Interaction of curcumin with lipid monolayers and liposomal bilayers

Curcumin shows huge potential as an anticancer and anti-inflammatory agent. However, to achieve a satisfactory bioavailability and stability of this compound, its liposomal form is preferable. Our detailed studies on the curcumin interaction with lipid membranes are aimed to obtain better understanding of the mechanism and eventually to improve the efficiency of curcumin delivery to cells. Egg yolk phosphatidylcholine (EYPC) one-component monolayers and bilayers, as well as mixed systems containing additionally dihexadecyl phosphate (DHP) and cholesterol, were studied. Curcumin binding constant to EYPC liposomes was determined based on two different methods: UV/Vis absorption and fluorescence measurements to be 4.26 × 10⁴ M⁻¹ and 3.79 × 10⁴ M⁻¹, respectively. The fluorescence quenching experiment revealed that curcumin locates in the hydrophobic region of EYPC liposomal bilayer. It was shown that curcumin impacts the size and stability of the liposomal carriers significantly. Loaded into the EYPC/DPH/cholesterol liposomal bilayer curcumin stabilizes the system proportionally to its content, while the EYPC/DPH system is destabilized upon drug loading. The three-component lipid composition of the liposome seems to be the most promising system for curcumin delivery. An interaction of free and liposomal curcumin with EYPC and mixed monolayers was also studied using Langmuir balance measurements. Monolayer systems were treated as a simple model of cell membrane. Condensing effect of curcumin on EYPC and EYPC/DHP monolayers and loosening influence on EYPC/DHP/chol ones were observed. It was also demonstrated that curcumin-loaded EYPC liposomes are more stable upon interaction with the model lipid membrane than the unloaded ones.     

Interaction of antimicrobial arginine-based cationic surfactants with liposomes and lipid monolayers

The present work examines the relationship between the antimicrobial activity of novel arginine-based cationic surfactants and the physicochemical process involved in the perturbation of the cell membrane. To this end, the interaction of these surfactants with two biomembrane models, namely, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar lipid vesicles (MLVs) and monolayers of DPPC, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG), and Escherichia coli total lipid extract, was investigated. For the sake of comparison, this study included two commercial antimicrobial agents, hexadecyltrimethylammonium bromide and chlorhexidine dihydrochloride. Changes in the thermotropic phase transition parameters of DPPC MLVs in the presence of the compounds were studied by differential scanning calorimetry analysis. The results show that variations in both the transition temperature (Tm) and the transition width at half-height of the heat absorption peak (deltaT1/2) were consistent with the antimicrobial activity of the compounds. Penetration kinetics and compression isotherm studies performed with DPPC, DPPG, and E. coli total lipid extract monolayers indicated that both steric hindrance effects and electrostatic forces explained the antimicrobial agent-lipid interaction. Overall, in DPPC monolayers single-chain surfactants had the highest penetration capacity, whereas gemini surfactants were the most active in DPPG systems. The compression isotherms showed an expansion of the monolayers compared with that of pure lipids, indicating an insertion of the compounds into the lipid molecules. Owing to their cationic character, they are incorporated better into the negatively charged DPPG than into zwitterionic DPPC lipid monolayers.     

Thermodynamics of monolayers formed by mixtures of phosphatidylcholine/phosphatidylserine

In this work we obtain the thermodynamic properties of mixed (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) PC and (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)) PS monolayers. Measurements of compressibility (isotherms, bulk modulus, and excess area per molecule) and surface potential show that the properties of monolayers at the air-water interface depend on the concentration of ions (Na(+) and K(+)) and the proportion of PS in the mixture. The dependence on PS arises because the molecule is originally bound to a Na(+) counterion; by increasing the concentration of ions the entropy changes, creating a favorable system for the bound counterions of PS to join the bulk, leaving a negatively charged molecule. This change leads to an increase in electrostatic repulsions which is reflected by the increase in area per molecule versus surface pressure and a higher surface potential. The results lead to the conclusion that this mixture of phospholipids follows a non ideal behavior and can help to understand the thermodynamic behavior of membranes made of binary mixtures of a zwitterionic and an anionic phospholipid with a bound counterion.     

