Effect of Altered Polar Headgroup of Phosphatidylethanolamines on Transbilayer Aminophospholipid Distribution in Sonicated Vesicles

The transbilayer aminophospholipid distributions in small unilamellar vesicles comprising of phosphatidylethanolamine or its analogs (bearing modifications in the polar headgroup) and egg hosphatidylcholine were ascertained using trinitrobenzenesulfonic acid as external membrane probe. These vesicles, containing 10-30 mol% phosphatidylethanolamine or its analogs, were formed by sonication and fractionated by centrifugation. Phosphatidylethanolamine at low concentrations (10 mol%) preferentially localized in the outer monolayer. This preference appeared to be reversed at higher phosphatidylethanolamine concentrations (30 mol%). Unlike this finding, phosphatidylethanolamine bearing ethyl, phenyl and benzyl substituents at the carbon atom adjacent to the amino group distributed mainly in the outer surface irrespective of their concentrations. Similar results were obtained when the phosphate and amino groups were separated by three methylene residues. These observations suggest that the effective polar headgroup volume and/or hydrogen-bonding capacity of phospholipids are the important factors that determine their distribution in small unilamellar vesicles.

Phase transition in dioctadecyldimethylammonium bromide and chloride vesicles prepared by different methods

The gel to liquid crystalline phase transition of the double-chained cationic dioctadecyldimethylammonium chloride and bromide (DODAX, X = Cl- or Br-) in aqueous vesicle dispersions prepared by non-sonication, sonication and extrusion has been investigated using high-sensitivity differential scanning calorimetry (DSC). The transition temperature (T-m) is a function of the preparation method, amphiphile concentration, vesicle curvature and nature of the counterion. DSC thermograms for DODAB and DODAC non-sonicated vesicle dispersions exhibit a single endothermic peak at T-m roughly independent of concentration up to 10 mM. Extrusion broadens the transition peak and shifts T-m downwards. Sonication, however, broadens slightly the transition peak and tends to shift T-m upwards suggesting that extrusion and sonication form vesicles with different characteristics. DODAC always exhibits higher T-m than DODAB irrespective of the preparation method. T-m changes as follows: T-m (sonicated) greater than or equal to T-m (non-sonicated) > T-m (extruded). Hysteresis of about 7 degrees C was observed for DODAB vesicle dispersions. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Effect of Sonication on the Thermotropic Behavior of DODAB Vesicles Studied by Fluorescence Probe Solubilization

The effect of sonication on fluorescence probe solubilization in cationic vesicles of dioctadecyldimethylammonium bromide (DODAB) was investigated by steady-state fluorescence of pyrene (Py), trans-diphenylpolyenes-diphenylbutadiene (DPB), diphenylhexatriene (DPH), and their corresponding 4,4'-dialkyl derivatives 4B4A and 4H4A fluorescence probes. The data indicate that sonication affects the bilayer polarity, the melting temperature (T (m)), and the cooperativity of the melting process due to changes in vesicle morphology. The effect of temperature on the fluorescence intensity and yielding I broken vertical bar(f) and anisotropy < r > shows that the ionizable probes 4B4A and 4H4A are solubilized close to the vesicle interfaces, whereas the non-ionizable DPH and DPB are deeper in the bilayers. Py solubilization indicates that sonicated vesicles exhibit less densely packed bilayers.

Interfacial concentrations of chloride and bromide and selectivity for ion exchange in vesicles prepared with dioctadecyldimethylammonium halides, lipids, and their mixtures

