糖和盐类物质对生物膜超分子结构稳定性影响的研究

用原子力显微镜(AFM)和小角X射线(SAXS)技术, 研究了NaCl、KCl、胆固醇、葡萄糖和蔗糖等与膜脂的相互作用. 研究发现它们能引起脂质膜超分子体系液晶态结构的变化. 葡萄糖和蔗糖对脂双层膜结构有稳定作用. 在NaCl溶液中制成的脂质膜, 随着NaCl浓度的增加, 它们的双层膜更稳定. 在KCl溶液中结果恰好相反. AFM研究发现液晶态脂双层膜结构与双亲性分子的结构、浓度以及介质的组分和pH等因素有关. 在1,2-反十八碳-3-磷脂酰乙醇胺(DEPE)液晶态中, 钠盐诱导形成Q²²⁹(Im3m)立方相. 油酸的含量对DEPE-PVP(聚乙烯吡咯烷酮)超分子结构也有一定的影响, 当油酸含量达到某一临界值时, 则发生从Im3m(Q²²⁹)到Pn3m(Q²²⁴)的转变. 胆固醇能促使形成Pn3m(Q²²⁴)和六角相H_II共存相. 研究结果表明, 生物膜超分子聚集体的氢键、分子van der Waals力、离子的静电力等这些弱相互作用的协同性、方向性和选择性, 可能决定着生物膜的结构和功能.     

溶菌酶晶体生长前期溶液中聚集体研究

用动态光散射法研究了不同浓度NaCl对溶菌酶晶体生长前期溶液中聚集体状态的影响,并将这些溶液中的聚集体吸附到硅片表面,用原子力显微镜进行了观察.结果表明,在NaCl浓度为0～0.5 mol·L^-1时,随着NaCl浓度的升高,溶液中大的聚集体逐渐消失,直至基本上只存在几纳米大小的聚集体.测量了相应条件下溶液的Zeta电势值以说明NaCl与溶菌酶之间的相互作用的变化情况.本文从溶液中无序聚集体的角度出发提出了判断晶体能否生长的一个可能的标准,并对动态光散射与原子力显微镜的结果进行了对比和分析.     

DOPC,DOPE和神经酰胺对鞘磷脂/胆固醇双层膜结构的影响

用LB技术和原子力显微镜(AFM)研究了1,2-二油酸甘油-3-磷脂酰胆碱(DOPC)、1,2-二油酸甘油-3-磷脂酰乙醇胺(DOPE)和神经酰胺(Ceramide)对鞘磷脂(SM)/胆固醇(Chol)结构的影响. 实验结果表明, 在表面压力较低时, 每种混合脂双层膜都呈现均匀分布的脂双层结构. 随着表面压力的增加, 形态发生了明显的变化: (1) SM/Chol二元组分双层膜形成均一的液态有序相微区结构, 衬底覆盖率达到80%; (2) DOPC的加入促使SM/Chol双层膜出现相分离现象, SM/Chol形成的液态有序相 “岛状” 微区结构漂浮在液态无序相的DOPC上部, 约占总面积的30%; (3) DOPE与SM/Chol形成的双层膜明显不同于DOPC/SM/Chol, 呈现出液态无序相、液态有序相及凝胶相3相共存的结构; (4) Ceramide诱导了SM/Chol双层膜结构发生重排, 两层脂分子间发生翻转形成囊泡结构, 部分神经酰胺从液态有序相中分离形成小颗粒结构. 在较高膜压下, 各系统都呈现出具有特定形态的双层膜结构. 分子官能团的成键能力决定了双层膜形态结构.     

电化学方法研究多粘菌素B与磷脂酰胆碱的相互作用

采用电化学方法研究了一种多肽类抗生素多粘菌素B(PMB)与模拟生物膜硫醇-磷脂酰胆碱杂化双层膜之间的相互作用。PMB可与磷脂酰胆碱发生强相互作用,破坏双层膜结构,从而使膜的通透性升高。PMB的浓度、酸度、与膜的作用时间及膜中胆固醇的存在均影响二者的作用程度。另外,膜的自修复实验表明,被PMB破坏的双层膜电极在一定程度上可以在KCl溶液中重新自组装,且自修复的程度和修复时间及与之相互作用的PMB的浓度有关。     

脱氧胆酸钠与支撑磷脂双层膜作用的电化学研究

以支撑磷脂双层膜(supported bilayer lipid membrane, s-BLM)作为生物膜模型, 采用循环伏安法和交流阻抗技术研究了脱氧胆酸钠(sodium deoxycholate, NaDC)与s-BLM的相互作用. 结果表明, NaDC能降低磷脂分子的有序性, 诱发s-BLM上形成孔洞或缺陷, 并且它们之间的这种相互作用对作用时间、NaDC溶液的浓度和pH值以及胆固醇的存在与否具有依赖性, 并且作用后的s-BLM在0.1 mol/L的KCl溶液中能够自我修复, 这表明NaDC与s-BLM的相互作用是可逆的.     