Fluorine containing poly(amide-imide)s: synthesis and formation of Langmuir-Blodgett monolayers

Soluble fluorine containing poly(amide-imide)s, PAI(1-4), were synthesized from diimide-dicarboxylic acid, 2,2-bis[N-(4-carboxyphenyl)-phthalimide-1,4-yl]hexafluoropropane with various diamines by direct polycondensation in N-methyl-2-pyrrolidone (NMP) containing CaCl₂ and using triphenyl phosphite and pyridine as condensing agents. The polymers were readily soluble in aprotic polar solvents such as NMP, N,N-dimethylacetamide, dimethyl sulfoxide and tetrahydrofuran. Their Langmuir monolayers were studied at the air/water interface. The monolayers were generally stable at the water surface and could be reproducibly transferred onto solid substrates to build up Langmuir-Blodgett (LB) multilayers. The LB mono- and multilayers were characterized by ultra-violet/visible spectroscopy (UV-Vis), surface plasmon resonance, atomic force microscopy.     

Analytical applications of gold electrodes modified with monolayers of thiolated cyclodextrins

The sequential method for the preparation of cyclodextrin monolayers is used to prepare modified electrodes responsive towards selected guest molecules: ferrocene, ibuprofen, methylene blue, dopamine and menadione. The inclusion into cyclodextrin cavities is monitored using cyclic voltammetry and the mediating role of the immobilized molecules towards solution species is shown on the example of dopamine oxidation.     

Role of the electrostatic interactions on the basic or acidic hydrolysis kinetics of poly-( , -lactide) monolayers

The hydrolysis kinetics of insoluble poly-( , -lactide) monolayers spread on basic or acidic aqueous subphase were followed by measuring simultaneously the decrease in the surface area at constant surface pressure and the evolution of the surface potential. An approach to analyse the role of the electrostatic interactions during the hydrolysis at alkaline pH, interpreting the surface potential data was developed. The theoretical predictions based on the idea of a random fragmentation of polymer molecules leading to the interfacial accumulation of charged insoluble products and solubilisation of small fragments describes well the experimental results. The reversibility of the hydrolysis/esterification reaction at acidic pH is taken into account.     

Rational construction of self-assembly azobenzene derivative monolayers with photoswitchable surface properties

hoto-responsive azobenzene (ABZ) derivatives with different end groups (R) were employed to construct selfassembled monolayers (SAMs) on silicon substrates. The SAMs based on hydrophilic (4-(4'-aminophenylazo) benzoic acid, ABZ-COOH) show excellent reversible photoswitching performance with a large contact angle change of 35° under optimized process.     

Identification of lectin activity in the hemolymph ofCastnia licus Drury,a sugar-cane giant borer (Lepidoptera-Castniidae)

Castnia licus Drury, in the larvae stage, is a major pest in the sugarcane industry of Northeastern Brazil (giant borer). A natural hemagglutination activity was detected in the larvae hemolymph of this insect and the activity have not been inhibited by tested carbohydrates. Fractionation with ammonium sulfate showed that the supernatant of the 15–30% fraction (S15-30%) had the lectin’s highest specific activity. Acidic and basic lectin activities were separated by chromatography on CM-cellulose; the fractionated molecular forms showed distinct electrophoresis mobilities.     

C60在卟啉衍生物Langmuir单层膜中的分散状态

研究了Ｃ６０在四苯基卟啉衍生物５，１０，１５，２０－四－对（癸酸α氧基）苯基卟啉（ＴＤＰＰ）及５，１０，１５，２０－四－对（乙酸α氧基）苯基卟啉（ＴＡＰＰ）单层膜中的分散状态。空气／Ｃｄ＾２＋水溶液界面上混合单层膜的π－Ａ等温线、混合膜与花生酸（ＡＡ）形成的交替多层膜的低角Ｘ射线衍射实验及混合单层ＬＢ膜的ＵＶ－Ｖｉｓ光谱表明，在ＴＤＰＰ／Ｃ６０（１：１）的混合单层膜中，Ｃ６０以单分子或（和）聚集体     

