Lipid bilayer permeation by neutral aluminum citrate and by three alpha-hydroxy carboxylic acids

Several groups have proposed that aluminum (Al) may permeate biological membranes as a neutral complex with citrate. We tested this hypothesis by measuring aluminum citrate flux across unilamellar phospholipid vesicles (liposomes). Results from two independent procedures show that lipid bilayer permeation by the neutral aluminum-citrate complex is slow (P approximately equal to 1 x 10(-11) cm.s-1). We then compared aluminum-citrate permeation with permeation by a series of alpha-hydroxy carboxylic acids and by trimethylcitrate. This comparison showed that the aluminum-citrate flux is limited by diffusion across the water/lipid interface. This is due to hydrogen bonding between water and the citrate carboxyl groups, and by hydration of the bound metal in the aqueous phase. By analogy with citric acid, steric hindrance of diffusion within the bilayer does not affect the permeation rate of aluminum citrate. Elevated tissue levels of Al in subjects fed a diet supplemented with citric acid and Al(OH)3 cannot be explained by lipid bilayer permeation of the neutral complex.     

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.     

Effect of the lipid phase transition on the kinetics of H+/OH− diffusion across phosphatidic acid bilayers

The kinetics of H⁺/OH^? diffusion across dimyristoyl phosphatidic acid bilayer membranes was measured by following the absorbance of the pH-sensitive indicator Cresol red ($\text{o-}\text{cresolsulfonphthalein}$) entrapped in single lamellar vesicles after rapidly changing the external pH in a stopped-flow apparatus. The H⁺/OH^?-permeability coefficient was found to be in the 10^?5 to 10^?3 cm·s^?1 range. The lipid phase transition has a strong influence on the permeation kinetics as the permeability coefficients in the liquid-crystalline phase are drastically higher. The permeability shows no maximum at the phase transition temperature as is the case for other ions, but displays a similar temperature dependence as water permeation. This is also reflected in the high activation energy of approx. 20 kcal/mol and supports the hypothesis (Nichols, J.W. and Deamer, D.W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2038–2042) of H⁺/OH^? permeation via hydrogen bonded water molecules. A second slower kinetic phase is also observed, where the permeation is obviously controlled by counterion diffusion. The temperature dependence of this slow process displays the for ion diffusion characteristic maximum in the permeability at the phase-transition temperature.     

Structure of the skin barrier and its modulation by vesicular formulations

The natural function of the skin is to protect the body from unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and elastic vesicles.     

Galactose oxidase action on galactose containing glycolipids--a fluorescence method

Features that alter the glycolipid sugar headgroup accessibility at the membrane interface have been studied in bilayer lipid model vesicles using a fluorescence technique with the enzyme galactose oxidase. The effects on oxidation caused by variation in the hydrophobic moiety of galactosylceramide or the membrane environment for galactosylceramide, monogalactosyldiacylglycerol and digalactosyldiacylglycerol were studied. For this study we combined the galactose oxidase method for determining the oxidizability of galactose containing glycolipids, and the fluorescence method for determining enzymatic hydrogen peroxide production. Exposed galactose residues with a free hydroxymethyl group at position 6 in the headgroup of glycolipids were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). Amplex Red reacts with hydrogen peroxide in the presence of horseradish peroxidase with a 1:1 stoichiometry to form resorufin. With this coupled enzyme approach it is also possible to determine the galactolipid transbilayer membrane distribution (inside-outside) in bilayer vesicles.     

