异丙酚与支撑双层类脂膜作用的电化学研究

以支撑双层类脂膜(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异丙酚与双层类脂膜的相互作用.结果表明,异丙酚能降低磷脂分子的有序性, 诱发s-BLM 上形成孔洞或缺陷, 且这种相互作用对作用时间、异丙酚浓度以及胆固醇的存在与否具有依赖性.     

Concentration and time dependant behavior of chlorpromazine interaction with supported bilayer lipid membrane

The interaction of chlorpromazine (CPZ) with supported bilayer lipid (dipalmitoyphosphatidylcholine) membrane (s-BLM) on the glassy carbon electrode (GCE) was investigated using cyclic voltammetry and ac impedance spectroscopy. The experimental data, based on the voltammetric response of Ru(NH₃)₆³⁺ associated with the oxidation of CPZ on the electrode, indicated that the interaction of CPZ with s-BLM was concentration and time dependant. The interaction between them could be divided into three stages by the concentration of CPZ: low, middle and high concentration. At the first stage, s-BLM was not affected by CPZ and the interaction was only a penetration of a small quantity of CPZ molecule into s-BLM. At the second stage, the defects formed in s-BLM due to the penetration of more CPZ molecule into s-BLM. At the last stage, a high CPZ:lipid ratio reached in s-BLM, resulting in the solubilization of s-BLM. The interaction time had different effect at three stages.     

模拟生物膜上铅离子毒性机理的研究

以玻碳电极支撑的磷脂双层膜(s-BLM)作为生物膜的模型,Fe(CN)63-/4-为探针分子,利用循环伏安法和交流阻抗技术研究了铅离子与s-BLM的相互作用,结果表明,铅离子与s-BLM之间可以发生比较强烈的相互作用,诱发s-BLM上形成离子通道,并且作用后的s-BLM在10 mmol/L的乙二胺四乙酸(EDTA)溶液中能够自我修复,这表明采用强的鳌合剂从机体组织中将铅夺取,撤走铅离子则通道关闭。     

Potassium ion transport by gramicidin and valinomycin across a Ag(1 1 1)-supported tethered bilayer lipid membrane

A tethered bilayer lipid membrane (tBLM) was prepared by anchoring a thiolipid monolayer to a Ag(1 1 1) single crystal from a thiolipid solution in ethanol; a diphytanoylphosphatidylcholine monolayer was then self-assembled on top of the thiolipid monolayer from a lipid solution in hexane or ethanol. The thickness of the mixed thiolipid|lipid bilayer was estimated at 7.5 ± 0.5 nm by scratching holes in the tBLM with an AFM tip and determining their height profile and the frequency distribution of height. The effect of the incorporation of gramicidin A and valinomycin upon K⁺ ion transport across the tBLM was investigated by electrochemical impedance spectroscopy. The position of the inflection point of the sigmoidal curve of the in-phase component of the electrode admittance against potential allowed the determination of an approximate scale of the absolute potential difference across the whole Ag(1 1 1)|(aqueous solution) interface, on the basis of a model of the electrified interface.     

Molecular Interaction of a New Antibacterial Polymer with a Supported Lipid Bilayer Measured by an in situ Label-Free Optical Technique

The interaction of the antibacterial polymer–branched poly(ethylene imine) substituted with quaternary ammonium groups, PEO and alkyl chains, PEI₂₅QI₅J₅A8₁₅–with a solid supported lipid bilayer was investigated using surface sensitive optical waveguide spectroscopy. The analysis of the optogeometrical parameters was extended developing a new composite layer model in which the structural and optical anisotropy of the molecular layers was taken into consideration. Following in situ the change of optical birefringence we were able to determine the composition of the lipid/polymer surface layer as well as the displacement of lipid bilayer by the antibacterial polymer without using additional labeling. Comparative assessment of the data of layer thickness and optical anisotropy helps to reveal the molecular mechanism of antibacterial effect of the polymer investigated.     

Supramolecular Amphiphiles Based on Pillar[5]arene and Meroterpenoids: Synthesis,Self-Association and Interaction with Floxuridine

In recent years, meroterpenoids have found wide biomedical application due to their synthetic availability, low toxicity, and biocompatibility. However, these compounds are not used in targeted drug delivery systems due to their high affinity for cell membranes, both healthy and in cancer cells. Using the approach of creating supramolecular amphiphiles, we have developed self-assembling systems based on water-soluble pillar[5]arene and synthetic meroterpenoids containing geraniol, myrtenol, farnesol, and phytol fragments. The resulting systems can be used as universal drug delivery systems. It was shown by turbidimetry that the obtained pillar[5]arene/synthetic meroterpenoid systems do not interact with the model cell membrane at pH = 7.4, but the associates are destroyed at pH = 4.1. In this case, the synthetic meroterpenoid is incorporated into the lipid bilayer of the model membrane. The characteristics of supramolecular self-assembly, association constants and stoichiometry of the most stable pillar[5]arene/synthetic meroterpenoid complexes were established by UV-vis spectroscopy and dynamic light scattering (DLS). It was shown that supramolecular amphiphiles based on pillar[5]arene/synthetic meroterpenoid systems form monodisperse associates in a wide range of concentrations. The inclusion of the antitumor drug 5-fluoro-2′-deoxyuridine (floxuridine) into the structure of the supramolecular associate was demonstrated by DLS, 19F, 2D DOSY NMR spectroscopy.     

