Molecular order and mobility within membranes made from fluorocarbon/fluorocarbon and fluorocarbon/hydrocarbon double-chain ether and amide phospholipids

The order of membranes formed by various ether- or amide-connected fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon double-chain phosphocholines which derive, respectively, from glycerol or diaminopropanol or serine, has been investigated using fluorescence anisotropy of TMA-DPH, and compared to that of membranes made from fluorinated or conventional ester-connected phosphatidylcholines. In the gel phase, membrane order is almost not affected by the molecular structure of the phospholipid. By contrast, in the liquid-crystalline state, it decreases on going (i) from membranes made from the fluorocarbon/fluorocarbon phospholipids to those formed by mixed fluorocarbon/hydrocarbon phospholipids, then to conventional ones, (the rigidifying effects of the fluorinated tails, their number and length is thus becoming noticeable) or (ii) from membranes made from fluorinated diamidopropano- or diamidoserinophosphocholines to those made from ester- or ether-connected glycerophosphocholines. The ester- versus ether-connection or 1,2- versus 1,3-connection of the chains on glycerol have no detectable impact on membrane order.     

Toward a second generation of fluorocarbon emulsions. Methodologies: synthesis of fluorocarbons, formulation, preparation and control of emulsions

The fluorocarbons to be used as intravascular gas carriers may be prepared: by fluorination of hydrocarbons, by means of gaseous fluorine or of metallic fluorides or by electrolysis in anhydrous hydrogen fluoride; by selective synthesis from small fluorinated molecules. Selective synthesis seems preferable because of the high purity of the compounds thus obtained, allowing fairly simple purification and detoxification procedures. The adequacy of fluorocarbons for intravascular use (limited by the values of some of their crucial physicochemical properties such as vapour pressure, gas dissolving power, viscosity) was found dependent of their molecular weight range, preferably between 460 and 540 dalton. Ionic balance, osmolarity, viscosity, mean particle size and particle size distribution are among the most important criteria to be respected in the preparation of biocompatible emulsions. Several physicochemical methods were developed for selecting the best ingredients and the best emulsification procedures and for evaluating the stability of the emulsions to storage and to thermal stress. The combination of these physicochemical measurements may be used as a methodology for the optimisation and control of fluorocarbon emulsions as well as a way of evaluating new ingredients and formulations.     

Fluorocarbon/lecithin emulsions: identification of EYP-coated fluorocarbon droplets and fluorocarbon-empty vesicles by freeze-fracture electron microscopy

Freeze-fracture has been used to examine perfluorodecalin/egg yolk phospholipid emulsions (70:8, w/v%) destined to be used as injectable oxygen carriers. The fluorocarbon displays a specific granular aspect upon freeze-fracture which makes it readily recognizable and allows the distinction between two populations of objects on the micrographs: fluorocarbon droplets and water-filled lipid vesicles.     

Phase behavior of fluorocarbon and hydrocarbon double-chain hydroxylated and galactosylated amphiphiles and bolaamphiphiles. Long-term shelf-stability of their liposomes.

This paper describes the morphological characterization, by freeze-fracture electron microscopy, and the thermotropic phase behavior, by differential scanning calorimetry and/or X-ray scattering, of aqueous dispersions of various hydroxylated and galactosylated double-chain amphiphiles and bolaamphiphiles, several of them containing one or two hydrophobic fluorocarbon chains. Colloidal systems are observed in water with the hydroxylated hydrocarbon or fluorocarbon bolaamphiphiles only when they are dispersed with a co-amphiphile such as rac-1,2-dimyristoylphosphatidylcholine (DMPC) or rac-1,2-distearoylphosphatidylcholine (DSPC). Liposomes are formed providing the relative content of bolaamphiphiles does not exceed 20% mol. Most of these liposomes can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution. The hydrocarbon galactosylated bolaamphiphile HO[C24][C12]Gal forms in water a lamellar phase (the gel to liquid-crystal phase transition is complete at 45 degrees C) and a Im3m cubic phase above 47 degrees C. The fluorocarbon HO[C24][F6C5]Gal analog displays a more complex and metastable phase behavior. The fluorinated non-bolaform galactosylated [F8C7][C16]AEGal and SerGal amphiphiles form lamellar phases in water. Low amounts (10% molar ratio) of the HO[C24][F6C5]Gal or HO[C24][C12]Gal bolaamphiphiles or of the single-headed [F8C7][C16]AEGal improve substantially the shelf-stability of reference phospholipon/cholesterol 2/1 liposomes. These liposomes when co-formulated with a single-headed amphiphile from the SerGal series are by far less stable.     

