基于磷脂膜的界面相互作用研究

磷脂是一类非常重要的生物分子,它是细胞膜的主要组成部分,同时也在诸多生命活动（如细胞激活、代谢维持和激素分泌等）中发挥着不可替代的作用。磷脂种类繁多,且具有自组装能力强、生物相容性好、无细胞毒性、易于获得等一系列优点。作为一种最典型的界面材料,磷脂膜特殊的双层结构及出色的生物学性能引起了科研人员的广泛关注,其能够模拟生物膜结构,有助于研究界面上的分子特征及作用行为。此外,磷脂可被用作生物医学材料,改性磷脂以及磷脂与纳米颗粒的复合物在肿瘤成像技术、药物靶向递送系统等方面具有良好的发展前景,显著促进了新型生物材料的开发与进步。本文先归纳了磷脂的分类,并比较了磷脂在不同基底的吸附行为;之后重点分析了磷脂膜界面的选择性识别功能以及与多肽、酶、蛋白质等生物分子之间的相互作用;最后对基于磷脂膜的生物材料在生物传感、药物研究和成像技术中的应用作出了展望。     

自组装ITO/双层磷脂膜的制备及其光电行为研究

在ITO(Indium-tin-oxide)导电玻璃电极上制备上自组装双层磷脂膜和经C60修饰的双层磷脂膜,研究了这种自组装双层磷脂膜的光电行为,考察了偏压、溶液中的给体和受体的浓度对自组装膜光电流强度的影响,讨论了C60分子对光电子跨膜传递过程的促进作用。     

磷脂膜色谱及其在药物跨膜转运评价中的应用

磷脂膜色谱是固态基质上的有序磷脂分子单层体系采用色谱学方法仿真药物与细胞膜相互作用过程,可用来评价药物的细胞膜渗透性和活性。硅胶表面上的磷脂单分子层模拟了单层细胞膜,因此药物的磷脂膜色谱保留行为可用于预测药物与细胞膜的相互作用。目前考察药物跨膜转运的模型主要有正辛醇/水系统、脂质体/水系统、反相色谱（ODS）以及磷脂膜色谱。与前述3种系统比较,磷脂膜色谱除了具有高效、简便等特点外,同时能模拟药物与生物膜之间疏水作用力以外的其他作用力,因此对磷脂膜色谱的研究也越来越深入。由于药物细胞膜渗透性对其有效性和安全性起着关键作用,因此磷脂膜色谱在新药研发早期阶段的介入可以有效地降低后期候选药物的淘汰率,提高新药的研发效率。该文就磷脂膜色谱的研究及在药物跨膜转运评价中的应用进行了综述。     

F-肌动蛋白与负电荷磷脂膜的相互作用研究

细胞膜的内膜含有大量的负电荷磷脂,研究F-肌动蛋白与负电荷磷脂的相互作用将有助于更深入地了解细胞骨架与细胞膜的体内相互作用机制.在金片和金电极上分别构建了负电荷磷脂的杂化双层磷脂膜,通过表面等离子体共振方法(SPR)和电化学阻抗技术研究了F-肌动蛋白与负电荷磷脂膜的相互作用.结果表明,F-肌动蛋白可以在没有中间联系蛋白的情况下,直接与负电荷磷脂膜发生相互作用.钙离子可以有效地促进它们的相互作用,表明钙离子在其中发挥了重要作用.高浓度的KCl显著抑制它们的相互作用,表明这种相互作用主要受静电作用影响.实验结果进一步证明在F-肌动蛋白与负电荷磷脂膜相互作用时,除了可以通过其它蛋白发生间接相互作用外,还可以与磷脂膜发生直接的相互作用.     

锆镁磷脂膜色谱固定相的制备及其在评价药物-膜相互作用中的应用

基于锆基质与磷脂之间强烈的路易斯酸碱作用,制备了锆镁磷脂膜色谱固定相,并使用红外光谱、X射线光电子能谱对该色谱固定相进行了表征;使用与体内环境类似的生理缓冲液体系为流动相,评价了该模拟生物膜色谱固定相预测药物膜渗透性的能力,结果表明药物在锆镁磷脂膜色谱中的保留(log Kmbm)与表观渗透率(log Papp)在预测药物的膜渗透性、跨膜吸收等方面具有非常好的相关性,相关系数为0.970,斜率接近1。通过理论推导,引入了直观、方便的热力学指标吉布斯自由能差值（Δ(ΔG°)）对药物-膜之间的相互作用强弱进行了评价。     

平板双分子层脂膜的制备及锌离子跨该膜的输运过程

制备了氧化胆固醇 卵磷脂(脑磷脂)平板双分子层脂膜,研究了膜配方对双分子层脂膜的稳定性和离子通透性的影响,得到了最佳制膜工艺,建立了锌离子跨卵 (脑 )磷脂膜的吸附 -扩散模型,其计算值与实验值基本吻合.     