Preparation,Characterization and99mTC Radiolabeling of Polyethylene glycol - coated Vesicles

Abstract

Poly (ethylene glycol) - coated lipid vesicles of average diameter ～200nm containing glutathione (GSH) were prepared by extrusion under pressure through polycarbonate filters, external GSH removed by chromatography on Sephadex G50, and the vesicles concentrated to a final lipid concentration of ～ 60 mM using Millipore 10,000 NMWL low-protein-binding regenerated cellulose ultrafiltration units. Vesicles were subsequently radiolabeled with diagnostic quantities of technetium-99m (^99mTc) - 740 Mbq (20 mCi) - using the ability of non-reduced hexamethylpropylene-amineoxime (HM-PAO) in the presence of tin (Sn) to transport ^99mTc across the lipid bilayer. The use of Millipore filters represents a gentle, relatively rapid and simple method to concentrate lipid vesicles without causing loss of entrapped marker or affecting the efficiency of subsequent radiolabeling. Once external GSH was removed, vesicles retained >97% of their contents over a period of ～ 4 months when stored at 4°C. The radiolabeling of the lipid vesicles using HM-PAO was unaffected by temperature, lipid composition or lipid phase state, but was critically dependent upon the ratios of HM-PAO, Sn and ^99mTc.     

Sorbitan ester niosomes for topical delivery of rofecoxib

The aim of the present investigation is to encapsulate rofecoxib in niosomes and incorporate the prepared niosomes into dermal gel base for sustained therapeutic action. Niosomes were prepared by lipid film hydration technique and were analyzed for size, entrapment efficiency and drug retention capacity. Niosomal vesicles were then incorporated into blank carbopol gel to form niosomal gel. The in vitro permeation study across pig skin was performed using Keshary-Chien glass diffusion cell. The size and entrapment efficiency of the niosomal vesicles increased with gradual increase in HLB value of nonionic surfactants used. Maximum drug entrapment was observed with Span 20 with HLB value of 8.6 and drug leakage from vesicles was less at refrigerated condition than at the room temperature. Higher proportion of cholesterol made the niosomal formulation more stable with high drug retention properties. The niosomal gel showed a prolong drug release behavior compared to plain drug gel. Differential scanning calorimetric study of drug loaded gel and pig skin after permeation study confirmed inertness of carbopol gel base toward rofecoxib and absence of drug metabolism in the skin during permeation study, respectively. The niosomal formulations were successfully prepared by lipid film hydration technique using cholesterol and Span as nonionic surfactant. Presence of cholesterol made niosomes more stable with high drug entrapment efficiency and retention properties. The lower flux value of niosomal gel as compared to plain drug gel across pig skin assured the prolong drug release behavior with sustained action.     

Temperature dependence of membrane ion conductance analyzed by using the amphiphilic anion 5/6-carboxyfluorescein

The rate of efflux of trapped 5/6-carboxyfluorescein from sealed lipid vesicles showed a marked dependence on (a) temperature, (b) phospholipid acyl chain composition, and (c) the nature of co-trapped counterions. When the dye was salted with sodium, at pH greater than 7, the rate of dye permeation showed a discrete maximum at the melting point of the lipid bilayer (Tc); in the case of membranes composed of dipalmitoylphosphatidylcholine, this discontinuity extended over a very broad temperature range, being detectable at least 10 degrees C above and below Tc. The peak in dye permeation rate was superimposed on a permeation profile that showed a simple exponential relationship to temperature. Studies with a homologous series of saturated lecithin bilayers revealed a consistent pattern of behavior: a logarithmic dependence of dye permeation rate on temperature with a superimposed discontinuity at Tc. For thin membranes (12-14-carbon acyl chains), the discontinuity was severe, exerting an influence over a very broad temperature range and leading to extremely high overall dye leakage rates. As the acyl chains were lengthened, the discontinuity became less pronounced, almost disappearing at a chain length of 20 carbons. In sharp contrast to these results, dye salted with N-methylglucamine [or with tris(hydroxymethyl)aminomethane] showed no efflux maximum at Tc, and base-line leakage rates were generally slower. When dye was salted with ammonium, efflux was too rapid to monitor, even at temperatures well below Tc. The results indicate that the rate of release of electrically charged dyes, such as 5/6-carboxyfluorescein, from sealed lipid vesicles can be tightly coupled to the counterion leakage rate and hence can provide an accurate and convenient assay of relative ion flux across phospholipid bilayers.     

