Photosensitive liposomes as 'cages' for laser-triggered solute delivery: the effect of bilayer cholesterol on kinetics of solute release

Liposomes containing acyl chains incorporating azobenzene chromophores have been investigated as potential 'caging' agents for fast solute release. On photolysis, trapped marker dye can be released from gel-phase liposomes within milliseconds. Solute release is markedly sensitive to the presence of cholesterol in the bilayer. Phospholipids bearing one saturated acyl chain and an azobenzene-substituted chain are ineffective as sensitisers unless cholesterol is present, while doubly substituted phospholipids sensitise release in its absence. Cholesterol markedly affects the temperature profile of solute release depending on the host phospholipid chain length. Solute release is not seen for lipid hosts with unsaturated acyl chains.     

Implications of a non-lamellar lipid phase for the tight junction stability. Part I: Influence of basic amino acids, pH and protamine on the bilayer-hexagonal II phase behaviour of PS-containing PE membranes.

Inverted lipid micelles have been proposed, among other biological functions, to constitute the structural basis of the so-called tight junctions, a special cell cell contact found in epithelia and endothelial, which act as a barrier for the paracellular solute passage. As a model system for the opening and closing of this gate, we investigated the formation of the inverted hexagonal phase (HII phase) in lipid bilayer systems consisting of egg phosphatidylethanolamine (egg PE) and mixed egg PE/bovine brain phosphatidylserine (BBPS) membranes. The formation of the HII phase was modulated by Ca2+ ions, pH, basic amino acids and protamine. The lamellar-HII phase transition temperature TH of pure egg PE membranes at pH 7.0 was lowered with increasing Ca2+ concentration. This effect was attenuated by the presence of 50 mM lysine methyl ester. In the mixed lipid system, this effect was also observed, but even more pronounced. However this effect could be compensated for by raising the Ca2+ concentration from 2 to 10 mM. This was not observed in the pure PE system. In the absence of Ca2+, lysine methyl ester and protamine lowered TH in both monocomponent and mixed lipid systems, whereas lysine caused the opposite effect. The pH-dependence of mixed lipid systems, which were investigated up to a BBPS content of 20 mol%, clearly shows that increasing PS content stabilizes the lamellar phase even at low pH. The results obtained with model membranes are discussed with respect to biological implications of the lamellar-HII phase transition for the modulation of tight junction stability.     

Alamethicin incorporation in lipid bilayers: a thermodynamic study

Interaction of the peptide antibiotic alamethicin with phospholipid vesicles has been monitored by changes in its circular dichroic and fluorescent properties. The data are consistent with an incorporation of the peptide in the lipid bilayer. Aggregation of alamethicin in the membrane phase is evident from a characteristic concentration dependence of the incorporation, reflecting the existence of a critical concentration. The data can be fully understood in terms of a theoretical approach that includes aggregation and thermodynamic nonideality. Thermodynamic parameters of the peptide-lipid interaction have been evaluated under a variety of conditions of temperature, ionic strength, and lipid type (saturated and unsaturated fatty acid chains). From the results obtained in this study, one can extrapolate to the incorporation behavior of alamethicin at low concentrations, as they are typical for measurements of conductance across planar lipid films. This leads to a simple explanation of the voltage-gating mechanism of alamethicin in a straightforward way.     

Thermodynamic elucidation of solute-induced lipid interdigitation phase: lipid interactions with hydrophobic versus amphipathic species.

Comparative thermodynamic studies on the interactions of aqueous dispersions of dipalmitoyl phosphatidylcholine (DPPC) bilayer vesicles with hydrophobic and amphipathic species were conducted to elucidate the nature of the solute-induced interdigitated lipid phase. Cyclohexanol, a strong hydrophobic species, lowers the temperature (tm) of the lipid main phase transition from the gel to the liquid-crystalline phase. Unlike ethanol (an amphipathic species), as reported previously, cyclohexanol does not exert a biphasic effect on tm (lowering tm at lower concentrations and raising tm at higher concentrations). At cyclohexanol greater than or equal to 15.4 mg/ml or 0.154 M, the thermogram of DPPC vesicles exhibits a small transition adjacent to the main phase transition but at a lower temperature. In contrast, ethanol does not promote such a small transition. Furthermore, the enthalpy (delta H) of the transition is increased in the presence of cyclohexanol. The sign of the enthalpy change (delta H-delta Ho) is positive and that of the free energy change (delta G-delta Go) is negative, a characteristic of solute-solute hydrophobic interaction. In contrast, DPPC bilayer vesicles exhibit both (delta H-delta Ho) and (delta G-delta Go) greater than 0 in the presence of ethanol in a concentration range where lipid vesicles exist in an interdigitated phase. To support the above distinct thermodynamic observations, fluorescence steady-state polarization (P) measurements were also performed. At the temperature below tm, the value of P decreases as cyclohexanol concentration increases, while a biphasic effect on P was found in the presence of ethanol. These findings support the postulation that the solute-induced interdigitated lipid phase requires the solute molecule to be amphipathic in nature.     