The local concentrations of chloride, Cl b, and bromide, Br b, in the interface of vesicles prepared with dioctadecyldimethylammonium chloride, DODAC, or bromide, DODAB, dipalmitoylphosphatidylcholine, DPPC, dimyristoylphosphatidylcholine, DMPC, and mixtures of DMPC, DPPC, and DODAC were determined by chemical trapping by analyzing product yields from spontaneous dediazoniation of vesicle-bound 2,6-dimethyl-4-hexadecylbenzenediazonium ion. The values of Cl b and Br b in DODAC and DODAB vesicles increase with vesicle size, in agreement with previous data showing that counterion dissociation decreases with vesicle size. Addition of tetramethylammonium chloride displaces bromide from the DODAB vesicular interface. The value for the selectivity constant for Br/Cl exchange at the DODAB vesicular interface obtained by chemical trapping was ∼2.0, well within values obtained for comparable amphiphiles. In vesicles of DPPC the values of Cl b were very sensitive to the nature of the cation and decreased in the order Ca 2+ > Mg 2+ > Li + > Na + > K + = Cs + = Rb + ≥ +. The effect of the cation becomes more important as temperature increases above the phase transition temperature, T m, of the lipid. The values of Cl b increased sigmoidally with the mol % of DODAC in vesicles prepared with DODAC/lipid mixtures. In sonicated vesicles prepared with DODAC and DMPC (or DPPC), the values of Cl b reach local concentrations measured for the pure amphiphile at 80 mol % DODAC. These results represent the first extensive study of local concentration of ions determined directly by chemical trapping in vesicles prepared with lipids, synthetic ampliiphiles, and their mixtures.

Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor

Sonicated vesicles of l-fatty acyl-2-w-(2-diazo-3.3,3-trifluoropropionoxy) fatty acyl sn-glycero-3-phosphorylcholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C-H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the sn-2 fatty acyl chain trails the sn-1 chain by 2-4 atoms.

Structural characterization of the interaction of the polyene antibiotic Amphotericin B with DODAB bicelles and vesicles

Amphotericin B (AmB) is widely used in the treatment of systemic fungal infections, despite its toxic effects. Nephrotoxicity, ascribed as the most serious toxic effect, has been related to the state of aggregation of the antibiotic. In search of the increase in AmB antifungal activity associated with low toxicity, several AmB-amphiphile formulations have been proposed. This work focuses on the structural characterization of a specific AmB formulation: AmB associated with sonicated dioctadecyl dimethylammonium bromide (DODAB) aggregates. Here, it was confirmed that sonicated DODAB dispersion is constituted by DODAB bicelles, and that monomeric AmB is much more soluble in bicelles than in DODAB vesicles. A new optical parameter is proposed for the estimation of the relative amount of amphiphile-bound monomeric AmB. With theoretical simulations of the spectra of spin labels incorporated in DODAB bicelles it was possible to prove that monomeric AmB binds preferentially to lipids located at the edges of DODAB bicelles, rigidifying them, and decreasing the polarity of the region. That special binding of monomeric AmB along the borders of bicelles, where the lipids are highly disorganized, could be used in the formulation of other carriers for the antibiotic, including mixtures of natural lipids which are known to form bicelles. (C) 2011 Elsevier B.V. All rights reserved.

Effect of vesicles of dimethyldioctadecylammonium chloride and phospholipids on the rate of decarboxylation of 6-nitrobenzisoxazole-3-carboxylate

Sonicated mixtures of dimethyldioctadecylammonium chloride (DODAC), egg phosphatidylcholine (PC), dimyristoyl phosphatidylcholine (DMPC), and dipalmitoyl phosphatidylcholine (DPPC) were used to analyze vesicle effects on the rate of decarboxylation of 6-nitrobenzisoxazol-3-carboxylic acid (Nboc). Electron microscopic images of the vesicles were obtained with trehalose, a know cryoprotector. Phase diagrams and phase transitions temperatures of the vesicle bilayers were determined. Nboc decarboxylation rates increased in the presence of vesicles prepared with both phospholipids and DODAC/phospholipid mixtures. Quantitative analysis of vesicular effects was done using pseudophase models. Phospholipids catalyzed up to 140-fold while the maximum catalysis by DODAC/lipid vesicles reached 800-fold. Acceleration depends on alkyl chain length, fatty acid insaturation of the lipids, and the DODAC/phospholipid molar ratio. Catalysis is not related to the liquid crystalline-gel state of the bilayer and may be related to the relative position of Nboc with respect to the interface.

