates that OLA molecules, in the presence of the long-chain SA, orient with the major axis almost perpendicular to the interface. Langmuir-Blodgett (LB) monolayers of pure SA and mixtures were further characterized by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR). AFM images of LB mixed monolayers evidenced microphase separation, not observable by BAM. The SA rich domains are 4-6A thicker than those rich in OLA. The FTIR spectra of mixed LB films on CaF2 substrates showed that OLA does not perturb the all-trans conformation of the SA long alkyl chains, up to a mole fraction of 0.4. The carbonyl-stretching band of OLA suggests that the carboxylic groups of neighbour OLA molecules are involved in hydrogen bonds, forming dimers, as in pure solid phase OLA. These interactions seem to prevail over the OLA-water hydrogen bonds.     

Differential scanning calorimetric studies of the thermotropic phase behavior of membranes composed of dipalmitoyllecithin and mixed-acid unsaturated lecithins

The lecithins 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) have been synthesized by reacylation of the appropriate lysolecithins with fatty acid anhydrides. These lecithins have been used to make model membranes in mixtures with dipalmitoyllecithin (DPPC), and phase diagrams of the two bilayer systems have been constructed. These diagrams show that there is essentially no gel-state miscibility in the POPC-DPPC bilayers at any composition, and that SOPC-DPPC bilayers show gel-state immiscibility at DPPC concentrations of less than 50 mol%, and partial miscibility above 50 mol% DPPC. Analysis of the POPC-DPPC phase diagram on the assumption of athermal solution in the liquid-crystalline phase shows that the two lipids mix nearly randomly above the phase transition. The liquidus curve of SOPC-DPPC bilayers showed deviations from calculated ideal behaviour, which indicated that there is a small excess tendency for the formation of pairs of like molecules in SOPC-DPPC bilayers in the liquid-crystalline phase. Thus, in the liquid-crystalline phase, SOPC and DPPC do not pack quite as well as do POPC and DPPC.     

Phospholipase D activity is regulated by product segregation and the structure formation of phosphatidic acid within model membranes

Phospholipase D from Streptomyces chromofuscus (scPLD) hydrolyzes phosphatidylcholines (PC) to produce choline and phosphatidic acid (PA), a lipid messenger molecule within biological membranes. To scrutinize the influence of membrane structure on scPLD activity, three different substrate-containing monolayers are used as model systems: pure dipalmitoylphosphatidylcholine (DPPC) as well as equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG). The activity of scPLD toward these monolayers is tested by infrared reflection-absorption spectroscopy and exhibits different dependencies on surface pressure. For pure DPPC, the catalytic turnover drastically drops above 20 mN/m. On addition of C(16)OH, this strong decrease starts at 5 mN/m. For the DPPC/DPG system, the reaction yield linearly decreases between 5 and 25 mN/m. The difference in scPLD activity is correlated to the phase state of the monolayers as examined by x-ray diffraction, Brewster angle microscopy, and atomic force microscopy. Because the additives C(16)OH and DPG mediate the miscibility of PC and PA, only a basal activity of scPLD is observed toward the mixed systems at higher surface pressures. At pure DPPC monolayers, scPLD is activated after the segregation of initially formed PA. Furthermore, scPLD is inhibited when the lipids in the PA-rich domains adopt an upright orientation. This phenomenon offers a self-regulating mechanism for the concentration of the second messenger PA within biological membranes.     