基于原子力显微镜研究阿霉素与不同种类细胞间的相互作用

原子力显微镜(AFM)因其制样简单以及高分辨成像特点,因此在药物与细胞相互作用的研究中具有越来越广泛的应用.Girasole等用AFM观察了在药物、低离子浓度等条件下的红细胞与正常红细胞之间表面微观结构的差别.     

电化学法研究多粘菌素B与磷脂酰丝氨酸的相互作用及与其他磷脂的比较

采用头基修饰的磷脂在金电极表面构建了稳定的磷脂双层膜,并使用该膜模拟生物膜对多肽类抗生素多粘菌素B(PMB)和磷脂酰丝氨酸的相互作用进行了研究.PMB可与磷脂酰丝氨酸发生相互作用,破坏双层膜结构,从而使膜的通透性升高.PMB的浓度、作用时间以及膜中胆固醇的存在均影响二者的作用程度.被PMB破坏的双层膜电极在一定程度上可在KC1溶液中重新自组装,且自修复的程度与修复时间和PMB的浓度有关.此外,比较了PMB和多种磷脂之间的作用程度,磷脂的头基、烃链的长度以及不饱和度均会影响二者间的相互作用.     

心磷脂蓝莓类结构的形成和演化

用共聚焦显微镜和光学显微镜研究了NaCl溶液中心磷脂蓝莓类结构的形成和演化过程. 研究发现, 在NaCl溶液(浓度0.005~0.25 mol/L)中, 心磷脂海绵相的表面首先生成半球形的层状相微结构元胞, 并逐渐呈蜂窝状六角密堆积排列. 溶液中NaCl浓度的轻微增加驱使层状相与海绵相的界面由表面向海绵相内部扩散, 溶液中微弱流场的存在也会影响微结构元胞的形貌, 最终形成蜂窝样网状结构、 蓝莓样包状结构和蓝莓样球状结构, 统称蓝莓类结构. 由于心磷脂在线粒体内膜的拓扑结构转变中具有重要作用, 因此相转变及微结构形态演化的研究有助于进一步理解其在线粒体功能发挥中的作用机理.     

DOPC/DPPC单层膜中分子间的相互作用及形态特征

利用Langmuir-Blodgett(LB)技术制备了不同表面压力下的1,2-二油酸-甘油-3-磷脂酰胆碱(DOPC)/1,2-二棕榈酸甘油-3-磷脂酰胆碱(DPPC)(摩尔比为1:1)和DOPC/DPPC/Chol(摩尔比为2:2:1)单层膜, 对单层膜内分子间的相互作用进行了热力学分析, 并用荧光显微镜和原子力显微镜对其形态进行了观测.热力学分析表明, DOPC与DPPC分子在单层膜结构中相互作用为排斥力, 诱导单层膜出现相变; DOPC, DPPC与胆固醇(Chol)间的相互作用均为吸引力, 当表面压力(π)大于18 mN/m时, DPPC与胆固醇的作用力大于DOPC.荧光显微镜观测表明, DOPC/DPPC单层膜出现明显相分离现象, 富含DPPC微区成“花形”结构, 且随着表面压力的升高微区逐渐增大, “花瓣”增多; 当胆固醇加入到DOPC/DPPC体系时, 单层膜相态由液相与凝胶相共存转变为液态无序相与液态有序相共存结构, 富含DPPC的微区形状从“花形”转变成“圆形”.原子力显微镜对单层膜的表征验证了荧光显微镜的观测结果, 表明胆固醇加入到DOPC/DPPC体系中对单层膜排列具有明显的影响, 压力和溶液状态等是影响脂膜结构的重要因素.     

