Temperature effect on thin lipid film elasticity and phase separation: insights from Langmuir monolayer and fluorescence microscopy techniques

Langmuir monolayer pressure isotherms and compressibility modulus measurements of phospholipid mixtures in several Langmuir monolayer systems at the air/water interface were investigated in this study. The ultimate aim was to carry out a comparison of the elasticity modulus for monolayers with different mixtures of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chicken egg yolk sphingomyelin (eSM), in the presence/absence of cholesterol (Chol). In particular, we were able to propose that the leading force beyond the phase separation into liquid expanded (LE-) and liquid condensed (LC-) phases emerges from the increasing barrier to incorporate DOPC molecules into a highly ordered LC-phase. In addition, our findings suggest that DOPC lipid molecules have a priority to incorporate in a disordered LE-phase, while DPPC and eSM prefer the ordered one. Also, Chol seems to split almost equally into both phases, indicating that Chol has no priority for either phase and there are no particular interactions between Chol and saturated lipid molecules.     

Tilt angle of lipid acyl chains in unilamellar vesicles determined by ellipsometric light scattering

Ellipsometric light scattering (ELS) at room temperature is applied to unilamellar vesicles (~50 nm radius) of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the gel phase and of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the liquid-crystaline phase. A high sensitivity of this technique to the local anisotropy is found. From the resulting local birefringence, a lower limit of (29 ±0.5)^○ for the average tilt angle of the lipid chains of DPPC with respect to the membrane normal is estimated. This tilt angle value is slightly lower than literature values for the tilt angle in oriented lipid multi-bilayers on solid substrates.     

Influence of membrane lipid composition on the activity of functionally reconstituted LmrA under high hydrostatic pressure

In cellular membranes, proteins and lipids are in sensitive macromolecular interaction influencing each other. To evaluate this interaction, the multi-drug transporter LmrA from Lactococcus lactis was functionally reconstituted in vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), DMPC+10 mol% cholesterol and the model raft mixture DOPC/1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (1:2:1) and in natural membrane lipids at 30 °C. The lateral structure and organization of these proteoliposomes were modulated using high hydrostatic pressure. A sharp pressure-induced fluid-to-gel phase transition is observed without an extended two-phase region. The possibility for lipid sorting, such as for DMPC/cholesterol bilayers, has an inhibitory effect on the LmrA activity. A fluid-like membrane phase over the whole pressure range with suitable hydrophobic matching, such as for DOPC, prevents the membrane protein from high-pressure inactivation up to 200 MPa. Under high-pressure conditions, highest LmrA activities, exceeding those at ambient pressure, are achieved for heterogeneous lipid matrices with a small hydrophobic mismatch and the ability of lipid sorting.     

Influence of (phospho)lipases on properties of mica supported phospholipid layers

The effect of enzymes: lipase from Candida cylindracea (L_Cc), phospholipase A₂ from hog pancreas (PLA₂) and phospholipase C from Bacillus cereus (PLC) to modulate wetting properties of solid supported phospholipid bilayers was studied via advancing and receding contact angle measurements of water, formamide and diiodomethane, and calculation of the surface free energy and its components from van Oss et al. (LWAB) and contact angle hysteresis (CAH) approaches. Simultaneously, topography of the studied layers was determined by Atomic Force Microscopy (AFM). The investigated lipid bilayers were transferred on mica plates from subphase of pure water by means of Langmuir-Blodgett and Langmuir-Schaefer techniques. The investigated phospolipid layers were: saturated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), unsaturated DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and their mixture DPPC/DOPC. The obtained results revealed that the lipid membrane degradation by the enzymes caused increase in its surface free energy due to the amphiphilic hydrolysis products, which may accumulate in the lipid bilayer. In result activity of the enzymes may increase and then break down the bilayer structure takes place. It is likely that after dissolution of the hydrolysis reaction products in the bulk phase, patches of bare mica surface are accessible, which contribute to the apparent surface free energy changes. Comparison of AFM images and the free energy changes of the layers gives better insight into changes of their properties. The observed gradual increase in the layer surface free energy allows controlling of the hydrolysis process to obtain the surfaces of defined properties.     

Role of non-lamellar-forming lipid in promotion of liposomal fusion

Membrane fusion is an important process in a wide range of cellular and sub-cellular activities. It is evident that during the intermediate stages of fusion some transitory non-bilayer configurations must appear within the lipid moiety. Using fluorescence techniques, we have studied here the process of aggregation and fusion of liposomes made of lipids, namely 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). When mixed together, the complete fusion between these two liposomes took around 44 h as both DPPC and DMPC favour lamellar configuration. When the mixture was incubated at 42°C the fusion process was completed after 23 h. But, when 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was added in the liposomal matrix the time for fusion was reduced to 21 h for mixture without incubation and 17 h when the mixture was incubated. This indicates that DPPE having a tendency to assume non-lamellar conformation, promoted destabilisation of the lamellar conformation within the liposome which facilitated the fusion between two apposing bilayers.     

