Negative tension induced by lipid uptake

Membrane fusion is an important process in cell biology. While the molecular mechanisms of fusion are actively studied at a very local scale, the consequences of fusion at a larger scale on the shape and stability of the membrane are still not explored. In this Letter, the evolution of the membrane tension during the fusion of positive small unilamellar vesicles with a negative giant unilamellar vesicle has been experimentally investigated and compared to an existing theoretical model. The tension has been deduced using videomicroscopy from the measurement of the fluctuation spectrum and of the time correlation function of the fluctuations. We show that fusion induces a strong decrease in the effective tension of the membrane which eventually reaches negative values. Under these conditions, we show that localized instabilities appear on the vesicle. The membrane finally collapses, forming dense lipid structures.     

Phase behavior of DODAB aqueous solution

Phase behavior of DODAB aqueous solution, prepared without sonication, was studied by adiabatic scanning calorimetry. Measurements revealed four phase transitions with the temperatures 35.2, 39.6, 44.6, and 52.4°C at heating and one transition at the temperature 40.4°C at cooling. The first three transitions at heating occur in unilamellar vesicles. The first and third transitions correspond to the subgel-gel and gelliquid phase transitions, corresponding enthalpy jumps are equal to 33 and 49 kJ/mol. The second transition appears after some aging and is similar to gel-ripple phase transition in a DPPC solution, with the enthalpy jump under the transition exceeding 7.4 kJ/mol. The transition occurs in unilamellar vesicles. The transition at the temperature 52.4°C occurs in another subsystem of the solution, which we believe to be multilamellar vesicles. The enthalpy jump at this transition is equal to 97 kJ/mol, and data analysis suggests that this is a subgel-liquid transition. The phase transition at cooling is the liquid-gel transition in unilamellar vesicles. During the measurements, a slow evolution of the solution occurs, consisting in a change of concentrations of unilamellar and multilamellar vesicles. This transformation mainly occurs at low temperatures.     

跨膜浓度梯度对渗透压囊泡循环活性的调控

本文通过分析囊泡在膨胀状态与孔隙状态的动力学行为,揭示调控膨胀拉伸能与孔隙势能的内在物理参量.建立递推微分方程的分析模型,该模型提供了膨胀-破裂循环特征量膨胀系数在各个循环中初始跨膜浓度梯度依赖性的定量信息.研究得出,通过增加初始跨膜浓度梯度,可加快膨胀系数增长速率(循环数为1时,初始跨膜浓度梯度增加3倍,膨胀系数增长速率增加2.65倍);随着循环数的增多,膨胀系数的增长由线性转变为非线性.此外,初始跨膜浓度梯度与循环次数密切相关,我们的模型计算预测增加初始跨膜浓度梯度可实现循环次数的增多.研究结果为靶组织以可编程方式释放活性生物治疗剂的发展提供了具有实际意义的理论依据.     

Direct Observation of Lipid Domains in Free-Standing Bilayers Using Two-Photon Excitation Fluorescence Microscopy

The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as the shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this review article, we address the most relevant findings reported from our laboratory regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) under two-photon excitation fluorescence microscopy is particularly addressed, especially, the possibility of obtaining information on the phase state of different lipid domains directly from the fluorescent images.     

Dissipative curvature fluctuations in bilayer vesicles: Coexistence of pure-bending and hybrid curvature-compression modes

We have studied the relaxation dynamics of shape fluctuations in unilamellar lipid vesicles by neutron spin echo (NSE). The presence of a hybrid curvature-compression mode coexisting with the usual bending one has been revealed in the experimental relaxation functions at high q . Differently to the conventional relaxation ∼ q ³ typical for bending modes, the hybrid mode was found to relax as ∼ q ² , which is compatible with a dissipation mechanism arising from intermonolayer friction. Complementary data obtained from flickering spectroscopy (FS) in giant unilamellar vesicles confirm the existence of both modes coexisting together. By combining NSE and FS data we have depicted the experimental bimodal dispersion diagram, which is found compatible with theoretical predictions for reliable values of the material parameters. From the present data two conventional dynamical methods (NSE and FS) have been shown to be suitable for measuring intermonolayer friction coefficients in bilayer vesicles.     

电制备巨囊泡的方法和机理探索

利用倒置显微镜研究电制备巨囊泡,并分析其形成和成长的动力学机理.由于磷脂头部的电特性,电场对水化的磷脂双层的静电力促使磷脂膜内双层之间分离.在动力学的作用下,双层两叶的不对称受力引发其弯曲、出芽、膨胀、封闭以及相互融合.结果表明,电场参数、干磷脂膜的均匀性、缓冲液以及温度等因素影响巨囊泡的粒径、形状和稳定性.     