Langmuir-Blodgett膜超分子SM/DOPC/Chol三元脂系的原子力显微镜观察

用原子力显微镜研究了胆固醇(Chol)对鞘磷脂(SM)/1,2-二油酸甘油-3-磷脂酰胆碱(DOPC)二元脂系统结构的影响和神经酰胺对SM/DOPC/Chol三元脂系统结构的影响. 实验发现, 在SM/DOPC二元脂系统中, 胆固醇和带饱和脂肪酸链的磷脂发生相互作用形成微区结构, 随着胆固醇含量的增加, 微区的面积逐渐增大, 形成了稳定的片层结构. 当把神经酰胺加入到等摩尔配比的SM/DOPC/Chol三元脂系统中时, 随着神经酰胺比例的增加, 先形成紧密的聚集态结构, 然后逐渐演变成具有特定微区的网状结构. 研究结果表明, 微区的形成主要是由分子不同的官能团之间的相互作用所决定, 这可能在细胞信号传导等生理活动中起到重要的作用.     

Cation selective ion channels formed by macrodiolide antibiotic elaiophylin in lipid bilayer membranes

The macrodiolide antibiotic elaiophylin (1) forms stable, long-lasting cation selective ion channels in planar lipid bilayer membranes prepared from soybean phosphatidylcholine. Current of the single ion channel displayed two sublevels corresponding to the two substates of the channel conductance: a slow substate, with about 5 s of mean dwell time in the open state at 40% level of the total amplitude conductance, and a fast substate of higher conductance with dwell times in the open and closed state of about 0.1 s. Amplitude conductances of the single ion channels in 200 mM of LiCl, NaCl, KCl, RbCl and CsCl were 75, 140, 220, 240 and 226 pS, and the conductance was linear function of the electrolyte concentration. Ratios of cation to anion permeabilities of the channel for NaCl and KCl were 8+/-2 and >24, respectively. A molecular model of the channel structure is suggested.     

Fluidity modulation of phospholipid bilayers by electrolyte ions: insights from fluorescence microscopy and microslit electrokinetic experiments

Fluidity and charging of supported bilayer lipid membranes (sBLMs) prepared from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) were studied by fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements at varying pH and ionic composition of the electrolyte. Measurements in neutral electrolytes (KCl, NaCl) revealed a strong correlation between the membrane fluidity and the membrane charging due to unsymmetrical water ion adsorption (OH(-) ? H(3)O(+)). The membrane fluidity significantly decreased below the isoelectric point of 3.9, suggesting a phase transition in the bilayer. The interactions of both chaotropic anions and strongly kosmotropic cations with the zwitterionic lipids were found to be related with nearly unhindered lipid mobility in the acidic pH range. While for the chaotropic anions the observed effect correlates with the increased negative net charge at low pH, no correlation was found between the changes in the membrane fluidity and charge in the presence of kosmotropic cations. We discuss the observed phenomena with respect to the interaction of the electrolyte ions with the lipid headgroup and the influence of this process on the headgroup orientation and hydration as well as on the lipid packaging.     

PAMAM dendrimer interactions with supported lipid bilayers: a kinetic and mechanistic investigation

The interaction kinetics of polyamidoamine (PAMAM) dendrimers with supported lipid bilayers of 1,2-sn-glycero-dimyristoylphosphocholine prepared by the vesicle deposition has been probed by optical waveguide lightmode spectroscopy and atomic force microscopy (AFM). In particular, the influence of PAMAM dendrimer generation (G2, G4, and G6) and concentration (1 to 100 nM) on the levels of adsorption and lipid bilayer removal have been determined as a function of time; hence interaction kinetics and mechanisms have been further elucidated. Dendrimer interaction kinetics with the lipid bilayer are concentration dependent in a complex manner, with net bilayer removal at 1 and 100 nM and net adsorption at 10 nM; these effects are irrespective of dendrimer generation. The pseudo first order rate constant for bilayer removal (at 1 and 100 nM) follows the order G6 > G4 > G2. In contrast, the pseudo first order rate constant for adsorption at 10 nM follows the order G2 > G4 > G6. AFM has confirmed expansion of lipid bilayer defects, hole formation, and adsorption to the bilayer or bilayer defects, and their concentration and generation dependence. These findings have implications when designing dendrimers for specific biopharmaceutical activities, e.g., as drugs, drug delivery vehicles, transfection agents, or antimicrobials.     