毒素蛋白ColicinE1与磷脂膜相互作用的荧光光谱研究临界摩尔比的测定

磷脂－蛋白相互作用的临界摩尔比是研究膜脂－蛋白相互作用的重要参数．本文利用荧光光谱技术首次测定了毒素蛋白ＣｏｌｉｃｉｎＥ１在不同条件下与不同磷脂膜相互作用的临界摩尔比并通过临界摩尔比的变化讨论了插膜蛋白与磷脂膜相互作用的规律，为进一步探讨毒素蛋白的插膜机制提供了重要的基础     

毒素蛋白Colicin E1与磷脂膜相互作用的荧光光谱研究——临界摩尔…

磷脂－蛋白相互作用的临界摩尔比是研究膜脂－蛋白相互作用的重要参数。本文利用荧光光谱技术首次测定了毒素蛋白Ｃｏｌｉｃｉｎ Ｅ１在不同条件下与不同磷脂膜相互作用的临界摩尔比并通过临界摩尔比的变化讨论了插膜蛋白与磷脂膜相互作用的规律，为进一步探讨毒素蛋白的插膜机制提供了重要的基础。     

溶质在磷脂膜色谱与正辛醇/水系统中的热力学分配行为

生物膜为液晶态磷脂双分子层结构, 其中蛋白质镶嵌在生物膜上, 处于脂质环境中, 因此药物膜的转运、 药物接近膜中蛋白质以及随后结合过程等均与药物和生物膜间的相互作用有着密切联系. 药物的膜/水分配系数(Km)是评价药物与生物膜间相互作用的定量参数, 为药物与生物膜间各种分子作用力的总和, 包括静电、 氢键和疏水等作用力及立体效应等[1,2]. 药物与生物膜间相互作用的评价系统一直是研究中的热点. 最初正辛醇/水系统为模型分配系统, 但是由于其不是理想的生物膜模拟相, 因此不能用来准确描述药物与生物膜间的相互作用. 最近出现的磷脂膜色谱可较好地模拟细胞膜有序磷脂层的空间环境, 因此在评价药物与生物膜间的相互作用、 预测药物跨膜转运以及生物活性上均明显优越于正辛醇/水系统[3]. 虽然我们已证明这两个系统在亲脂性测量尺度上存在明显差别, 但是并没有说明溶质与两个生物膜模拟相的相互作用机制的差别[4]. 本文考察了温度对溶质分子在这两个分配系统中分配的影响, 并从溶质分配过程中的熵变和焓变的角度对这两个分配系统进行了比较.     

制菌霉素与固体支撑磷脂膜的相互作用

为了更好地了解制菌霉素的作用机理, 本文利用表面等离子体共振(SPR)和交流阻抗两种技术, 考察了制菌霉素与不含固醇的固体支撑纯磷脂膜的相互作用, 结果发现, 制菌霉素可与纯磷脂膜相互作用, 并可能在膜上形成微孔.     

Graphene oxide assisted electromembrane extraction with gas chromatography for the determination of methamphetamine as a model analyte in hair and urine samples

In the present study, graphene oxide reinforced two‐phase electromembrane extraction (EME) coupled with gas chromatography was applied for the determination of methamphetamine as a model analyte in biological samples. The presence of graphene oxide in the hollow fiber wall can increase the effective surface area, interactions with analyte and polarity of support liquid membrane that leads to an enhancement in the analyte migration. To investigate the influence of the presence of graphene oxide in the support liquid membrane on the extraction efficiency, a comparative study was performed between graphene oxide and graphene oxide/EME methods. The extraction parameters such as type of organic solvent, pH of the donor phase, stirring speed, time, voltage, salt addition and the concentration of graphene oxide were optimized. Under the optimum conditions, the proposed microextraction technique provided low limit of detection (2.4 ng/mL), high preconcentration factor (195–198) and high relative recovery (95–98.5%). Finally, the method was successfully employed for the determination of methamphetamine in urine and hair samples.     