Bilayer structures in dioctadecyldimethylammonium bromide/oleic acid dispersions

This paper reports on the properties of bilayers composed of dioctadecyldimethylammonium bromide (DODAB) and oleic acid (OA) at various molar ratios. The mole fraction of OA, X_OA, was varied in the range of 0–1 and the total lipid content was constant and equal to 10 mM. The DODAB/OA dispersions were extruded at a temperature higher than that of the gel–liquid transition of DODAB. The morphology of bilayer structures formed in the dispersions was inspected using a cryogenic transmission electron microscopy (cryo-TEM) and a differential interference contrast microscopy (DIC). The observations revealed that the incorporation of OA into DODAB bilayer results in a decrease of the membrane curvature. Anisotropy measurements using 1,6-diphenylhexatriene (DPH) as a rotator probe demonstrated that the DODAB/OA membrane microviscosity decreased considerably for X_OA > 0.4. The thermal behavior of DODAB/OA membranes has been studied by differential scanning calorimetry (DSC). In the case of the systems in which X_OA < 0.8, the DODAB/OA membranes are in the gel phase at room temperature. Additionally, Langmuir monolayer experiments of the DODAB/OA mixtures showed that due to the electrostatic interactions between the oppositely charged head groups of DODAB and OA they get close to each other, which results in a decrease of the mean area per molecule. The results were next discussed based on the packing parameter concept. The reduction of the mean area per head group (a) in the DODAB/OA systems leads to subsequent increase in the so-called packing parameter (S), which governs the morphology of surfactant aggregates.     

Lateral phase separation in interfacial films of pulmonary surfactant.

To determine if lateral phase separation occurs in films of pulmonary surfactant, we used epifluorescence microscopy and Brewster angle microscopy (BAM) to study spread films of calf lung surfactant extract (CLSE). Both microscopic methods demonstrated that compression produced domains of liquid-condensed lipids surrounded by a liquid-expanded film. The temperature dependence of the pressure at which domains first emerged for CLSE paralleled the behavior of its most prevalent component, dipalmitoyl phosphatidylcholine (DPPC), although the domains appeared at pressures 8-10 mN/m higher than for DPPC over the range of 20-37 degrees C. The total area occupied by the domains at room temperature increased to a maximum value at 35 mN/m during compression. The area of domains reached 25 +/- 5% of the interface, which corresponds to the predicted area of DPPC in the monolayer. At pressures above 35 mN/m, however, both epifluorescence and BAM showed that the area of the domains decreased dramatically. These studies therefore demonstrate a pressure-dependent gap in the miscibility of surfactant constituents. The monolayers separate into two phases during compression but remain largely miscible at higher and lower surface pressures.     

Modifying dipalmitoylphosphatidylcholine monolayers by n-hexadecanol and dipalmitoylglycerol

The monolayer structure of pure dipalmitoylphosphatidylcholine (DPPC) and equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG) are studied by the film balance technique and grazing incidence X-ray diffraction measurements. At 20 degrees C, the binary systems exhibit complete miscibility. In contrast to pure DPPC monolayers, a condensing effect is observed in the presence of both non-phospholipid additives; but the phase transition behavior differs. The tilt angle of the hydrocarbon chains in the DPPC/C(16)OH mixture is significantly smaller than in pure DPPC monolayers. The tilt of the chains is even further reduced in the mixed monolayer of DPPC/DPG. A comparison of the three systems reveals distinct structural features such as phase state, chain tilt, and molecular area over a wide range of surface pressures. Therefore, these monolayers provide a highly suitable model to investigate the influence of structural parameters on biological processes occurring at the membrane surface, e.g. enzymatic reactions and adsorption events.     

The effect of methylation of phosphatidylethanolamine on the behaviour of lipid monolayers at the air-water interface

Monolayers of DPPE and its N-methylated derivatives including DPPC have been investigated at 23 and 37 degrees C using a modified Langmuir-Wilhelmy surface balance. The monolayers have been subjected to dynamic compression and expansion, and some characteristics of the surfaces have been determined. The minimum surface tension attained by surfaces containing the lipids (maximum surface pressures sustained by the films) depended on the extent of methylation of the head group. Monolayers of DPPE or N-MeDPPE collapsed at surface tensions of 12-16 mN.m-1, whereas those containing N,N-diMeDPPE and DPPC could be compressed to near zero surface tension. The areas per molecule occupied by these lipids under high compression varied slightly and not systematically with head-group methylation. Monolayers containing mixtures of DPPC and DPPE were also studied under the same conditions. The monolayers showed some deviation from the behaviour expected if they were to have characteristics of ideally mixed systems. The minimum surface tensions attained suggested that monolayers containing 50 mol% or more DPPC might be further enriched during compression by some selective exclusion of the DPPE. At high surface pressures, some positive deviations in nominal areas per molecule from that expected for ideal mixing were observed in the monolayers made with 50 mol% or more DPPC. These deviations might be caused by packing disruptions associated with the explosion of lipid from the films.     