蜂毒肽浓度和磷脂组分对巨囊泡泄露的影响

蜂毒肽作为一种广谱抗菌肽已经得到广泛认知,用蜂毒肽构建载药体系攻击癌细胞研究正在兴起.基于仿生物膜模型探索其破坏机理,可以避免潜在活性细胞过程的影响.在此,我们选用细胞尺寸的单层巨囊泡膜模型,可在光学显微镜下直接观察和操作,获得仿正常细胞膜和仿癌细胞膜在不同蜂毒肽浓度刺激下的响应.研究得出,低浓度蜂毒肽诱导囊泡泄露实验表明中性磷脂囊泡以孔模式为主泄露,负电性磷脂囊泡以爆裂模式为主泄露;高浓度蜂毒肽诱导囊泡泄露实验表明负电性磷脂相较于中性磷脂可延迟蜂毒肽作用效果;蜂毒肽色氨酸残基荧光光谱表明囊泡膜表面蜂毒肽吸附量以及泄露模式依赖于磷脂组分.此外,推断了蜂毒肽对不同组分磷脂膜的破坏作用模型.研究为蜂毒肽在肿瘤细胞的作用机制及其衍生物的优化设计提供参考.     

电制备巨囊泡的方法和机理探索

利用倒置显微镜研究电制备巨囊泡, 并分析其形成和成长的动力学机理. 由于磷脂头部的电特性, 电场对水化的磷脂双层的静电力促使磷脂膜内双层之间分离. 在动力学的作用下, 双层两叶的不对称受力引发其弯曲、出芽、膨胀、封闭以及相互融合. 结果表明, 电场参数、干磷脂膜的均匀性、缓冲液以及温度等因素影响巨囊泡的粒径、形状和稳定性.     

Advantages of statistical analysis of giant vesicle flickering for bending elasticity measurements

We show how to greatly improve precision when determining bending elasticity of giant unilamellar vesicles. Taking advantage of the well-known quasi-spherical model of liposome flickering, we analyze the full probability distributions of the configurational fluctuations instead of limiting the analysis to the second moment measurements only as usually done in previously published works. This leads to objective criteria to reject vesicles that do not behave according to the model. As a result, the confidence in the bending elasticity determination of individual vesicles that fit the model is improved and, consequently, the reproducibility of this measurement for a given membrane system. This approach uncovers new possibilities for bending elasticity studies like detection of minute influences by solutes in the buffer or into the membrane. In the same way, we are now able to detect the inhomogeneous behavior of giant vesicle systems such as the hazardous production of peroxide in bilayers containing fluorescent dyes.     

多聚赖氨酸诱导的负电性磷脂巨囊泡形变

利用荧光显微技术表征了多聚赖氨酸诱导的负电性磷脂巨囊泡的动力学响应行为.研究发现,多聚赖氨酸可吸附至二油酰磷脂酰胆碱和二油酰磷脂酸混合磷脂巨囊泡的表面,诱导其发生粘连、出"绳"及破裂现象.分析认为,在低盐环境中,膜形变由多聚赖氨酸吸附于二油酰磷脂酸富集区引起的膜两叶应力不对称,以及静电相互作用等因素产生.研究结果对基于聚合物-巨囊泡体系的药物输运控释、细胞形变、微控反应和基因治疗等方面的研究提供有价值的支持.     

Transition from small to big charged unilamellar vesicles

The transition from small to big unilamellar vesicles predicted by a Poisson-Boltzmann Cell Model for the thermodynamics of a dilute phase of unilamellar charged vesicles is characterized. The radius as a function of experimental parameters is calculated and the coexistence region of small and big vesicles is identified. We further investigate the physical meaning in terms of simplified models, which allow for an identification of the role of parameters like the surface charge density or the Debye-length. Connections to experiments are discussed. Received: 20 October 1997 / Received in final form: 9 February 1998 / Accepted: 9 March 1998     

Stokes instability in inhomogeneous membranes: application to lipoprotein suction of cholesterol-enriched domains

We examine the time-dependent distortion of a nearly circular viscous domain in an infinite viscous sheet when suction occurs. Suction, the driving force of the instability, can occur everywhere in the two phases separated by an interface. The model assumes a two-dimensional Stokes flow; the selection of the wavelength at short times is determined by a variational procedure. Contrary to the viscous fingering instability, undulations of the boundary may be observed for enough pumping, whatever the sign of the viscosity contrast between the two fluids involved. We apply our model to the suction by lipoproteins of cholesterol-enriched domains in giant unilamellar vesicles. Comparison of the number of undulations given by the model and by the experiments gives reasonable values of physical quantities such as the viscosities of the domains.     

Optical measurement of cell membrane tension

Using a novel noncontact technique based on optical interferometry, we quantify the nanoscale thermal fluctuations of red blood cells (RBCs) and giant unilamellar vesicles (GUVs). The measurements reveal a nonvanishing tension coefficient for RBCs, which increases as cells transition from a discocytic shape to a spherical shape. The tension coefficient measured for GUVs is, however, a factor of 4-24 smaller. By contrast, the bending moduli for cells and vesicles have similar values. This is consistent with the cytoskeleton confinement model, in which the cytoskeleton inhibits membrane fluctuations [Gov et al., Phys. Rev. Lett. 90, 228101, (2003).     