Surface phase behavior and microstructure of lipid/PEG-emulsifier monolayer-coated microbubbles

Langmuir trough methods and fluorescence microscopy were combined to investigate the phase behavior and microstructure of monolayer shells coating micron-scale bubbles (microbubbles) typically used in biomedical applications. The monolayer shell consisted of a homologous series of saturated acyl chain phospholipids and an emulsifier containing a single hydrophobic stearate chain and polyethylene glycol (PEG) head group. PEG-emulsifier was fully miscible with expanded phase lipids and phase separated from condensed phase lipids. Phase coexistence was observed in the form of dark condensed phase lipid domains surrounded by a sea of bright, emulsifier-rich expanded phase. A rich assortment of condensed phase area fractions and domain morphologies, including networks and other novel structures, were observed in each batch of microbubbles. Network domains were reproduced in Langmuir monolayers under conditions of heating–cooling followed by compression–expansion, as well as in microbubble shells that underwent surface flow with slight compression. Domain size decreased with increased cooling rate through the phase transition temperature, and domain branching increased with lipid acyl chain length at high cooling rates. Squeeze-out of the emulsifier at a surface pressure near 35 mN/m was indicated by a plateau in Langmuir isotherms and directly visualized with fluorescence microscopy, although collapse of the solid lipid domains occurred at much higher surface pressures. Compression of the monolayer past the PEG-emulsifier squeeze-out surface pressure resulted in a dark shell composed entirely of lipid. Under certain conditions, the PEG-emulsifier was reincorporated upon subsequent expansion. Factors that affect shell formation and evolution, as well as implications for the rational design of microbubbles in medical applications, are discussed.     

Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

A Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au–cystamine–glutaraldehyde–lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 ± 0.2) × 10⁴ M^− 1 for Jacalin–Fetuin and (4.5 ± 0.1) × 10² M^− 1 for ConA–maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin–carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides.     

Surface thermodynamics study of monolayers formed with heteroacid phospholipids of biological interest

The interaction of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), two of the major components in biological membranes, were investigated using the monolayer technique at the air–water interface. The pressure–area isotherms indicate that both phospholipids are miscible through all range of compositions. POPE–POPC form stable mixtures, with a minimum for the Gibbs energy of mixing at X_POPC = 0.4. A virial equation of state was fitted to the experimental values. Positive values found for the second virial coefficient indicate repulsion between POPC and POPE. The interaction parameter was evaluated which indicated that a corresponding decrease in the repulsion occurs when POPC molar fraction is low. This effect suggests the existence of hydrogen bonds between POPE and the water beneath the interface.     

Interaction of iron(III) tetrasulphonated phthalocyanine with cytochrome c oxidase apoprotein

A complex of bovine cytochrome c oxidase protein with iron(III) tetrasulphonated phthalocyanine in place of hemes has been prepared. Its structure and properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, potentiometric measurements and polypeptide fragments examination. The visible absorption spectrum of Fe^III-apo-oxidase shows the main intense peak at 657 nm and weaker one at 700 nm. Molecular weight estimation demonstrated that one mole of the complex includes three functional units of MW 130,000 per unit. Spectroscopic examination of dithionite reduced Fe^III-apo-oxidase suggests the open crevice structure of the subunits containing iron tetrasulphonated phthalocyanine which is supported by the results of circular dichroism studies. Deep conformational changes of the protein upon displacement of hemes a and a₃ are not reversed upon Fe^IIIL⁺ incorporation into the protein. The molar ratio of the protein to Fe^IIIL in the complex (MW 130,000) was found to be 1:2. In the reduced form Fe^IIIL-apo-oxidase reacts with CN⁻, N^-₃ imidazole and molecular oxygen. Oxygen binding is irreversible, which indicates that the oxygen adduct is not of the oxyhemoglobin type. Electrophoretic and gel filtration studies of the SDS-urea dissociation products of cytochrome c oxidase and its phthalocyanine derivative suggest that Fe^IIIL and hemes a and a₃ are located on the same polypeptide fragments of the protein. Fe^IIIL-apo-oxidase is reduced by ferrous cytochrome c in agreement with their midpoint potentials which are 315.5 and 260 mV, respectively. However, the rate of the reaction of Fe^IIIL-apo-oxidase with ferrous cytochrome c is markedly lower than that of the native cytochrome c oxidase suggesting different mechanisms for this process in both cases.     