甲基对硫磷与牛血清白蛋白相互作用的光谱研究

[目的]为了解释有机磷农药—甲基对硫磷对牛血清白蛋白(BSA)的作用与致毒机理。[方法]采用紫外和荧光光谱法研究甲基对硫磷与BSA的相互作用。[结论]甲基对硫磷能与BSA相互作用,其作用程度和方式与浓度、酸度和离子强度等因素有关。甲基对硫磷对BSA的荧光猝灭方式为静态猝灭,通过计算得到结合位点数(n)和结合常数(K),且结合距离(r)小于7 nm。二者的作用力为氢键或范德华力。甲基对硫磷使BSA构象发生变化,且对酪氨酸残基的影响较大。     

活性艳蓝KN-R与DNA相互作用的光谱研究

采用紫外光谱和荧光光谱法研究了活性艳蓝KN-R(RBB KN-R)与DNA的相互作用.结果表明,RBBKN-R与DNA能够发生相互作用,其作用方式和程度取决于RBB KN-R浓度、离子强度、酸度和磷酸根浓度等因素.随着RBB KN-R浓度的增加,RBB KN-R-DNA的紫外光谱表现为增色效应,荧光发生猝灭.RBB K...     

Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy

Cell membranes are typically very complex, consisting of a multitude of different lipids and proteins. Supported lipid bilayers are widely used as model systems to study biological membranes. Atomic force microscopy and force spectroscopy techniques are nanoscale methods that are successfully used to study supported lipid bilayers. These methods, especially force spectroscopy, require the reliable preparation of supported lipid bilayers with extended coverage. The unreliability and a lack of a complete understanding of the vesicle fusion process though have held back progress in this promising field. We document here robust protocols for the formation of fluid phase DOPC and gel phase DPPC bilayers on mica. Insights into the most crucial experimental parameters and a comparison between DOPC and DPPC preparation are presented. Finally, we demonstrate force spectroscopy measurements on DOPC surfaces and measure rupture forces and bilayer depths that agree well with X-ray diffraction data. We also believe our approach to decomposing the force-distance curves into depth sub-components provides a more reliable method for characterising the depth of fluid phase lipid bilayers, particularly in comparison with typical image analysis approaches.     

Preparation and characterization of chitosan-grafted multiwalled carbon nanotubes and their electrochemical properties

A chitosan (CS)-grafted multiwalled carbon nanotubes (MWCNT) composite (CS-MWCNT) was prepared via covalently grafting a biocompatible polymer CS onto the surfaces of MWCNT and was characterized by Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis), and transmission electron microscopy (TEM). The interaction between MWCNT and CS was investigated and explained according to the FTIR and UV-vis results. More evidence for the presence of the CS-MWCNT was confirmed by TEM images. In the TEM images, a core-shell structure with nanotubes in the center and a CS layer lying around them was observed. The electrochemical properties of the CS-MWCNT were characterized by an electrodeposition method that involves depositing it on a Au electrode. The porous microstructure of the CS-MWCNT modified electrode was observed by scanning electron microscopy. The excellent electrocatalytic ability of the CS-MWCNT modified electrode towards hydrogen peroxide is applicable to the development of oxidase-based amperometric biosensors.     

Membrane-Associated Nucleobase-Functionalized β-Peptides (β-PNAs) Affecting Membrane Support and Lipid Composition

Protein-membrane interactions are essential to maintain membrane integrity and control membrane morphology and composition. Cytoskeletal proteins in particular are known to interact to a high degree with lipid bilayers and to line the cytoplasmic side of the plasma membrane with an extensive network structure. In order to gain a better mechanistical understanding of the protein–membrane interplay and possible membrane signaling, we started to develop a model system based on β-peptide nucleic acids (β-PNAs). These β-peptides are known to form stable hydrogen-bonded aggregates due to their helical secondary structure, which serve to pre-organize the attached nucleobases. After optimization of the β-PNA solid-phase peptide synthesis and validation of helix formation, the ability of the novel β-PNAs to dimerize and interact with lipid bilayers was investigated by both fluorescence and circular dichroism spectroscopy. It was shown that duplex formation occurs rapidly and with high specificity and could also be detected on the surfaces of the lipid bilayers. Hereby, the potential of a β-PNA-based peptide system to mimic membrane-associated protein networks could be demonstrated.     

