Vesicles with charged domains

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region. (C) 2010 Elsevier B.V. All rights reserved.

Measurement of forces between and within bilayers of neutral phospholipids

Phospholipids in water form lamellar phases made up of alternating layers of water and bimolecular lipid leaflets. Three complementary methods, osmotic, mechanical, and vapour pressures, were used to measure the work of removing water from lamellar phases composed of frozen dipalmitoylphosphatidylcholine ( DPPC ), melted DPPC, egg phosphatidylethanolamine or equimolar mixtures of DPPC and cholesterol ( DPPC/CHOL ), Concurrently the structural changes that resulted from this water removal were measured using X-ray diffraction. The work was divided into that which forces the bilayers together ( F ) and that which compresses the molecules together within the bilayers ( F )# A large repulsive force exists between bilayers composed of each of the lipids studied and this force increases exponentially as bilayer separation is decreased. F is affected by the nature of the head groups, conformation of the acyl chains and heterogeneity of these chains. In general all of the melted phosphatidylcholines ( melted DPPC, egg lecithin and DPPC/CHOL ) have large equilibrium separations in excess water resulting from large repulsive hydration forces between these bilayers. By comparison, egg PE has an increased attractive force, and frozen DPPC has a decreased hydration force; each results in smaller separations in water for these two lipids. The chemical potentials of the water between the bilayers for all these lipids lie on a continuum, indicating that interbilayer water cannot be characterized by two discrete states, usually referred to as "bound" or "non**bound". For all lipids studied a maximum of 25 % of the total work done on the system goes into deforming the bilayers. The method used here viii to separate repulsion from deformation, developed for us by v. A. Parsegian, provides a unique method for the measurement of lateral pressure of a bilayer and its modulus of deformability ( Y ). Lateral pressure is affected by the nature of the head group, conformation and heterogeneity of the acyl chains. For small changes in molecular surface area ( A ) near equilibrium, both melted and frozen DPPC have similar values for the deformability modulus. Thus in this regime it requires about the same force to change the angle of tilt of frozen chains as it does to compress the fluid bilayer. The introduction of cholesterol into bilayers of DPPC reduces dramatically the lateral pressure of the bilayers over a large range of molecular surface areas ( A ). The variation in the magnitude of bilayer repulsion with different phospholipids provides a basis for the mechanism of lipid segregation in mixed lipid systems and suggests that interacting heterogeneous membranes may influence or modulate the composition of the opposing membrane. The measurements of deformabilities of bilayers provides a direct comparison of them with the properties of monolayers.

Structural effects of neutral lipids on the divalent cation-induced interactions of phosphatidylserine - containing bilayers

As Ca2+ and phosphatidylserine (PS) are known to induce the adhesion of bilayer vesicles and form collapsed multibilayer structures in vitro, it was the aim of this study to examine how that interaction and the resultant structures might be modified by neutral lipid species. X-ray diffraction data from multilamellar systems suggest that phosphatidylcholine (PC) and diacylglycerol (DG) might be in the collapsed phase up to a concentration of -30 mole % and that above this concentration these neutral lipids may modify Ca2+-induced bilayer interactions. Using large unilamellar vesicles and long incubations in excess Ca2+ to ensure equilibration, similar preliminary results were again obtained with PC, and also with phosphatidylethanolamine (PE). A combination of X-ray diffraction, thin-layer chromatography, density gradient centrifugation and freeze-fracture electron microscopy, used in conjunction with an osmotic stress technique, showed that (i) -30 mole % PC can be accomodated in the Ca(DOPS)2 phase; and (ii) higher PC levels modify Ca2+-induced bilayer interactions resulting in single lamellar phases of larger dimension and reduced tendency for REV collapse. Importantly, the data suggest that PC is dehydrated during the rapid collapse process leading. to Ca(DOPS)2 formation and exists with this dehydrated phase. Similar results were obtained using PS isolated from bovine brain. Preliminary studies using two different phosphatidylethanolamine (PE) species indicated accomodation by Ca(DOPS)2 of -25-30 mole 0/0 PE and bulk phase separation, of species favouring a non-bilayer phase, at higher levels. Significantly, all PS/PE vesicles appear to undergo a complete Ca2+-induced collapse, even with contents of up to 90 mole % PE. These data suggest that PE may have an important role in fusion mechanisms in vivo. In sum the data lend both structural and stoichiometric evidence for th~ existence of laterally segregated neutral lipid molecules within the same bilayers as PS domains exposed to Ca2+.