Reaction of ozone with phospholipid vesicles and human erythrocyte ghosts

Phospholipid vesicles (unilamellar) and liposomes (multilamellar) made from egg phosphatidylcholine reacted similarly with ozone, producing hydrogen peroxide and malonaldehyde. On the basis of amount of ozone reacted, there was a 20% yield of hydrogen peroxide and 2.4% yield of malonaldehyde. The reactivity of the egg phosphatidylcholine membranes was a function of exposed membrane surface area. Large amounts of ozone caused no change in erythrocyte ghost phospholipid, fatty acid, or cholesterol composition. Thiobarbituric acid-positive material and conjugated dienes were present in very small quantities, suggesting some lipid oxidation which was below the limits of chromatographic detection. Ozone inhibited glyceraldehyde 3-phosphate dehydrogenase more than (Na⁺ + K⁺) adenosine triphosphate in exposed unsealed erythrocyte ghosts. The (Na⁺ + K⁺) adenosine triphosphatase activity sensitive to ozone was the ouabain-insensitive activity. Acetylcholinesterase activity was not significantly inhibited.     

The mechanism of ferrocytochrome C oxidation by a horseradish isoperoxidase.

The oxidation of ferrocytochrome c catalysed by highly purified horse-radish isoperoxidase P2 was studied kinetically. To take into account the low turnover number of the enzyme and the tendency to autocatalytic oxidation of ferrocytochrome c, experimental conditions were used which prevented us from using the steady-state treatment. According to kinetic results reported by several authors, a kinetic scheme involving a ternary complex between the enzyme and the substrates was postulated and simulated on a hybrid computer. By assuming that the interaction of peroxidase with hydrogen peroxide is much faster than the interaction with ferrocytochrome c, one can verify that this scheme explains the fact that initial velocity does not vary in relation to the hydrogen peroxide concentration and that a sudden change of slope occurs in the kinetic curve for an initial hydrogen peroxide/ferrocytochrome c ratio lower than 0.5.     

THE PRODUCTION OF HYDROGEN PEROXIDE BY BLUE-GREEN ALGAE: A SURVEY1

A survey of 38 axenic isolates of blue-green algae indicated that over half the isolates produced hydrogen peroxide under defined growth conditions. Three kinetic profiles for the formation of hydrogen peroxide, were observed; these are described. The possible site or sites of hydrogen peroxide formation remain unknown.     

Direct observation of release of cytochrome c from lipid-encapsulated protein by peroxide and superoxide: a possible mechanism for drug-induced apoptosis

Release of cytochrome c from inside lipid vesicles and from inside proteoliposomes formed by cytochrome c oxidase has been studied by spectrophotometric methods. The protein encapsulated inside vesicles did not form complex with sodium azide solution added externally. Both hydrogen peroxide and superoxide were found to cause release of cytochrome c from the lipid encapsulated protein, which was detected from the distinct spectral changes due to the formation of the azide complex of cytochrome c in the solution. Cytochrome c encapsulated inside proteoliposomes containing cytochrome c oxidase (CcO) did not release the cytochrome c during enzymatic turnover of CcO. The anticancer drug, doxorubicin, was found to inhibit the biochemical function of cytochrome c oxidase and release of cytochrome c was observed from the proteoliposome encapsulating the protein during the enzymatic turnover in the presence of doxorubicin. The results indicated that the inhibition of enzymatic activity by doxorubicin possibly leads to the formation of reactive oxygen species, which induce the release of cytochrome c from inside to outside of the membrane.     