Hydrogenation of chalcones using hydrogen permeating through a Pd and palladized Pd electrodes

The hydrogenation of benzalacetone and benzalacetophenone was carried out using atomic hydrogen permeating through a palladium membrane. A two-compartment cell separated by a Pd sheet or a palladized Pd (Pd/Pd black) sheet electrode was employed. The reduction products were identified by (GC) gas chromatography, UV-vis absorption spectroscopy and NMR spectroscopy. The carbon-carbon double bond was hydrogenated and the benzylacetone and benzylacetophenone were obtained as products using palladium catalyst. The current efficiency for hydrogenation reaction increases when the current density for water electrolysis decreases and depends on the initial chalcone concentration. It is over 90% at the concentration of 10 mmol L⁻¹. The hydrogen absorption and diffusion into and through a palladium membrane electrode has been studied by using an electrochemical impedance spectroscopy method. The impedance results would indicate that the hydrogen permeated through the membrane is consumed by the chalcone during the hydrogenation process keeping as the permeable boundary condition in the outer side of the Pd membrane the hydrogen activity almost zero. The hydrogen entering the metal through an adsorbed state and the rate of hydrogen absorption is diffusion-controlled.     

Fluorescence Anisotropy,FT-IR Spectroscopy and 31-P NMR Studies on the Interaction of Paclitaxel with Lipid Bilayers

To understand the bilayer interaction with paclitaxel, fluorescence polarization, Fourier transform infrared spectroscopy (FT-IR) and 31-phosphorus nuclear magnetic resonance (31P-NMR) studies were performed on paclitaxel bearing liposomes. Fluorescence anisotropy of three probes namely, 1,6-diphenyl-1,3,5-hexatriene (DPH), 12-(9-anthroyloxy) stearic acid (12AS) and 8-anilino-1-naphthalene sulfonate (ANS) were monitored as a function of paclitaxel concentration in the unsaturated bilayers. The incorporation of paclitaxel decreased anisotropy of 12AS and ANS probes, while slightly increased anisotropy of DPH. Paclitaxel has a fluidizing effect in the upper region of the bilayer whereas the hydrophobic core is slightly rigidized. FT-IR spectroscopy showed an increase in the asymmetric and symmetric methylene stretching frequencies, splitting of methylene scissoring band and broadening of carbonyl stretching mode. These studies collectively ascertained that paclitaxel mainly occupies the cooperativity region and interact with the interfacial region of unsaturated bilayers and induces fluidity in the headgroup region of bilayer. At higher loadings (>3 mol%), paclitaxel might gradually tend to accumulate at the interface and eventually partition out of bilayer as a result of solute exclusion phenomenon, resulting in crystallization; seed non-bilayer phases, as revealed by 31P-NMR, thereby destabilizing liposomal formulations. In general, any membrane component which has a rigidization effect will decrease, while that with a fluidizing effect will increase, with a bearing on headgroup interactions, partitioning of paclitaxel into bilayers and stability of the liposomes.     

Mechanism of cellular uptake of genotoxic silica nanoparticles

ABSTRACT: Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations 1 mug/ml, but DNA damage was evident at 0.1 mug/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 mug/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4 C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal microbalance experiments on tethered bilayer lipid membrane systems show that the nanoparticles strongly bind to lipid membranes, forming an adherent monolayer on the membrane. Leakage assays on large unilamellar vesicles (400 nm diameter) indicate that binding of the silica NPs transiently disrupts the vesicles which rapidly self-seal. We suggest that an adhesive interaction between silica nanoparticles and lipid membranes could cause passive cellular uptake of the particles.     

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.     

Mechanisms underlying the effects of membrane fouling on the nanofiltration of trace organic contaminants

The influence of membrane fouling on the retention of four trace organic contaminants - namely sulfamethoxazole, ibuprofen, carbamazepine, and triclosan - by nanofiltration membranes was investigated in this study. Humic acid, alginate, bovine serum albumin, and silica colloids were selected as model foulants to simulate various organic fractions and colloidal matter that are found in secondary treated effluent and surface water. The effects of membrane fouling on the separation process was delineated by comparing retention values of clean and fouled membranes and relate them to the membrane properties (under both clean and fouled conditions) as well as physicochemical characteristics of the trace organic contaminants. Membrane fouling was dependent on the physicochemical properties of the model foulants. Initial foulant-membrane interaction could probably be a major factor governing the process of membrane fouling particularly by the organic foulants. Such membrane-foulant interaction was also a dominating factor governing the effects of membrane fouling on the membrane separation efficacy. In good agreement with our previous study (Nghiem and Hawkes, 2007 [1]), the effects of fouling on retention were found to be membrane pore size dependent. In addition, results reported here suggest that these effects could also be foulant dependent. It was probable that the influence of membrane fouling on trace organic retention could be governed by four distinctive mechanisms: modification of the membrane charge surface, pore blocking, cake enhanced concentration polarisation, and modification of the membrane hydrophobicity. The presence of the fouling layer could affect the retention behavior of charged solutes by altering the membrane surface charge density. While the effect of surface charge modification was clear for inorganic salts, it was less obvious for the negatively charged pharmaceutical species (sulfamethoxazole and ibuprofen) examined in this investigation, possibly due to the interference of the pore blocking mechanism. Evidence of the cake enhanced concentration polarisation effect was quite clear, particularly under colloidal fouling conditions. In addition, organic fouling could also interfere with the solute-membrane interaction, and therefore, exerted considerable influence on the separation process of the hydrophobic trace organic contaminant triclosan.     