Growth of giant membrane lobes mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces

We report on the growth of giant membrane lobes that is mechanically driven by wetting fronts of phospholipid membranes at water-solid interfaces and a strategy to control the two-dimensional structure of the membrane lobes on a solid surface. The growth of giant membrane lobes was observed on a single-lipid bilayer which spread from a lump of phospholipid deposited on a silica-glass substrate or an oxidized silicon wafer in aqueous solutions of NaCl, KCl, MgCl2, or CaCl2 at relatively high salt concentrations. Most of the membrane lobes were very flat unilamellar tubes elongating from the lump of phospholipid, and their length reached 1 mm in 5 h. Experimental findings clearly indicate that the membrane lobes are adherent to the surface of the single-lipid bilayer and are mechanically elongated from the lump of phospholipid by the sliding motion of the single-lipid bilayer. We could control the two-dimensional structure of the membrane lobes on the substrate by controlling the spreading direction of the single-lipid bilayer using Pt micropatterns that were deposited on the smooth surface of the oxidized silicon wafer.     

Characterization of the physical properties of model biomembranes at the nanometer scale with the atomic force microscope

Interaction forces and topography of mixed phospholipid-glycolipid bilayers were investigated by atomic force microscopy (AFM) in aqueous conditions with probes functionalized with self-assembled monolayers terminating in hydroxy groups. Short-range repulsive forces were measured between the hydroxy-terminated probe and the surface of the two-dimensional (2-D) solid-like domains of distearoyl-phosphatidylethanolamine (DSPE) and digalactosyldiglyceride (DGDG). The form and range of the short-range repulsive force indicated that repulsive hydration/steric forces dominate the interaction at separation distances of 0.3-1.0 nm after which the probe makes mechanical contact with the bilayers. At loads < 5 nN the bilayer was elastically deformed by the probe, while at higher loads plastic deformation of the bilayer was observed. Surprisingly, a short-range repulsive force was not observed at the surface of the 2-D liquid-like dioleoylphosphatidylethanolamine (DOPE) film, despite the identical head groups of DOPE and DSPE. This provides direct evidence for the influence of the structure and mechanical properties of lipid bilayers on their interaction forces, an effect which may be a major importance in the control of biological processes such as cell adhesion and membrane fusion. The step height measured between lipid domains in the AFM topographic images was larger than could be accounted for by the thickness and mechanical properties of the molecules. A direct correlation was observed between the repulsive force range over the lipid domains and the topographic contrast, which provides direct insight into the fundamental mechanisms of AFM imaging in aqueous solutions. This study demonstrates that chemically modified AFM probes can be used in combination with patterned lipid bilayers as a novel and powerful approach to characterize the nanometer scale chemical and physical properties of heterogeneous biosurfaces such as cell membranes.     

Control of the structure of self-spreading lipid membrane by changing electrolyte concentration

We have controlled the structure of self-spreading lipid bilayer membranes prepared on surface-oxidized silicon substrates by changing electrolyte concentration. Analysis of the fluorescence intensity, considering the optical interference effect, clarified the stacking structure of the lipid membrane. By varying the electrolyte concentration, we can vary the number of single multilamellar lobes adsorbed on the underlying self-spreading bilayer. This dependence of the stacking ability on the electrolyte concentration was investigated on the basis of changes in the bilayer-lobe interaction energies, including van der Waals, electrostatic double layer, and hydration interaction energies. Theoretical estimation suggests that the observed electrolyte concentration dependence can be explained by the combination of the van der Waals attractive interaction energy and the repulsive double-layer interaction energy.     

Study of frictional properties of a phospholipid bilayer in a liquid environment with lateral force microscopy as a function of NaCl concentration

Friction properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-supported planar bilayers deposited on mica were tested in a liquid environment by lateral force microscopy. The presence of these bilayers was detected by imaging and force measurements with atomic force microscopy. To test how the presence of NaCl affects the frictional properties of the phospholipid bilayers, four DMPC bilayers were prepared on mica in saline media ranging from 0 to 0.1 M NaCl. Changes in the lateral vs vertical force curves were recorded as a function of NaCl concentration and related to structural changes induced in the DMPC bilayer by electrolyte ions. Three friction regimes were observed as the vertical force exerted by the tip on the bilayer increased. To relate the friction response to the structure of the DMPC bilayer, topographic images were recorded at the same time as friction data. Ions in solution screened charges present in DMPC polar heads, leading to more compact bilayers. As a consequence, the vertical force at which the bilayer broke during friction experiments increased with NaCl concentration. In addition, the topographic images showed that low-NaCl-concentration bilayers recover more easily due to the low cohesion between phospholipid molecules.     

Agar-supported lipid bilayers — basic structures for biosensor design. Electrical and mechanical properties

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel.

In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.