Photoisomerisable cholesterol derivatives as photo-trigger of liposomes: effect of lipid polarity, temperature, incorporation ratio, and cholesterol

Three cholesterol derivatives containing an azobenzene moiety with different polarities were designed and synthesized (AB lipids 1 to 3). The effects of structure, temperature and incorporation ratio on liposomes were studied, with the results showing that the polarity in 4-substituent and in some cases, 4'-substituent may be important for their incorporation feasibility and photoisomerizability in liposomes. Liposomes incorporated with AB lipid 3 could release multi-pulsatilely upon UV and visible light irradiation both in gel state and liquid crystal state of liposomes. An increase in the incorporation ratio of AB lipid 3 enhanced the amount of drug released greatly. Unlike other azobenzene photo-triggers reported, AB lipid 3 did not increase the spontaneous release of liposomes. Furthermore, cholesterol suppressed the spontaneous release of liposomes.     

Synergistic effects of temperature and plant quality,on development time,size and lipid in Eccritotarsus eichhorniae

Body size is an important biotic factor in evolutionary ecology, since it affects all aspects of insect physiology, life history and, consequently, fitness in ectothermic insects and how species adapt with their environment. It has been linked to temperature, with lower temperatures resulting in larger size. In this study, we tested the combined impact of temperature and plant quality on the body size, and development time from egg to adult of Eccritotarsus eichhorniae (Hemiptera: Miridae), an herbivorous insect used as a biological control agent against the invasive aquatic weed, water hyacinth Eichhornia crassipes (Pontederiaceae). We quantified insect size in individuals exposed to three temperatures (20, 25 and 30°C) combined with three qualities of host plant (high, medium and low) by calculating development time and measuring four traits: tibia length, forewing length, dry body mass and lipid content, and we also determined the wing loading index. The development time, dry body mass and lipid content decreased linearly with increasing temperature and decreasing plant quality. The decrease in size was the greatest when high temperature interacted with low plant quality. Smaller individuals had proportionately less lipid content. Wing loading decreased significantly with lower quality of host plant, resulting in individuals likely to have theoretically higher flight ability. The results support the temperature-size rule (TSR) and that plant quality could influence the relationship between development time and the TSR. Results also provide novel evidence for a possible food quality-size rule for both sexes.     

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.     

Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 microM and 19 microM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 microM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (<5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other "single-chain" lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

Protective effect of L-arginine against lipid peroxidation in goat epididymal spermatozoa

L-arginine plays an important role in physiology of spermatozoa and is shown to enhance the metabolism of these cells. We report here the effect of L-arginine on membrane lipid peroxidation of goat epididymal spermatozoa. Both natural peroxidation as well as that induced by UV radiation, freezing and oxidizing agents have been studied. Irrespective of the nature of induction of peroxidation, L-arginine reduces the extent of lipid peroxidation in a concentration dependent manner. Both L-arginine and alpha-tocopherol act synergistically in protecting against lipid peroxidation induced by the above methods. Thus, in order to provide protection against lipid peroxidation, L-arginine may be added in media used to preserve spermatozoa.     

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide

We have reported here on the structural polymorphism of lipid A, the "endotoxic principle" of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg2+] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70 degrees C. These data were correlated with measurements of the beta----alpha phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges--in the absence of Mg2(+)-from 45 degrees C at high to 56 degrees C at low water content, and-at an equimolar content of Mg2(+)-from 52 degrees C at high to 59 degrees C at low water concentrations. In the gel phase-in which the lipid A acyl chains are more disordered than those from saturated phospholipids-cubic phases are adopted at high water content (greater than 60%) and at high [lipid A]:[Mg2+] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal HII state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg2+ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.     

Interactions between anaesthetics and lipid mixtures amines

The efefct of a number of amine anaesthetics related to procaine on the temperature of lipid phase transitions has been studied using chlorophyll α as a fluorescence probe. The amines cause a reduction in the temperature of the phase transition of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanol-amine and of mixtures of these lipids. The binding of charged amines causes a build up of positive charge on the membranes, limiting the binding. Incorporation of negative charge into the lipid bilayers causes a considerable increase in the binding of the charged amines, and the effect is reversed by addition of Ca²⁺. Anaesthesia is suggested to arise from an increase in the proportion of lipid in the liquid crystalline phase, resulting in a conformational change in the sodium channel. Effects of the tertiary amines on nerve conduction can be understood if the negatively charged lipid in the membrane is concentrated around the sodium channel: positively charged anaesthetics will have a greater effect when applied to the inside of a nerve because of the low Ca²⁺ concentration inside the nerve.     