Supramolecular assemblies of rifampicin and cationic bilayers: preparation, characterization and micobactericidal activity

Background: Cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide (DODAB) have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents. Methods: Rifampicin (RIF) or isoniazid (ISO) interacted with DODAB bilayer fragments (BF) or large vesicles (LV). Dispersions were evaluated by dynamic light-scattering for zeta-average diameter (Dz) and zeta-potential (zeta) analysis; dialysis for determination of drug entrapment efficiency; plating and CFU counting for determination of cell viability of Mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration (MBC) and synergism index for DODAB/drug combinations. Results: DODAB alone killed micobacteria over a range of micromolar concentrations. RIF aggregates in water solution were solubilised by DODAB BF. RIF was incorporated in DODAB bilayers at high percentiles in contrast to the leaky behavior of ISO. Combination DODAB/RIF yielded MBCs of 2/2 and 4/0.007 mu g/mL against Mycobacterium smegmatis or Mycobacterium tuberculosis, respectively. Synergism indexes equal to 0.5 or 1.0, indicated synergism against the former and independent action, against the latter species. Conclusions: In vitro, DODAB acted effectively both as micobactericidal agent and carrier for rifampicin. The novel assemblies at reduced doses may become valuable against tuberculosis.

Silica-based cationic bilayers as immunoadjuvants

Background: Silica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design. Results: Firstly, the silica/DODAB assembly was characterized at 1 mM NaCl, pH 6.3 or 5 mM Tris. HCl, pH 7.4 and 0.1 mg/ml silica over a range of DODAB concentrations (0.001-1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.05 mM DODAB is enough to produce cationic bilayer-covered particles with good colloid stability. Secondly, conditions for maximal adsorption of bovine serum albumin (BSA) or a recombinant, heat-shock protein from Mycobacterium leprae (18 kDa-hsp) onto DODAB-covered or onto bare silica were determined. At maximal antigen adsorption, cellular immune responses in vivo from delayed-type hypersensitivity reactions determined by foot-pad swelling tests (DTH) and cytokines analysis evidenced the superior performance of the silica/DODAB adjuvant as compared to alum or antigens alone whereas humoral response from IgG in serum was equal to the one elicited by alum as adjuvant. Conclusion: Cationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum, better performance regarding cellular immune responses and employs very low, micromolar doses of cationic and toxic synthetic lipid.

Evidence for zero-differential resistance states in electronic bilayers

We observe zero-differential resistance states at low temperatures and moderate direct currents in a bilayer electron system formed by a wide quantum well. Several regions of vanishing resistance evolve from the inverted peaks of magneto-intersubband oscillations as the current increases. The experiment, supported by a theoretical analysis, suggests that the origin of this phenomenon is based on instability of homogeneous current flow under conditions of negative differential resistivity, which leads to formation of current domains in our sample, similar to the case of single-layer systems.

Dibucaine effects on structural and elastic properties of lipid bilayers

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPQ were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes. (C) 2008 Elsevier B.V. All rights reserved.

Surface miscibility of EPC/DOTAP/DOPE in binary and ternary mixed monolayers

Surface pressure (pi)-molecular area (A) curves were used to characterize the packing of pseudo-ternary mixed Langmuir monolayers of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and L-alpha-dioleoyl phosphatidylethanolamine (DOPE). This pseudo-ternary mixture EPC/DOPE/DOTAP has been successfully employed in liposome formulations designed for DNA non-viral vectors. Pseudo-binary mixtures were also studied as a control. Miscibility behavior was inferred from pi-A curves applying the additivity rule by calculating the excess free energy of mixture (Delta G(Exc)). The interaction between the lipids was also deduced from the surface compressional modulus (C(s)(-1)). The deviation from ideality shows dependence on the lipid polar head type and monolayer composition. For lower DOPE concentrations, the forces are predominantly attractive. However, if the monolayer is DOPE rich, the DOTAP presence disturbs the PE-PE intermolecular interaction and the net interaction is then repulsive. The ternary monolayer EPC/DOPE/DOTAP presented itself in two configurations, modulated by the DOPE content, in a similar behavior to the DOPE/DOTAP monolayers. These results contribute to the understanding of the lipid interactions and packing in self-assembled systems associated with the in vitro and in vivo stability of liposomes. (C) 2010 Elsevier B.V. All rights reserved.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Quantitative x-ray photoelectron spectroscopy study of Al/AlOx bilayers