Influence of the physical state of phospholipid monolayers on protein binding

Langmuir monolayers were used to characterize the influence of the physical state of phospholipid monolayers on the binding of protein Retinis Pigmentosa 2 (RP2). The binding parameters of RP2 (maximum insertion pressure (MIP), synergy and ΔΠ(0)) in monolayers were thus analyzed in the presence of phospholipids bearing increasing fatty acyl chain lengths at temperatures where their liquid-expanded (LE), liquid-condensed (LC), or solid-condensed (SC) states can be individually observed. The data show that a larger value of synergy is observed in the LC/SC states than in the LE state, independent of the fatty acyl chain length of phospholipids. Moreover, both the MIP and the ΔΠ(0) increase with the fatty acyl chain length when phospholipids are in the LC/SC state, whereas those binding parameters remain almost unchanged when phospholipids are in the LE state. This effect of the phospholipid physical state on the binding of RP2 was further demonstrated by measurements performed in the presence of a phospholipid monolayer showing a phase transition from the LE to the LC state at room temperature. The data collected are showing that very similar values of MIP but very different values of synergy and ΔΠ(0) are obtained in the LE (below the phase transition) and LC (above the phase transition) states. In addition, the binding parameters of RP2 in the LE (below the phase transition) as well as in the LC (above the phase transition) states were found to be indistinguishable from those where single LC and LE states are respectively observed. The preference of RP2 for binding phospholipids in the LC state was then confirmed by the observation of a large modification of the shape of the LC domains in the phase transition. Therefore, protein binding parameters can be strongly influenced by the physical state of phospholipid monolayers. Moreover, measurements performed with the α/β domain of RP2 strongly suggest that the β helix of RP2 plays a major role in the preferential binding of this protein to phospholipids in the LC state.     

Magnetic graphene oxide modified by imidazole‐based ionic liquids for the magnetic‐based solid‐phase extraction of polysaccharides from brown alga

Magnetic graphene oxide was modified by four imidazole‐based ionic liquids to synthesize materials for the extraction of polysaccharides by magnetic solid‐phase extraction. Fucoidan and laminarin were chosen as the representative polysaccharides owing to their excellent pharmaceutical value and availability. Fourier transform infrared spectroscopy, field‐emission scanning electron microscopy, and thermogravimetric analysis were applied to characterize the synthesized materials. Single‐factor experiments showed that the extraction efficiency of polysaccharides was affected by the amount of ionic liquids for modification, solid–liquid ratio of brown alga and ethanol, the stirring time of brown alga and ionic liquid‐modified magnetic graphene oxide materials, and amount of 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide materials added to the brown alga sample solution. The results indicated that 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide possessed better extraction ability than graphene oxide, magnetic graphene oxide, and other three ionic‐liquid‐modified magnetic graphene oxide materials. The highest extraction recoveries of fucoidan and laminarin extracted by 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide were 93.3 and 87.2%, respectively. In addition, solid materials could be separated and reused easily owing to their magnetic properties.     

Morphological and Thermal Properties of Vesicular Phospholipid Gels Studied by DSC,Rheometry and Electron Microscopy

Semisolid phospholipid preparations have been well known for several years and are still investigated as drug carrier systems, e.g. for potential cancer therapy. They may be applied parenterally as semisolid vesicular phospholipid gels suitable as implants for sustained drug release or as liposomal preparations after redisperging the stable storage form. Due to enhanced stability, mixtures of hydrated phospholipids and cholesterol are more suitable than natural unsaturated phospholipids. In order to describe characteristics of vesicular phospholipid gels, only a few techniques may be useful. Especially the structure of the semisolid preparation is not yet completely understood. We tried to get some more information about these systems by using a combination of freeze-fracture electron microscopy, differential scanning calorimetry and rheometry to elucidate, on the one hand, the inner structure or homogeneity and, on the other, the thermotropic phase transition of the three-dimensional lipid network and the temperature dependency of the fluidity/viscosity of the samples. Using freeze-fracture electron microscopy we found coexisting phospholipid domains of lamellar sheets and vesicular structures. With the help of differential scanning calorimetry the reasons for the different phase behaviour were elucidated. Rheometric measurements show increased intermediate viscosity at the thermotropic phase transition of the lipid bilayers, possibly induced by interacting membrane defects. This revised version was published online in August 2006 with corrections to the Cover Date.     