Fragment and vesicle structures in sonicated dispersions of dioctadecyldimethylammonium bromide

Dynamic light scattering has been used to investigate sonicated aqueous dispersions of dioctadecyldimethylammonium bromide (DODAB). The hydrodynamic radius (R-H) of the scattering particles and the mean scattering intensity (I) have been monitored as functions of the DODAB concentration and temperature (T). In the dilute regime, the relaxation time distribution of the sonicated dispersion of DODAB is bimodal with the slow mode dominating the distribution. The slow and fast modes are respectively characteristic of vesicles and bilayer fragments with R-H values of 22 and 8.5 nm (25 degrees C) and 20 and 6 nm (50 degrees C), respectively. The total scattered intensity initially decreased with temperature up to 45 degrees C (T-c), above which it was constant; identical behavior was observed for the slow mode intensity, but the fast mode intensity was constant with temperature change, showing that T-c is a property of the vesicles and not of the bilayer fragments. At T-c the slow vesicle mode becomes narrower whereas the fast fragment mode shows no change. on aging, the dispersion showed a slow transition from bimodal to a rather broad single-modal relaxation time distribution. The corresponding R-H was 33.8 nm when measured 10 months after preparation. These results suggest that aqueous sonicated dispersions of DODAB are metastable.

Phase behavior of synthetic amphiphile vesicles investigated by calorimetry and fluorescence methods

The understanding of biological membranes may be improved by investigating physical properties of vesicles from natural or synthetic amphiphiles. The application of vesicles as mimetic agents depends on the knowledgment of their structure and properties. Vesicles having different curvature and size may be obtained using different preparation protocols. We have used differential scanning calorimetry (DSC) and steady-state fluorescence to investigate the gel to liquid-crystal phase transition of vesicles prepared by sonication (SUV) and non-sonication (GUV) of the synthetic dioctadecyldimethylammonium bromide (DODAB) in aqueous solution. DSC thermograms for a non-sonicated dispersion show a well-defined pre- and main transition corresponding to two narrow peaks at 36 and 45°C in the first upscan, while in a second upscan, only the main peak was observed. The sharpness of the peaks indicate a cooperative phase behavior for GUV. For a sonicated DODAB dispersion, the first upscan shows a third peak at 40.3°C, whereas for the second upscan the peaks are not well-defined, indicating a less cooperative phase behavior. Alternatively, the fluorescence quantum yield (Φ f) and the anisotropy (r) of trans, trans, trans-1-[4-(3-carboxypropyl)-phenyl]-6-[4-butylphenyl]-1,3,5-hexatriene (4H4A) and the ratio I 1/I 3 of the first to the third vibronic peaks of the pyrene emission spectrum as function of temperature are used as well to describe the phase behavior of DODAB sonicated and non-sonicated dispersions. It is in good agreement with the DSC results that the cooperativity of the thermotropic process is diminished under sonication of the DODAB dispersion, meaning that sonication changes from homogeneous to heterogeneous populations of the amphiphile aggregates. The pre- and main transitions obtained from these techniques are in fairly good accord with results from the literature.

Direct Visualization Of The Action Of Triton X-100 On Giant Vesicles Of Erythrocyte Membrane Lipids.

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

On The Distinct Molecular Architectures Of Dipping- And Spray-lbl Films Containing Lipid Vesicles.

The introduction of spraying procedures to fabricate layer-by-layer (LbL) films has brought new possibilities for the control of molecular architectures and for making the LbL technique compliant with industrial processes. In this study we show that significantly distinct architectures are produced for dipping and spray-LbL films of the same components, which included DODAB/DPPG vesicles. The films differed notably in their thickness and stratified nature. The electrical response of the two types of films to aqueous solutions containing erythrosin was also different. With multidimensional projections we showed that the impedance for the DODAB/DPPG spray-LbL film is more sensitive to changes in concentration, being therefore more promising as sensing units. Furthermore, with surface-enhanced Raman scattering (SERS) we could ascribe the high sensitivity of the LbL films to adsorption of erythrosin.

Preparation of PVP hydrogel nanoparticles using lecithin vesicles

Hydrogels micro, sub-micro and nanoparticles are of great interest for drug encapsulation and delivery or as embolotherapic agents. In this work it is described the preparation of nano and sub-microparticles of pre-formed, high molecular weight and monomer free poly(N-vinyl-2-pyrrolidone) encapsulated inside the core of lecithin vesicles. The hydrogel particles are formed with a very narrow diameter distribution, of about 800 nm, and a moderate swelling ratio, of approximately 10.

Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells. Conclusions/Significance: Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.

Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid head-group. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.