表面增强拉曼散射(SERS)光谱研究含偶氮基长链脂肪酸薄膜

表面增强拉曼散射（ＳＥＲＳ）光谱研究含偶氮基长链脂肪酸薄膜吴玉清赵冰徐蔚青陶艳春吴英李伯符（吉林大学超分子结构与谱学开放实验室１３００２３）ＳｕｒｆａｃｅＥｎｈａｎｃｅｄＲａｍａｎＳｐｅｃｔｒｏｓｃｏｐｉｃＳｔｕｄｙｏｆＡｚｏｂｅｎｚｅｎｅＣｏｎｔａ...     

二次谐波光谱和布鲁斯特角显微镜研究二棕榈酰磷脂酰胆碱和维生素B2的相互作用

二棕榈酰磷脂酰胆碱（DPPC）分子在气液界面上形成的Langmuir膜是一种重要的生物膜模拟体系,其手性结构及其与外来物质的相互作用一直是相关学科研究的前沿问题。维生素B₂（VB₂）是人体中一种重要的营养物质,它在代谢障碍引起的脂质沉积性类疾病中有大量的实例应用,经常在一些特殊的临床症状中有出乎意料的治疗奇效。目前,VB₂如何参与到膜上生物事件的过程和细胞乃至生命的作用过程中的研究报道较少,特别是VB₂分子与磷脂分子靶标的立体相互作用,其可能发生的手性分子识别现象会在许多生物事件中起着关键作用。综合二次谐波-线二色光谱（SHG-LD）、Langmuir膜天平和布鲁斯特角显微镜(BAM)技术初步研究了VB₂和DPPC分子在气液界面上的相互作用,分别从气液界面上介观水平和宏观水平上互补表征脂质分子在气液界面上的分子骨架自组装的结构。压缩等温线发现纯水界面L-DPPC和D-DPPC液态扩展相/液态凝聚相(LE/LC)共存阶段的膜压几乎不变,race-DPPC的共存相膜压区域稍微缩短,VB₂水溶液界面上race-DPPC的LE/LC共存相消失。此外,弹性模量研究表明VB₂分子可以提高L-DPPC单分子层膜的弹性模量,但降低D-DPPC和race-DPPC单层膜的弹性模量。结合SHG-LD研究发现,在膜压13 mN·m-1下,L-DPPC在纯水和VB₂水溶液界面上表面手性过量值（DCE）保持不变。与纯水界面相比较,D-DPPC在VB₂水溶液上DCE值出现反转,而race-DPPC的DCE值则不随亚相改变而变化。相同膜压下,BAM观察到单一手性相互作用使得L-DPPC和D-DPPC在纯水界面上各自组装成不同枝臂弯曲方向的手性三叶草微畴(microdomain)。VB₂诱导D-DPPC微畴,使其直径增大1～2倍。同时,VB₂也诱导了race-DPPC单层膜上近似圆形状的微畴伸展,并长出了三条有曲率的枝臂。对此可以解释为VB₂降低了非单一手性相互作用的能量,使得race-DPPC出现手性相分离。与此同时,VB₂也诱导了race-DPPC单层膜微畴的手性结构发生变化。该研究有助于理解VB₂调节磷脂膜横向组织结构的分子机理,在细胞膜界面发生的过程中,脂层单层的二维特性和生物分子之间的相互作用可能决定了生物分子的亲和力。     

Visualization of the langmuir-blodgett film structure in liquid-crystal cells

The use of liquid-crystal (LC) cells for the visualization of the Langmuir-Blodgett (LB) film structure is described. Images appearing in LC cells upon LB film application are presented. The images are analyzed in relation to the LB film structure (application conditions) and to the relief formed by indium tin oxide (ITO) electrodes.     

Behavior of lysozyme adsorbed onto biological liquid crystal lipid monolayer at the air/water interface

The interaction between proteins and lipids is one of the basic problems of modern biochemistry and biophysics.The purpose of this study is to compare the penetration degree of lysozyme into 1,2-diapalmitoyl-sn-glycero-3-phosphocholine(DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethano-lamine(DPPE) by analyzing the data of surface pressure–area(π–A) isotherms and surface pressure–time(π–T) curves.Lysozyme can penetrate into both DPPC and DPPE monolayers because of the increase of surface pressure at an initial pressure of 15 m N/m.However,the changes of DPPE are larger than DPPC,indicating stronger interaction of lysozyme with DPPE than DPPC.The reason may be due to the different head groups and phase state of DPPC and DPPE monolayers at the surface pressure of 15 m N/m.Atomic force microscopy reveals that lysozyme was absorbed by DPPC and DPPE monolayers,which leads to self-aggregation and self-assembly,forming irregular multimers and conical multimeric.Through analysis,we think that the process of polymer formation is similar to the aggregation mechanism of amyloid fibers.     