Prodan fluorescence detects the bilayer packing of asymmetric phospholipids

The bilayer phase behavior of asymmetric phospholipids, palmitoylstearoylphosphatidylcholine (PSPC) and stearoylpalmitoylphosphatidylcholine (SPPC), with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under high pressure. The results were compared with those of a symmetric phospholipid, diheptadecanoyl PC (C17PC). The difference in phase transitions of the PSPC and SPPC bilayers and in thermodynamic quantities of the transitions was hardly observed between LMV and GMV as the case of the C17PC bilayer. On the other hand, the Prodan fluorescence showed clear differences between LMV and GMV of the asymmetric PC bilayers. From the second derivative of Prodan fluorescence spectra, the three dimensional image plots in which we can clearly see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under high pressure. We revealed from the plots that the bilayer packing is significantly dependent on not only the vesicle size but also the acyl-chain asymmetry of PC molecule in addition to the phase states. It was found that the packing of the gel phases of the asymmetric PC bilayers is weaker than that of the symmetric PC bilayer, and the size of vesicle affects the packing of the interdigitated gel phase the most markedly among three gel phases. This study suggests that the Prodan molecules can detect the effect of vesicle size on the phase states for the asymmetric PC bilayers, and they become a useful indicator for various membrane properties, especially bilayer interdigitation.     

Deep eutectic solvents skeleton typed molecularly imprinted chitosan microsphere coated magnetic graphene oxide for solid‐phase microextraction of chlorophenols from environmental water

Novel molecularly imprinted chitosan microspheres were prepared on the surface of magnetic graphene oxide, with deep eutectic solvents both as a functional monomer and template. The prepared molecularly imprinted chitosan microspheres‐magnetic graphene oxide was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, Brunauer‐Emmett‐Teller surface area, thermogravimetric analysis were subsequently combined with solid‐phase micro‐extraction for simultaneous separation and enrichment of the extraction of chlorophenols from environmental water. Factors affecting the extraction efficiency of chlorophenols were optimized using response surface methodology. The actual extraction capacities under the optimal conditions (liquid to solid ratio = 3, cycles of adsorption/desorption = 5, 40°C extraction temperature, and extraction time for 35 min) were 86.90 mg/g. Compared to the traditional materials, the molecularly imprinted chitosan microspheres‐magnetic graphene oxide produced higher selectivity and extraction capacity.     

Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation

The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.     

Separation and quantification by high-performance liquid chromatography with light scattering detection of the main wheat flour phospholipids during dough mixing in the presence of phospholipase

Phospholipids (PL) are minor components of wheat flour involved in baking quality and exogenous phospholipids are used as emulsifiers giving better loaf volume and crumb grain. Few biochemical data are available on the phospholipid evolution during mixing, probably because of the time-consuming methods proposed for their extraction, separation and quantification. In the present study, the extraction, separation and quantification of the main wheat flour phospholipids were carried out. Total lipids (2% dry mass of wheat flour) were extracted from flour or dough by a mixture of chloroform-methanol-water (1:1:1 (v/v)). The phospholipids were separated from the lipid extract on silica cartridge by solid-phase extraction (SPE) procedure under a 1.5-4 mmHg vacuum, at a 0.8 mL min(-1) flow rate (1 mmHg = 133.322 Pa). The recovery of the lipid extract was 100%, whereas the SPE yield for the PLs was 50%. The resulting fraction was then submitted to HPLC with evaporative light scattering detection on a Diol stationary phase allowing the separation and quantification of each class of phospholipids, in less than 16 min. The developed method allowed to quantify the phospholipid amounts from eight wheat flours as well as their evolution during mixing in the presence of phospholipase.     

Determination of phospholipids in dairy products by SPE/HPLC/ELSD

The aim of this work was to evaluate the performance of different methods for both milk lipid extraction and phospholipids separation. As far as the lipid extraction procedure is concerned, the Folch method showed a higher phospholipid recovery with respect to the Rose-Gottlieb method. Different SPE cartridges and solvent phases were tested to carry out the separation of phospholipids from fat. The yield of extraction was evaluated by isolating phospholipids from both milk fat and synthetic fat; Standard Addition Method was applied as well. The isolation of the phospholipids by SPE silica column and subsequent analysis by HPLC/ELSD was shown to be an accurate and reproducible analytical method for the determination of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine and sphingomyelin in milk fat extracted by Folch method.