Bleaching in coral reef anthozoans: effects of irradiance,ultraviolet radiation,and temperature on the activities of protective enzymes against active oxygen

Recent widespread bleaching of coral reef anthozoans has been observed on the Great Barrier Reef, the Pacific coast of Panama, and in the Caribbean Sea. Bleaching events have been correlated with anomalously high sea surface temperatures which are presumed to cause the expulsion of zooxanthellae from their hosts. Our experimental results show that increases in temperature significantly reduce the total number of zooxanthellae per polyp. At the same time temperature, irradiance (photosynthetically active radiation=PAR), and ultraviolet radiation (UV) independently increase the activities of the enzymes superoxide dismutase, catalase, and ascorbate peroxidase within the zooxanthellae of the zoanthid Palythoa caribaeorum. Enzyme activities within the host are only suggestive of similar changes. These enzymes are responsible for detoxifying active forms of oxygen, and their elevated activities indirectly indicate an increase in the production of active oxygen species by increases in these environmental factors. Historically, bleaching has been attributed to changes in temperature, salinity, and UV. Increases in temperature or highly energetic UV radiation can increase the flux of active forms of oxygen, particularly at the elevated oxygen concentrations that prevail in the tissues during photosynthesis, with oxygen toxicity potentially mediating the bleaching event. Additionally, the concentration of UV absorbing compounds within the symbiosis is inversely related to temperature, potentially increasing exposure of the host and zooxanthellae to the direct effects of UV.     

Effect of lipid physical state on the rate of peroxidation of liposomes.

The effect of cholesterol on the rate of peroxidation of arachidonic acid and 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) in dimyristoylphosphatidylcholine (DMPC) liposomes was examined above and below the phase transition temperature (Tm) of the lipid. The rate of peroxidation of arachidonic acid was more rapid below the phase transition temperature of the host lipid. At a temperature below the Tm (4 degrees C), increasing concentrations of cholesterol reduced the rate of peroxidation of arachidonic acid as judged by the production of thiobarbituric acid reactive substances. Above Tm (37 degrees C), cholesterol increased the rate of peroxidation of the fatty acid. Similarly, PAPC was peroxidized more rapidly at 4 degrees C than at 37 degrees C. However, cholesterol had little effect on the rate of peroxidation of PAPC at 4 degrees C. The rate of peroxidation of arachidonic acid was related to the lipid bilayer fluidity as judged by fluorescence anisotropy measurements of diphenylhexatriene. The rate of peroxidation increased slowly with increasing rigidity of the probe environment when the bilayer was relatively fluid and more rapidly as the environment became more rigid. The increase in the rate of peroxidation of arachidonic acid in the less fluid host lipid was unrelated to differences in iron binding or to transfer of arachidonic acid to the aqueous phase. Decreasing the concentration of arachidonic acid in DMPC to less than 2 mol% dramatically decreased the rate of peroxidation at 4 degrees C, suggesting that formation of clusters of fatty acids at 4 degrees C is required for rapid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, approximately 40-45 degrees C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer "frustration" which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a "frustrated" bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex-membrane interactions and DNA release energetics.     

Real-time measurement of solute partitioning to lipid monolayers

The interaction of a peripheral protein with a lipid-water interface can show a pronounced dependence on the composition and two-dimensional packing density of the lipids that comprise the interface. We report a novel optical method for measuring the adsorption of macromolecules, such as proteins and nucleic acids, and smaller solutes, such as drugs, to lipid monolayers at the gas-liquid interface. Using fluorescence emission from proteins and a small molecule, we demonstrate that the emissions from these solutes when in the aqueous phase and when associated with the monolayer can be temporally separated. Such separation allows measurement of the extent of solute adsorption, spectral characterization of the adsorbed solute, and characterization of lipid organization using adsorption kinetics. The method does not require, but is compatible with, the solute having different spectral properties in the bulk and surface phases. Indeed, if optical signals from adsorbed and soluble solute are the same or their relationship is known, absolute surface excess of adsorbed solute can be calculated without independent calibration. With appropriate instrumental configuration, the method should be adaptable for screening solutes for interaction with planar monolayers having both well-defined composition and adjustable lipid packing density.     

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.     

Interdigitated hydrocarbon chain packing causes the biphasic transition behavior in lipid/alcohol suspensions

It has been shown recently by Rowe ((1983) Biochemistry 22, 3299-3305) that ethanol has a 'biphasic' effect on the transition temperature (Tm) of phosphatidylcholine bilayers, reducing Tm at low concentrations but increasing Tm at high concentrations. Our X-ray diffraction data show that this reversal of Tm is a consequence of the induction of an unusual gel phase, where the lipid hydrocarbon chains from apposing monolayers fully interpenetrate or interdigitate. The properties of this interdigitated phase also explain the lipid chain length dependence of the reversal in the Tm versus ethanol concentration curves and the narrow width of the transition at high ethanol concentrations, as well as spectroscopic and calorimetric data from lipid suspensions containing other drugs such as methanol, benzyl alcohol, phenyl ethanol, and chlorpromazine.