Acoustic diagnosis for porous medium with circular cylindrical pores

Acoustic transmission coefficient and phase velocity of a Lucite slab with circular cylindrical pores with a nonrigid pore frame were experimentally and theoretically investigated. For theoretical investigation a new phenomenological model, the modified Biot-Attenborough (MBA) model, was proposed. The MBA model takes into account both the first kind and the second kind of waves introduced by Biot. It also separately considers viscous and thermal effects with three new phenomenological parameters: boundary, phase velocity, and impedance parameters. The theoretical estimation with three phenomenological parameters shows reasonably good agreement with the experimental data. The physical characteristics of porous medium such as porosity and pore size can be inversely analyzed in terms of the acoustic data such as the transmission coefficient and phase velocity as the functions of porosity and frequency. This makes acoustic diagnosis possible for noninvasively investigating physical characteristics of porous media such as bones and ocean sediments.     

Shear-induced deformation of surfactant multilamellar vesicles

Surfactant multilamellar vesicles (SMLVs) play a key role in the formulation of many industrial products, such as detergents, foodstuff, and cosmetics. In this Letter, we present the first quantitative investigation of the flow behavior of single SMLVs in a shearing parallel plate apparatus. We found that SMLVs are deformed and oriented by the action of shear flow while keeping constant volume and exhibit complex dynamic modes (i.e., tumbling, breathing, and tank treading). This behavior can be explained in terms of an excess area (as compared to a sphere of the same volume) and of microstructural defects, which were observed by 3D shape reconstruction through confocal microscopy. Furthermore, the deformation and orientation of SMLVs scale with radius R in analogy with emulsion droplets and elastic capsules (instead of R(3), such as in unilamellar vesicles). A possible application of the physical insight provided by this Letter is in the rationale design of processing methods of surfactant-based systems.     

Transition from unilamellar to bilamellar vesicles induced by an amphiphilic biopolymer

We report some unusual structural transitions upon the addition of an amphiphilic biopolymer to unilamellar surfactant vesicles. The polymer is a hydrophobically modified chitosan and it embeds its hydrophobes in vesicle bilayers. We study vesicle-polymer mixtures using small-angle neutron scattering (SANS) and cryotransmission electron microscopy (cryo-TEM). When low amounts of the polymer are added to unilamellar vesicles of ca. 120 nm diameter, the vesicle size decreases by about 50%. Upon further addition of polymer, lamellar peaks are observed in the SANS spectra at high scattering vectors. We show that these spectra correspond to a co-existence of unilamellar and bilamellar vesicles. The transition to bilamellar vesicles as well as the changes in unilamellar vesicle size are further confirmed by cryo-TEM. A mechanism for the polymer-induced transitions in vesicle morphology is proposed.     

Organization in lipid membranes containing cholesterol

A fundamental attribute of raft formation in cell membranes is lateral separation of lipids into coexisting liquid phases. Using fluorescence microscopy, we observe spontaneous lateral separation in free-floating giant unilamellar vesicles. We record coexisting liquid domains over a range of composition and temperature significantly wider than previously reported. Furthermore, we establish correlations between miscibility in bilayers and in monolayers. For example, the same lipid mixtures that produce liquid domains in bilayer membranes produce two upper miscibility critical points in the phase diagrams of monolayers.     

Transport of ions and solvent in confined media

A new theoretical approach is used to model the transport properties of a cation-exchange membrane. By using the Navier-Stokes equation related to the Poisson-Boltzmann relation, it is thus possible to determine the solvent velocity in a membrane pore, and the influence of electroosmosis on the transport properties of the polymer. The variation of the transport coefficients with salt concentration in the membrane pore was modeled as for simple electrolytes: taking electrophoretic interactions and relaxation effect into account, we used MSA analytical expressions. We have investigated membrane conductivity and electrophoretic sodium mobility measurements when the membrane was equilibrated with NaCl solution. Good agreement was found between the experimental results and our theoretical model.     

Interaction of polyelectrolyte coated beads with phospholipid vesicles

Colloidal particles coated by polyelectrolyte multilayers of alternatingly positive and negative charge are shown to interact strongly with lipid vesicles. We have studied two cases: (i) the interaction between beads and small unilamellar vesicles (vesicles diameter smaller than the particles one), where we found evidence for coating of the beads with lipid bi- or multilayers in the form of an increase in bead diameter and changes in the beads surface potential; (ii) the interaction of beads with giant vesicles (vesicles larger than the particles), where we observed by fluorescence microscopy the spreading of the vesicle on the bead manipulated with an optical tweezer. Giant fluctuations of the vesicles are suppressed due to the adhesion of the vesicle to the bead and direct observation of the coating process shows that lipid coverage is not limited to the direct vesicle-bead contact area, but is rather extended to the entire bead. To cite this article: A. Fery et al., C. R. Physique 4 (2003).