Introducing a new Element - Fluorine -Into the Liposomal Membrane

Abstract

Amphiphiles with fluorinated hydrophobic tails constitute new, distinctively different components for membranes, liposomes, tubules and other self-aggregated supramolecular systems. Fluorinated liposomes (F-liposomes) can also be obtained from combinations of standard phospholipids with mixed fluorocarbon-hydrocarbon amphiphiles. The fluorinated moieties are considerably more hydrophobic than their hydrocarbon counterparts; they have also a larger cross section, are more rigid, and are lipophobic as well. As a result, fluorinated amphiphiles show enhanced propensity to self-assemble, lead to increased membrane ordering and stability, and their stacking creates a teflon-like repellent film within the liposomal membrane, which can significantly increase drug encapsulation stability. The fluorinated chains also impact on behavior in biological media and particle recognition, as exemplified by reduced hemolytic and detergent activity, prolonged intravascular persistence, or slower enzymatic hydrolysis of phospholipids.     

Cholesterol oxidation on fluorocarbon emulsion surface leads to the formation of 7-peroxycholesterol

Formation of biologically active oxidized derivatives of cholesterol as a result of its oxidation on the surface of fluorocarbon emulsions was studied. A single product of cholesterol oxidation, 7-peroxycholesterol, was found. It was shown that 7-peroxycholesterol and its derivative 7-keto-cholesterol inhibit the rosette formation between human T-lymphocytes and sheep erythrocytes. These substances exert a strong cytostatic action on the growth of procaryotic and eucaryotic cell cultures. Thus, oxidative modification of blood plasma components on the surface of fluorocarbon emulsion particles with the formation of highly active compounds must be taken into account when using the fluorocarbon emulsions in medicine.     

Incorporation of semi-fluorinated alkanes in the bilayer of small unilamellar vesicles of phosphatidylserine: impact on fusion kinetics

Semi-fluorinated alkanes C(n)F(2n+1)C(m)H(2m+1) (FnHm) can be co-dispersed with standard phospholipids to form 'fluorinated' vesicles, i.e. vesicles with an internal fluorinated film within their bilayer membrane. This paper reports the effect of the presence of such FnHm diblocks in phosphatidylserine (PS)-based small unilamellar vesicles (SUVs) on their kinetics of fusion. Fusion was induced by calcium ions and monitored by the terbium/dipicolinic acid assay. The diblocks were composed of a 10-carbon long linear hydrocarbon segment and of a linear fluorocarbon segment of four, six or eight carbon atoms. We found that the incorporation of FnHm in the PS membrane considerably modifies the kinetics of the process of fusion, with Ca(2+) concentration having a much more limited effect on the fluorinated vesicles. Both the rates of fusion and the rates of release of the internal content, as evaluated by the release of 5,6-carboxyfluorescein, were much lower for the fluorinated SUVs than for those based on phosphatidylserine alone, the highest effect being obtained for F6H10 with a 10 times slower rate of fusion and a 40-fold reduction in the release of content. FnHm molecules are proposed to have a dual action: by hindering fusion and release by creating an inert, hydrophobic and lipophobic fluorinated film in the core of the membrane, and by stabilizing the membrane by increasing van der Waals interactions in the hydrocarbon region.     

Microvascular gas embolization clearance following perfluorocarbon administration.

Effective treatment of vascular gas embolism may be possible with emulsified fluorocarbon compounds. We tested the hypothesis that a fluorocarbon emulsion delivered before gas embolization would enhance bubble motion through the vasculature, favoring more rapid clearance. Air microbubbles were injected into the rat cremaster microcirculation in six groups of rats receiving Perftoran, an emulsified fluorocarbon, or saline immediately before, 2 h before, or after bubble injection. Embolism dimensions and dynamics were observed by using intravital microscopy. Surface area at lodging was equal between groups. Bubbles having smaller volume embolized smaller diameter vessels in the Perftoran pretreatment groups. A higher incidence of bubble dislodgement and larger distal displacement occurred in these two groups, with a 36% decrease in the time to bubble clearance and restoration of blood flow. Intravascular emulsified fluorocarbon administration before gas embolization affected initial bubble conformation, increased bubble dislodgement, and resulted in bubble displacement further into the periphery of the microcirculation. These dynamic events did not occur if embolization preceded fluorocarbon administration.     