Lateral diffusion in equimolar mixtures of natural sphingomyelins with dioleoylphosphatidylcholine

Cellular membranes of mammals are composed of a complex assembly of diverse phospholipids. Sphingomyelin (SM) and phosphatidylcholine (PC) are important lipids of eukaryotic cellular membranes and neuronal tissues, and presumably participate in the formation of membrane domains, known as "rafts," through intermolecular interaction and lateral microphase decomposition. In these two-dimensional membrane systems, lateral diffusion of lipids is an essential dynamic factor, which might even be indicative of lipid phase separation process. Here, we used pulsed field gradient nuclear magnetic resonance to study lateral diffusion of lipid components in macroscopically oriented bilayers composed of equimolar mixtures of natural SMs of egg yolk, bovine brain, bovine milk and dipalmitoylphosphatidylcholine (DPPC) with dioleoylphosphatidylcholine (DOPC). In addition, differential scanning calorimetry was used as a complementary technique to characterize the phase state of the lipid bilayers. In fully liquid bilayers, the lateral diffusion coefficients in both DOPC/DPPC and DOPC/SM systems exhibit mean values of the pure bilayers. For DOPC/SM bilayer system, this behavior can be explained by a model where most SM molecules form short-lived lateral domains with preferential SM-SM interactions occurring within them. However, for bilayers in the presence of their low-temperature gel phase, lateral diffusion becomes complicated and cannot simply be understood solely by a simple change in the liquid phase decomposition.     

Langmuir and Langmuir-Blodgett (LB) films of tetrapyridyl metalloporphyrins

We report on the formation of Langmuir films of 5,10,15,20-tetra(4-pyridyl) 21H,23H-porphine, hereafter named tetrapyridyl porphyrins with distinct central ions (2H⁺, Zn²⁺, Cu²⁺, Ni²⁺). The films were characterized with surface pressure and surface potential isotherms and in situ UV-vis absorbance. The measurements indicated strong aggregation of porphyrin monomers at the air-water interface, with a red shift of the Soret band in comparison with the spectrum obtained from CHCl₃ solutions. The shift was larger for the non-substituted H₂TPyP, and depended on the metal ion. Significantly, aggregation occurred right after spreading of the Langmuir film, with no further shifts in the UV-vis spectra upon compression of the film, or even after transferring them onto solid substrates in the form of Langmuir-Blodgett (LB) films. The buildup of LB films from H₂TPyP and ZnTPyP was monitored with UV-vis spectroscopy, indicating an equal amount of material deposited in each deposition step. Using FTIR in the transmission and reflection modes, we inferred that the H₂TPyP molecules exhibit no preferential orientation in the LB films, while for ZnTPyP there is preferential orientation, with the porphyrin molecules anchored to the substrate by the lateral pyridyl groups.     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

摘要：巨囊泡作为细胞的简化模型,其分相与出芽机理及动力学规律已引起许多领域科学家的关注。在富含胆固醇的典型生物膜体系如二棕榈酰磷脂酰胆碱DPPC(2-dihexadecanoyl-rac-glycero-3phosphocholine)/二油酰磷脂酰胆碱DOPC(dioleoyl-phosphatidylcholine)/胆固醇(Chol)的三组分形成的巨囊泡作为模型,从高温退火至低温会发生相分离,形成微畴。实验中借助荧光显微镜观察生物膜体系侧向分离的相结构图。实验发现,体系各组分的不同会影响磷脂膜的相结构和膜内微畴的成长,固定 DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇参入量的增加而变大。最后运用理论进一步分析了微畴的成长机理。     

Formation of thin luminescent Eu3+-LB films by in situ coordination with 2,3,5,6-tetra(2′-pyridyl)pyrazine and 1-octadecanol in pure and mixed Langmuir monolayers

The in situ complexation between 2,3,5,6-tetra(2-pyridyl)pyrazine (tppz) molecules and europium ions at the air–liquid interface by means of mixed 1-octadecanol Langmuir films is reported. These films were transferred to solid supports by means of the Langmuir–Blodgett (LB) technique. The EDS maps attested the homogeneity of the LB films as well as the presence of the europium ions. The mixed alcohol/tppz LB film contained a larger amount of europium ions as compared to the pure octadecanol LB film. This work reports the production of a thin luminescent Eu³⁺ film containing europium ions using only alcohol molecules as ligands—an unexpected result, since it is well known that there is an occurrence of non-radiative deactivation of excited europium by hydroxyl groups. Europium ion multiple binding sites were detected from lifetime decay measurements of these films in the presence of tppz molecules.     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