Chiral recognition of D-kyotorphin by lipidic membranes: relevance toward improved analgesic efficiency

D-Kyotorphin (D-KTP), the most potent isomer of the endorphin-like dipeptide kyotorphin (KTP), is a good drug candidate for the treatment of chronic pain and is thought to be involved in receptor-mediated processes. According to the "membrane catalysis" model, ligands interact with membrane lipids to attain high local concentrations in the receptor vicinity and to adopt the necessary conformation for docking. Therefore, the interaction and recognition of D-KTP by membranes is potentially important to its increased analgesic effect. In spite of the neutral net charge of D-KTP at pH 7.4, fluorescence spectroscopy reveals that the interaction with large unilamellar vesicles is more extensive than was observed for KTP. The tyrosine residue interacts extensively with rigid membranes, with a location and well-defined orientation in the bilayer. This suggests not only that D-KTP meets the structural constraints needed for receptor-ligand interaction in a manner similar to that of KTP, but also that the stronger membrane interaction and ability to discriminate rigid membrane domains might contribute to its improved analgesic effect.     

电解质对卵磷脂双分子层电化学行为的影响

实验利用自组装技术在铂电极上制备支撑的卵磷脂双层膜（s-BLM）作为生物膜的模型,利用循环伏安法（cV）和电化学阻抗谱（EIS）对由电解质KCI引起的s-BLM通透性的变化进行了研究。结果表明,KCI可与s-BLM发生较强的相互作用,导致s—BLM表面磷脂分子的有序排列受到影响,产生一些离子通道,增加了对探针分子电流响应,同时降低了s—BLM的电阻。     

Flow Alignment of Extracellular Vesicles: Structure and Orientation of Membrane‐Associated Bio‐macromolecules Studied with Polarized Light

Extracellular vesicles (EVs) are currently in scientific focus, as they have great potential to revolutionize the diagnosis and therapy of various diseases. However, numerous aspects of these species are still poorly understood, and thus, additional insight into their molecular‐level properties, membrane–protein interactions, and membrane rigidity is still needed. We here demonstrate the use of red‐blood‐cell‐derived EVs (REVs) that polarized light spectroscopy techniques, linear and circular dichroism, can provide molecular‐level structural information on these systems. Flow‐linear dichroism (flow‐LD) measurements show that EVs can be oriented by shear force and indicate that hemoglobin molecules are associated to the lipid bilayer in freshly released REVs. During storage, this interaction ceases; this is coupled to major protein conformational changes relative to the initial state. Further on, the degree of orientation gives insight into vesicle rigidity, which decreases in time parallel to changes in protein conformation. Overall, we propose that both linear dichroism and circular dichroism spectroscopy can provide simple, rapid, yet efficient ways to track changes in the membrane–protein interactions of EV components at the molecular level, which may also give insight into processes occurring during vesiculation.     

Evaluation of the Physico-Chemical Properties of Liposomes Assembled from Bioconjugates of Anisic Acid with Phosphatidylcholine

The aim of this work was the evaluation of the physico-chemical properties of a new type of liposomes that are composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine. In particular, the impact of modified anisic acid phospholipids on the thermotropic parameters of liposomes was determined, which is crucial for using them as potential carriers of active substances in cancer therapies. Their properties were determined using three biophysical methods, namely differential scanning calorimetry (DSC), steady-state fluorimetry and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Moreover, temperature studies of liposomes composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine provided information about the phase transition, fluidity regarding chain order, hydration and dynamics. The DSC results show that the main phase transition peak for conjugates of anisic acid with phosphatidylcholine molecules was broadened and shifted to a lower temperature in a concentration- and structure-dependent manner. The ATR-FTIR results and the results of measurements conducted using fluorescent probes located at different regions in the lipid bilayer are in line with DSC. The results show that the new bioconjugates with phosphatidylcholine have a significant impact on the physico-chemical properties of a membrane and cause a decrease in the temperature of the main phase transition. The consequence of this is greater fluidity of the lipid bilayer.