The plasticins: membrane adsorption, lipid disorders, and biological activity

The present study investigates the relationships between structural polymorphism, adsorption onto membrane mimetic support, lipid disturbance, and biological activity of bactericidal 23-residue, glycine-leucine-rich dermaseptin orthologues from the Phyllomedusinae frog skin, the "plasticins". Biological activities were evaluated using the membrane models DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) for prokaryotic membranes and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) for eukaryotic membranes. We performed a conformational analysis of plasticins by molecular simulations and spectroscopic methods and analyzed phospholipid perturbations by infrared spectroscopy. Adsorption onto synthetic model membranes was quantified by surface plasmon resonance. Biological assays including antimicrobial and membrane potential-dissipating activities, together with hemolytic tests and imaging analysis of cytotoxicity, were carried out to clarify the peptide-membrane interactions. Two major groups were distinguished: (i) Neutral plasticins revealed the presence of strong beta-structures with the zwitterionic or anionic phospholipid vesicles. They were weakly adsorbed in the range of antibacterial activity concentrations (micromolar). Nevertheless, for millimolar concentrations, they caused perturbations at the interface peptide-DMPG vesicles and in the bilayer alkyl chains, suggesting insertion into bacterial membranes. (ii) Cationic plasticins revealed multiple conformational transitions, including destabilized helix states, beta-structures, and disordered states. Peptide-lipid complex densities depended on hydrophobic bond strengths. The most soluble cationic plasticins were strongly adsorbed, with stable peptide-lipid interactions inducing noticeable perturbations of bilayer alkyl chains, pointing out possible insertion into bacterial membranes. In contrast, cytotoxic plasticins were less adsorbed, with less stable peptide-lipid interactions causing membrane dehydration, formation of peptide-membrane hydrogen bonds, and little disturbances of lipid alkyl chains. These characteristics could be compatible with their putative action on intracellular targets leading to apoptosis.     

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.     

The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure

Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains.     

Seeing spots: complex phase behavior in simple membranes

Liquid domains in model lipid bilayers are frequently studied as models of raft domains in cell plasma membranes. Micron-scale liquid domains are easily produced in vesicles composed of ternary mixtures of a high melting temperature lipid, a low melting temperature lipid, and cholesterol. Here, we describe the rich phase behavior observed in binary and ternary systems. We then discuss experimental challenges inherent in mapping phase diagrams of even simple lipid systems. For example, miscibility behavior varies with lipid type, lipid ratio, lipid oxidation, and level of impurity. Liquid domains are often circular, but can become noncircular when membranes are near critical points. Finally, we reflect on applications of phase diagrams in model systems to rafts in cell membranes.     

Fluorinated and hemifluorinated surfactants derived from maltose: synthesis and application to handling membrane proteins in aqueous solution

Two novel fluorinated surfactants have been obtained by grafting by radical reaction either a fluorocarbon or an ethyl end-capped fluorocarbon chain onto the double bond of beta-D-allyl maltose. The two compounds thus obtained form polydisperse aggregates in water. They can keep membrane proteins water-soluble, but the protein/surfactant complexes are polydisperse, which affects neither the native state nor the stability of the proteins.     

Nonequilibrium behavior in supported lipid membranes containing cholesterol

We investigate lateral organization of lipid domains in vesicles versus supported membranes and monolayers. The lipid mixtures used are predominantly DOPC/DPPC/Chol and DOPC/BSM/Chol, which have been previously shown to produce coexisting liquid phases in vesicles and monolayers. In a monolayer at an air-water interface, these lipids have miscibility transition pressures of approximately 12-15 mN/m, which can rise to 32 mN/m if the monolayer is exposed to air. Lipid monolayers can be transferred by Langmuir-Sch?fer deposition onto either silanized glass or existing Langmuir-Blodgett supported monolayers. Micron-scale domains are present in the transferred lipids only if they were present in the original monolayer before deposition. This result is valid for transfers at 32 mN/m and also at lower pressures. Domains transferred to glass supports differ from liquid domains in vesicles because they are static, do not align in registration across leaflets, and do not reappear after temperature is cycled. Similar static domains are found for vesicles ruptured onto glass surfaces. Although supported membranes on glass capture some aspects of vesicles in equilibrium (e.g., gel-liquid transition temperatures and diffusion rates of individual lipids), the collective behavior of lipids in large liquid domains is poorly reproduced.     

Liposomes from polymerizable phospholipids

The synthesis and characterization of a great variety of single and double chain phospholipids containing the diacetylene and butadiene moiety is described. These substances can be dispersed in water by ultrasonication and the resulting vesicles can be photopolymerized with the retention of their original structure. Absorption spectra of the polymerized diacetylenic lipids show significant differences depending on the molecular structure of the monomers. By the polymerization reaction, the gel to liquid crystalline phase transition is suppressed, which does not correspond to the properties of biological membranes. Evidence for enhanced stability of polymerized vesicles is given by treatment with ethanol and detergents showing that trapped markers are released to a much smaller extent than in the case of unpolymerized vesicles. Diacetylenic lipids show a pronounced hysteresis of the phase transition. If the membrane of supercooled vesicles crystallizes, all trapped marker is released within seconds. Possibilities for overcoming this extreme rigidity of the membranes are discussed.     