Interaction of Egg-Sphingomyelin with DOPC in Langmuir Monolayers

Lipid rafts are a dynamic microdomain structure found in recent years, enriched in sphin-golipids, cholesterol and particular proteins. The change of structure and function of lipid rafts could result in many diseases. In this work, the monolayer miscibility behavior of mixed systems of Egg-Sphingomyelin (ESM) with 1, 2-dioleoyl-sn-glycero-3-phosphocholine was in-vestigated in terms of mean surface area per molecule and excess molecular area ΔA_ex at certain surface pressure, surface pressure and excess surface pressure Δπ_ex at certain mean molecular area. The stability and compressibility of the mixed monolayers was assessed by the parameters of surface excess Gibbs free energy ΔG_ex, excess Helmholtz energy ΔH_ex and elasticity. Thermodynamic analysis indicates ΔA_ex and Δπe_ex in the binary systems with positive deviations from the ideal behavior, suggesting repulsive interaction. The max-imum of ΔG_ex and ΔH_ex was at the molar fraction of ESM of 0.6, demonstrating the mixed monolayer was more unstable. The repulsive interaction induced phase separation in the monolayer     

Mixed monolayers composed of PC/PS/Chol. Interaction with opioid molecules

The miscibility of phosphatidylserine, phosphatidylcholine, and cholesterol in monolayers were studied. The influence of sodium and calcium ions in this system was determined. The compression isotherms of mixed monolayers of the above cited three components spread on subphases containing opiate molecules are elucidated. Moreover, the penetration kinetics of opiate molecules in these mixed monolayers was also recorded. The results show that the presence of cholesterol always lowers the penetration of opioid molecules; this effect is weaken for meperidine, the most hydrophobic of the molecules assayed.Abreviations PS phosphatidylserine - PC phosphatidylcholine - PI phosphatidylinositol - Chol cholesterol     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

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.     

Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.     

Action of Humicola lanuginosa lipase on long-chain lipid substrates: 1. Hydrolysis of monoolein monolayers

The hydrolysis of 1-monoolein (MO) monomolecular films by Humicola lanuginosa lipase (HLL) was followed by measuring the simultaneous decrease with time in the film area and the surface potential using a ‘zero order trough’ at constant surface pressure (Verger and de Haas, Chem. Phys. Lipids 10 (1973) 127). The decrease with time in the film area reflects both the reduction in the area per molecule as well as the solubilization of the substrate and the product molecules during the transformation of the substrate MO into product of oleic acid (OA). The surface potential changes were interpreted as the results of the accumulation at the interface of negatively charged OA. The surface concentration of OA transiently present at the interface was determined by the surface pressure and the surface potential measurements on the basis of a developed kinetic model. In the proposed model we have taken into account the product and substrate solubilization rates in the presence of β-cyclodextrin (β-CD) as well as the flux supplied progressively by the moving barrier from the reservoir to the reaction compartment in order to keep the constant surface pressure. Values of the global kinetic constant Q_m were obtained. The selective lipolytic product acceptor, β-CD, accelerated considerably the hydrolytic process.     

两性霉素B与鞘磷脂单分子层的相互作用

选取哺乳动物生物膜中的重要脂质分子鞘磷脂(SM)作为单分子膜的基本组分, 采用Langmuir-Blodgett(LB)膜技术研究了不同比例的两性霉素B/鞘磷脂单层膜的表面压力-平均分子面积(π-A)曲线以及基于π-A曲线的混合性分析, 同时通过原子力显微镜(AFM)研究了其表面形态的变化. 结果表明, 组分间的摩尔比和表面压力对混合单层膜稳定性、混合性以及分子间相互作用具有重要影响.