Rate of solute incorporation to liposomes evaluated from encapsulated enzymes activities

There are numerous studies on systems comprising an enzyme encapsulated in unilamellar liposomes and its substrate initially present in the external aqueous media. Most of these studies are focused on enzyme stability and activity in a restricted media. However, the rate of the process is also determined by the capacity of the substrate to permeate towards the liposome inner pool. In spite of this, there are few studies aimed at a quantitative evaluation of the substrate permeation rate and its lifetime inside the liposome pool. In the present work, we describe, in terms of a very simple mechanism, the permeation of glucose and hydrogen peroxide in DPPC unilamellar liposomes. To this aim, we evaluated the rate of the process employing encapsulated glucose oxidase and catalase in the kinetic diffusion controlled limit. Under this condition, the rate of the process becomes zero order in the enzyme and allows a direct evaluation of the rate constant for the permeation process and the lifetime of a substrate molecule incorporated into the liposome inner pool.     

Rapid purification of clathrin-coated vesicles by free-flow electrophoresis

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.     

The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles

Band 3 protein has been incorporated into lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine or total erythrocyte lipids by means of a Triton X-100 Bio-Beads method, with an additional sonication step prior to the removal of the detergent. This methods results, for both types of band 3 lipid vesicles, in rather homogeneous vesicles with comparable protein content and vesicle trap. Freeze-fracture electron microscopy revealed that band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have considerably more intramembrane particles as compared to the band 3-erythrocyte lipid vesicles. The dimensions of the nonspecific permeation pathways present in the band 3-lipid vesicles were measured using an influx assay procedure for nonelectrolytes of different size, in which the vesicles were sampled and subsequently freed from nonenclosed labeled permeant by means of gel-filtration. The band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles have nonspecific permeation pathways (pores), with diameters of up to 60 A. In contrast, the band 3-total erythrocyte lipid vesicles are more homogeneous and show much smaller nonspecific permeation pathways, having a diameter of about 12 A. These results suggest that the nonspecific permeability of the band 3-lipid vesicles is strongly lipid-dependent. Increase in specific anion permeability expected as a consequence of the presence of band 3 in the erythrocyte lipid vesicles was found to be very limited. However, stereospecific, phloretin-inhibitable D-glucose permeability could clearly be demonstrated in these vesicles. The difference of the nonspecific permeability of the band 3-egg phosphatidylcholine-bovine heart phosphatidylserine vesicles and band 3-erythrocyte lipid vesicles, is discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce formation of pores.     

Hydrogen Peroxide Stimulates Exosomal Cathepsin B Regulation of the Receptor for Advanced Glycation End‐Products (RAGE)

Exosomes are nano‐sized vesicles that are secreted into the extracellular environment. These vesicles contain various biological effector molecules that can regulate intracellular signaling pathways in recipient cells. The aim of this study was to examine a correlation between exosomal cathepsin B activity and the receptor for advanced glycation end‐products (RAGE). Type 1 alveolar epithelial (R3/1) cells were treated with or without hydrogen peroxide and exosomes isolated from the cell conditioned media were characterized by NanoSight analysis. Lipidomic and proteomic analysis showed exosomes released from R3/1 cells exposed to oxidative stress induced by hydrogen peroxide or vehicle differ in their lipid and protein content, respectively. Cathepsin B activity was detected in exosomes isolated from hydrogen peroxide treated cells. The mRNA and protein expression of RAGE increased in cultured R3/1 cells treated with exosomes containing active cathepsin B while depletion of exosomal cathepsin B attenuated RAGE mRNA and protein expression. These results suggest exosomal cathepsin B regulates RAGE in type 1 alveolar cells under conditions of oxidative stress. J. Cell. Biochem. 119: 599–606, 2018. © 2017 Wiley Periodicals, Inc.     