The effect of long-chain bases on polysialic acid-mediated membrane interactions

Negatively-charged polysialic acid (polySia) chains are usually membrane-bound and are often expressed on the surface of neuroinvasive bacterial cells, neural cells, and tumor cells. PolySia can mediate both repulsive and attractive cis interactions between membrane components, and trans interactions between membranes. Positively-charged long-chain bases are widely present in cells, are often localized in membranes and can function as bioactive lipids. Here we use Langmuir monolayer technique, fluorescence spectroscopy and electron microscopy of lipid vesicles to study the role of a simple long-chain base, octadecylamine (ODA), in both cis and trans interactions mediated by polySia in model membranes composed of ODA and dioleoylphospatidycholine (DOPC). When added free to an aqueous solution, polySia increases the collapse pressure of ODA/DOPC monolayers, reduces the effect of ODA on the limiting molecular area, inverses the values of excess area per molecule and of excess free energy of mixing from positive to negative, and induces fusion of ODA/DOPC vesicles. These results suggest that a polySia chain can act as a multi-bridge that mediates cis interactions between different components of a lipid membrane, disrupts membrane aggregates, and mediates trans interactions between lipids in apposing membranes. These observations imply that polySia in cellular systems can act in a similar way.     

Effects of visible and UV light on the characteristics and properties of crude oil-in-water (O/W) emulsions

The effects of visible and UV light on the characteristics and properties of Prudhoe Bay (PB) and South Louisiana (SL) emulsions were investigated to better understand the role of sunlight on the fate of spilled crude oils that form emulsions with a dispersant in the aquatic environment. Before irradiation, crude oil emulsions showed the presence of dispersed crude oil micelles in a continuous water phase and crude oil components floating on the surface. The crude oil micelles decreased in size with irradiation, but emulsions retained their high degree of polydispersity. UV irradiation reduced the stability of emulsions more effectively than visible light. The reduction of micelles size caused the viscosity of emulsions to increase and melting point to decrease. Further, irradiation increased acid concentrations and induced ion formation which lowered the pH and increased the conductivity of emulsions, respectively. Ni and Fe in PB emulsions were extracted from crude oil with UV irradiation, which may provide an efficient process for metal removal. The emulsions were stable toward freeze/thaw cycles and their melting temperatures generally decreased with irradiation. Evidence of ˙OH production existed when emulsions were exposed to UV but not to visible light. The presence of H(2)O(2) enhanced the photodegradation of crude oil. Overall, the changes in emulsion properties were attributed to direct photodegradation and photooxidation of crude oil components.     

Effect of organic solvents on oxygen mass transfer in multiphase systems: Application to bioreactors in environmental protection

The absorption of oxygen in aqueous–organic solvent emulsions was studied in a laboratory-scale bubble reactor at a constant gas flow rate. The organic and the gas phases were dispersed in the continuous aqueous phase. Volumetric mass transfer coefficients (k_La) of oxygen between air and water were measured experimentally using a dynamic method. It was assumed that the gas phase contacts preferentially the water phase. It was found that addition of silicone oils hinders oxygen mass transfer compared to air–water systems whereas the addition of decane, hexadecane and perfluorocarbon PFC40 has no significant influence. By and large, the results show that, for experimental conditions (organic liquid hold-up ≤10% and solubility ratio ≤10), the k_La values of oxygen determined in binary air–water systems can be used for multiphase (gas–liquid–liquid) reactor design with applications in environmental protection (water and air treatment processes).     

Size analysis and stability study of lipid vesicles by high-performance gel exclusion chromatography, turbidity, and dynamic light scattering

Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 micron or by filtration through 0.8-micron cellulosic or 0.22-micron polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 micron, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF "tortuous" ones being less efficient than "straight bores" polycarbonate ones; (ii) mixed EPC/EPA REVs sequentially extruded down through 0.2-micron polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC/EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC/EPA (mole ratio: 91/9) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.     

Stability of microbubbles prepared by co-axial electrohydrodynamic atomisation

Previous studies have indicated that microbubbles prepared by co-axial electrohydrodynamic atomisation (CEHDA) are less stable than those prepared by other methods such as sonication and microfluidic techniques. The aim of this investigation was to determine the reasons for this observation and how this might be addressed in future work. Microbubbles were prepared by CEHDA using (i) a glycerol–air system, (ii) a glycerol–Tween 80–air system and (iii) a glycerol–zirconia–air system and also by simple agitation of (i) and (ii), in order to compare the effect upon the dissolution rate of microbubbles of different materials and processing methods. Both theoretical examination and the experimental results indicated that all three quantities were important in controlling the rate of microbubble dissolution, namely: surface tension at the gas/liquid interface, the effective diffusivity of gas through this interface and the initial concentration of gas dissolved in the surrounding liquid. However, it was the difference in gas concentration in the surrounding liquid that was indicated as the primary reason for the differences in stability observed with different processing methods. It was concluded, therefore, that improved stability could be achieved for microbubbles prepared using CEHDA by saturating the collecting fluid with gas and/or maintaining a high concentration of microbubbles during collection.