An x-ray photoelectron spectroscopy (XPS) analysis of Nb/Al wedge bilayers, oxidized by both plasma and natural oxidation, is reported. The main goal is to show that the oxidation state¿i.e., O:(oxidize)Al ratio¿, structure and thickness of the surface oxide layer, as well as the thickness of the metallic Al leftover, as functions of the oxidation procedure, can be quantitatively evaluated from the XPS spectra. This is relevant to the detailed characterization of the insulating barriers in (magnetic) tunnel junctions

Electron paramagnetic resonance (EPR) spectral components of spin-labeled lipids in saturated phospholipid bilayers: effect of cholesterol

Electron paramagnetic resonance (EPR) spectroscopy was used to study the main structural accommodations of spin labels in bilayers of saturated phosphatidylcholines with acyl chain lengths ranging from 16 to 22 carbon atoms. EPR spectra allowed the identification of two distinct spectral components in thermodynamic equilibrium at temperatures below and above the main phase transition. An accurate analysis of EPR spectra, using two fitting programs, enabled determination of the thermodynamic profile for these major probe accommodations. Focusing the analysis on two-component EPR spectra of a spin-labeled lipid, the influence of 40 mol % cholesterol in DPPC was studied.

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellataroot extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Measurement of repulsive forces between charged phospholipid bilayers

Electrostatic forces between membranes containing charged lipids were assumed to play an important role in influencing interactions between membranes long before quantitative measurements of such forces were available. ~ur measurements were designed to measure electrostatic forces between layers of lecithin charged with lipi~s carrying ionizable head groups. These experiments have shown that the interactions between charged lipid bila.yere are dominated by electrostatic forces only at separations greater than 30 A. At smaller separations the repulsion between charged bilayers is dominated by strong hydration forces. The net repulsive force between egg lecithin bilayers containing various amounts of cherged lipids (phosphatidylglycerol (PG) 5,10 ano 50 mole%, phosphatidyli. nosi tol (PI) 10 mole% and sodium oleate (Na-Ol) 3,5 and 10 mole%, where mole% gives the ratio of the number of moles' of .charged lipid to the total number of moles of all lipids present in the sample) was stuoied with the help ('If the osmotic streas technique described by LeNeveu et aI, (1977). Also, the forces between pure PG were j_nvestigated in the same manner. The results have been plotted showing variation of force as a function of bilay- _ er separation dw• All curVes 90 obtained called force curves, were found to be similar in sha.pe, showing two distinct regions, one when dw<.30 A is a region cf very rapid iiivariation of force with separation ( it is the region dominated by hydre,tion force) and second when dw> 40 A is a region of very slow variation of force with separB.tion ( it is the region dominated by the electrostatic force). Between these two regions there exists a transition area in which, in most systems studied, a phase separation of lipids into fractions containing different amounts of charged groups, was observed. A qualitative analysis showed that our results were v/ell described by the simple electrostatic double -le.yer theory. For quantitative agreement between measured and calculated force curves however, the charge density for the calculations had to be taken as half of that given by the number density of charged lipids present in the lecithin bilayers. It is not clear at the moment what causes such low apparent degree of ionization among the charged head groups, and further study is needed in this area.

Simultaneous extraction of order parameters and orientational distribution fuctions from 31[superscript]P NMR spectra of magnetically partially oriented phospholipid bilayers

Order parameter profiles extracted from the NMR spectra of model membranes are a valuable source of information about their structure and molecular motions. To al1alyze powder spectra the de-Pake-ing (numerical deconvolution) ~echnique can be used, but it assumes a random (spherical) dist.ribution of orientations in the sample. Multilamellar vesicles are known to deform and orient in the strong magnetic fields of NMR magnets, producing non-spherical orientation distributions. A recently developed technique for simultaneously extracting the anisotropies of the system as well as the orientation distributions is applied to the analysis of partially magnetically oriented 31p NMR spectra of phospholipids. A mixture of synthetic lipids, POPE and POPG, is analyzed to measure distortion of multilamellar vesicles in a magnetic field. In the analysis three models describing the shape of the distorted vesicles are examined. Ellipsoids of rotation with a semiaxis ratio of about 1.14 are found to provide a good approximation of the shape of the distorted vesicles. This is in reasonable agreement with published experimental work. All three models yield clearly non-spherical orientational distributions, as well as a precise measure of the anisotropy of the chemical shift. Noise in the experimental data prevented the analysis from concluding which of the three models is the best approximation. A discretization scheme for finding stability in the algorithm is outlined