Characterization of DODAB/DPPC vesicles

Dioctadecyldimethylammonium bromide (DODAB)/dipalmitoylphosphatidylcholine (DPPC) large and cationic vesicles obtained by vortexing a lipid film in aqueous solution and above the mean phase transition temperature (T(m)) are characterized by means of determination of phase behaviour, size distribution, zeta-potential analysis and colloid stability. The effect of increasing % DODAB over the 0-100% range was a nonmonotonic phase behaviour. At 50% DODAB, the mean phase transition temperature and the colloid stability were at maximum. There is an intimate relationship between stability of the bilayer structure and colloid stability. In 1, 50 and 150mM NaCl, the colloid stability for pure DPPC or pure DODAB vesicles was very low as observed by sedimentation or flocculation, respectively. In contrast, at 50% DODAB, remarkable colloid stability was achieved in 1, 50 or 150mM NaCl for the DODAB/DPPC composite vesicles. Vesicle size decreased but the zeta-potential remained constant with % DODAB, due to a decrease of counterion binding with vesicle size. This might be important for several biotechnological applications currently being attempted with cationic bilayer systems.     

Phase separation in monolayers of pulmonary surfactant phospholipids at the air-water interface: composition and structure.

The phase behavior of monolayers containing the complete set of purified phospholipids (PPL) obtained from calf surfactant was investigated as a model for understanding the phase transitions that precede compression of pulmonary surfactant to high surface pressure. During compression, both fluorescence microscopy and Brewster angle microscopy (BAM) distinguished domains that separated from the surrounding film. Quantitative analysis of BAM grayscales indicated optical thicknesses for the PPL domains that were similar to the liquid condensed phase for dipalmitoyl phosphatidylcholine (DPPC), the most abundant component of pulmonary surfactant, and higher and less variable with surface pressure than for the surrounding film. BAM also showed the optical anisotropy that indicates long-range orientational order of tilted lipid chains for the domains, but not for the surrounding film. Fluorescence microscopy shows that addition of DPPC to the PPL increased the area of the domains. At fixed surface pressures from 20-40 mN/m, the total area of each phase grew in proportion with the mol fraction of DPPC. This constant variation allowed analysis of the DPPC mol fraction in each phase, construction of a simple phase diagram, and calculation of the molecular area for each phase. Our results indicate that the phase surrounding the domains is more expanded and compressible, and contains reduced amounts of DPPC in addition to the other phospholipids. The domains contain a mol fraction for DPPC of at least 96%.     

Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films

Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 wt%, and 10 wt% of both) under conditions of cyclic compression and expansion. Matrix-assisted laser desorption/ionization (MALDI) spectroscopy of R-SP-B and F-SP-C indicated that the proteins were intact and labeled with the appropriate fluorescent probe. The monolayers were compressed and expanded for four cycles at an initial rate of 0.64 A2 x mol(-1) x s(-1) (333 mm2 x s x [-1]) up to a surface pressure pi approximately 65 mN/m, and pi-area per residue (pi-A) isotherms at 22 +/- 1 degrees C were obtained. The monolayers were microscopically observed for the fluorescence emission of the individual proteins present in the film lipid matrix, and their visual features were video recorded for image analysis. The pi-A isotherms of the DPPC/protein monolayers showed characteristic "squeeze out" effects at pi approximately 43 mN/m for R-SP-B and 55 mN/m for F-SP-C, as had previously been observed for monolayers of the native proteins in DPPC. Both proteins associated with the expanded (fluid) phase of DPPC monolayers remained in or associated with the monolayers at high pi (approximately 65 mN/m) and redispersed in the monolayer upon its reexpansion. At comparable pi and area/molecule of the lipid, the proteins reduced the amounts of condensed (gel-like) phase of DPPC monolayers, with F-SP-C having a greater effect on a weight basis than did R-SP-B. In any one of the lipid/protein monolayers the amounts of the DPPC in condensed phase were the same at equivalent pi during compression and expansion and from cycle to cycle. This indicated that only minor loss of components from these systems occurred between compression-expansion cycles. This study indicates that hydrophobic PS proteins associate with the fluid phase of DPPC in films, some proteins remain at high surface pressures in the films, and such lipid-protein films can still attain high pi during compression.     

Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.     

Spatial variation in the molecular tilt orientational order within the solid domains of phase-separated, mixed dialkylphosphatidylcholine monolayers

The miscibility of the solid-phase-forming distearoylphosphatidylcholine (DSPC) and the fluid-phase-forming dilauroylphosphatidylcholine (DLPC) at the air/water interface was investigated by the Langmuir film balance. Surface pressure-area isotherms suggest that mixtures containing 25.0-62.5-mol% DLPC (range of composition investigated) are phase-separated. The lateral structure of the DSPC/DLPC monolayers was imaged by Brewster angle microscopy (BAM) as a function of the surface pressure. Quasi-circular condensed domains appeared at pressures between 0 and 0.5mN m(-1), and these structures were already fully developed at approximately 1mN m(-1). Further compression of the monolayers above 1mN m(-1) merely brought the domains closer together. The mixed monolayers consisted of solid domains of DSPC, approximately 3-20 micro in size, in a fluid matrix of DLPC. BAM and the phase contrast mode of intermittent-contact atomic force microscopy (AFM) revealed that the quasi-circular DSPC domains are divided into segments of different reflectivities (BAM) or phase shift (AFM) that arise from abrupt changes in the long-range orientational order of the tilted hydrocarbon chains. The DSPC domains in DSPC/DLPC internally exhibited star and cardioid textures that were heretofore only reported for single-component lipid monolayers in the phase coexistence region.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: I. Monolayers of pulmonary surfactant protein SP-B and phospholipids.

The effects of pulmonary surfactant protein SP-B on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and a mixture of DPPC:DPPG (7:3, mol:mol) were studied using spread films at the air-water interface. The addition of SP-B to the phospholipid monolayers gave positive deviations from additivity of the mean areas in the films. At low protein concentrations (less than 45% amino acid residues which corresponds to 0.5 mol% or 10 weight% SP-B) monolayers of SP-B/DPPC, SP-B/DPPG and SP-B/(DPPC:DPPG) collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At higher concentrations of SP-B in the protein-lipid monolayers, kink points appeared in the isotherms at about 40-45 mN.m-1, implying possible exclusion of material from the films, hence, changes in the original monolayer compositions. Calculated analyses of the monolayer compositions as a function of surface pressure indicated that nearly pure SP-B, associated with small amounts of phospholipid (2-3 lipid molecules per SP-B dimer), was lost from SP-B/DPPC, SP-B/DPPG, and SP-B/(DPPC:DPPG) films at surface pressures higher than 40-45 mN.m-1. The results are consistent with a low effectiveness of SP-B in removing saturated phospholipids, DPPC or DPPG, from the spread SP-B/phospholipid films.     

The interaction of meso-tetraphenylporphyrin with phospholipid monolayers

In this article, we investigate the interaction of meso-tetraphenylporphyrin (TPP) with phospholipid monolayers. Pure TPP molecules form films at the air-water interface with large extension of aggregation, which is confirmed by UV-vis spectra of transferred monolayers. For mixed films of TPP with dipalmitoyl phosphatidyl choline (DPPC) or dipalmitoyl phosphatidyl glycerol (DPPG), on the other hand, aggregation is only significant at high surface pressures or high concentrations of TPP (above 0.1 molar ratio). This was observed via Brewster angle microscopy (BAM) for the Langmuir films and UV-vis spectroscopy for transferred layers onto solid substrates. TPP indeed causes the DPPC and DPPG monolayers to expand, especially at the liquid-expanded to liquid-condensed phase transition for DPPC. The effects from TPP cannot be explained using purely geometrical considerations, as the area per TPP molecule obtained from the isotherms is at least twice the expected value from the literature. Therefore, interaction between TPP and DPPC or DPPG should be cooperative, so that more phospholipid molecules are affected than just the first neighbors to a TPP molecule.     

Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity

Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GUV lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2±0.5mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state.