Lipid peroxidation initiated by superoxide-dependent hydroxyl radicals using complexed iron and hydrogen peroxide

Iron salts stimulate lipid peroxidation by decomposing lipid peroxides to produce alkoxyl and peroxyl radicals which initiate further oxidation. In aqueous solution ferrous salts produce OH. radicals, a reactive species able to abstract hydrogen atoms from unsaturated fatty acids, and so can initiate lipid peroxidation. When iron salts are added to lipids, containing variable amounts of lipid peroxide, the former reaction is favoured and OH. radicals contribute little to the observed rate of peroxidation. When iron is complexed with EDTA, however, lipid peroxide decomposition is prevented, but the complex reacts with hydrogen peroxide to form OH. radicals which are seen to initiate lipid peroxidation. Superoxide radicals appear to play an important part in reducing the iron complex.     

Transmembrane movement of heme

Evidence for CO-heme partitioning into and across lipid bilayers was obtained by kinetic and chromatographic studies. Biphasic time courses were observed when CO-heme was rapidly mixed with unilamellar lipid vesicles in a stopped-flow spectrometer. The initial rapid phase depended linearly on lipid concentration and was assigned to heme partitioning between the external solvent phase and the outer lipid layer of the membranes. The rate of the second, much slower phase was independent of both heme and lipid concentration. The fraction of absorbance change associated with this slower phase increased with increasing heme to lipid ratios and reached a maximum of approximately 45%. A similar slow phase was observed when membrane-bound heme was reacted with apomyoglobin. In the presence of excess globin, all of the CO-heme was extracted from the membranes to form native CO myoglobin. Under these conditions, the fractional amount of absorbance change associated with the slow dissociation phase was approximately 45%, regardless of the heme to lipid ratio. These results suggest strongly that the slow phases represent transmembrane movement of heme, from the outer to the inner lipid layer in the association reactions and from the inner to the outer layer in dissociation reactions. The temperature dependence of the rate of CO-heme binding to the outer lipid layer was markedly different from that of transmembrane movement. The rate of the latter, slower process decreased greatly with increasing acyl chain length, whereas the rate of the initial binding process varied little with vesicle composition, as long as the membranes were examined above their melting temperatures. Finally, the two kinetically distinct bound heme fractions could be isolated directly by column chromatography.     

Diffusion and Catalysis Processes in Unilamellar Liposomes

Abstract

This research concerns the study of enzyme loaded unilamellar vesicles as bioreactors. The plant enzyme Ascorbic Acid Oxidase (AAO) was entrapped in unilamellar liposomes.

Kinetic study shows that the enzyme activity is largely criptic and that the enzyme in liposomes is partially protected against proteolysis. In order to describe the reactivity of such a system, a kinetic model was developed which accounts both for the enzyme location and for the substrate diffusion across the lipid membrane. For the building of the kinetic model we have used information derived from previous studies of freeze-fracture and label fracture microscopy, which have shown that the protein is located both inside the aqueous core and across the lipid membrane of the vesicles.     

Modeling of enzymatic reactions in vesicles: the case of alpha-chymotrypsin

The kinetic behavior of the alpha-chymotrypsin-catalyzed hydrolysis of the two p-nitroanilide substrates succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) and benzoyl-L-Tyr-p-nitroanilide (Bz-Tyr-pNA) was modeled and simulated for two different systems, namely for an aqueous solution and for a vesicle system, which was composed of phospholipid vesicles containing entrapped alpha-chymotrypsin. In the case of the vesicles, the substrate was added to the bulk, exovesicular aqueous phase. The experimentally determined time-dependence of product (p-nitroaniline) formation was modeled by considering the kinetic behavior of the enzyme and-in the case of vesicles-the substrate permeability across the bilayer membrane. In aqueous solution-without vesicles-the kinetic constants kcat and KS (respectively KM) were determined from fitting the model to experimental data of batch product concentration-time curves. The results were in good agreement with the corresponding values obtained from initial velocity measurements. For the vesicle system, using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), simulation showed that the substrate permeation across the bilayer was rate limiting. Using experimental data, we could obtain the substrate permeability coefficient for Bz-Tyr-pNA by parametric fitting as 2. 45 x 10(-7) cm/s.