巨囊泡作为细胞的简化模型,其相分离与出芽动力学规律已引起许多领域科学家的关注.本实验采用DPPC/DOPC/Chol的三组分形成的巨囊泡作为模型,借助荧光显微镜观察该三组分体系侧向分离的相结构图,并对微畴的成长过程作了系统的观察研究和理论分析.实验发现:从高温的均相区域淬灭到低温的分相区域,膜表面发生侧向分离形成微畴.体系内胆固醇的掺入量的多少会影响磷脂膜的相结构和膜内微畴的成长,固定DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇掺入量的增加而变大.     

Hydrated cholesterol: Phospholipid domains probed by synchrotron radiation

X-ray scattering experiments on mixed films of cholesterol and phospholipids at air-water and Si solid-water interfaces were undertaken to glean information on pathological crystallization of cholesterol bilayers. Grazing-incidence X-ray diffraction patterns at the air-water interface of various cholesterol:dipalmitoyl-phosphatidylcholine (Ch:DPPC) monolayer mixtures compressed beyond monolayer collapse yielded the established 10×7.5 Å^2 Ch bilayer motif, for Ch:DPPC molar ratios higher than 2.5:1. Attempts to obtain a diffraction signal from various Ch:phospholipid film mixtures at the Si solid-water interface, indicative of the presence of the Ch bilayer motif, were unsuccessful. Only after removal of sufficient water from the cell was a weak diffraction signal obtained suggestive of a cholesterol film two bilayers thick. Off-specular X-ray reflectivity measurements made on a 1.75:1 mixture of Ch and bovine cardiac phosphatidylcholine (BCPC) deposited as a bilayer on a Si wafer and placed in a cell filled with water yielded positive results. The derived electron density profile showed the presence of a bilayer mixture consistent with a phase separation of cholesterol and BCPC, and possible formation of a crystalline cholesterol bilayer within the hydrated mixed bilayer, but not a proof thereof.     

Miscibility phase diagrams of giant vesicles containing sphingomyelin

Saturated sphingomyelin (SM) lipids are implicated in lipid rafts in cell plasma membranes. Here we use fluorescence microscopy to observe coexisting liquid domains in vesicles containing SM, an unsaturated phosphatidylcholine lipid (either DOPC or POPC), and cholesterol. We note similar phase behavior in a model membrane mixture without SM (DOPC/DPPC/Chol), but find no micron-scale liquid domains in membranes of POPC/PSM/Chol. We delineate the onset of solid phases below the miscibility transition temperature, and detail indirect evidence for a three-phase coexistence of one solid and two liquid phases.     

Langmuir–Blodgett films and optical second-harmonic generation of a crowned [60]fulleropyrrolidine

The Langmuir and Langmuir–Blodgett (LB) films of a novel crowned [60]fullero-pyrrolidine (CFP) were produced in different conditions. Macroscopic second-harmonic generation of the LB film was investigated by means of AM1 Hamiltonian as well as experiments. The monolayer LB film displayed a periodic fringe pattern. A linear dependence of second-harmonic intensity on the number of layers was observed. The second-order molecular susceptibility χ⁽²⁾ and hyperpolarizability β were evaluated to be 3.2×10⁻⁸ and 8.3×10⁻²⁹ esu. Received: 21 October 1999 / Revised version: 30 March 2000 / Published online: 2 August 2000     

Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.     

1-羟基-5-十二烷氧基-萘LB膜结构和取向的光谱研究

用红外透射,反射光谱及紫外可见吸收光谱方法研究了１－羟基－５－十二烷基－萘ＬＢ膜及溶液状态下的结构,分子取向。研究结果表明分子在溶液中主要是以单体形式存在;在ＬＢ膜中是以聚集体形式存在。     

Adsorption of a water soluble cationic dye into a cationic Langmuir monolayer

This communication reports the successful adsorption of a water soluble cationic fluorescent dye Rhodamine B (RhB) into a cationic Langmuir monolayer of Octadecylamine (ODA). Anionic nano clay platelets Hectorite played an important role in the process of adsorption. Surface pressure vs. area per molecule (π–A) isotherms were studied to monitor the adsorption process. In-situ fluorescence Imaging Microscopic (FIM) technique was employed to visualize the domain structures formed at the air–water interface. Atomic Force Microscopic (AFM) image of the monolayer Langmuir–Blodgett (LB) films were taken to study the morphology and ultrastructure of the film. Detailed spectroscopic investigations were carried out on the mono- and multilayer Langmuir–Blodgett (LB) films.