Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.     

Effect of phosphatidylcholine bilayer thickness and molecular order on the binding of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides (AMPs) interact directly with bacterial membrane lipids. Thus, changes in the lipid composition of bacterial membranes can have profound effects on the activity of AMPs. In order to understand the effect of bilayer thickness and molecular order on the activity of AMPs, the interaction of maculatin 1.1 (Mac1.1) with phosphatidylcholine (PC) model membranes composed of different monounsaturated acyl chain lengths between 14 and 22 carbons was characterised by dual polarisation interferometry (DPI) and 31P and 1H solid-state NMR techniques. The thickness and bilayer order of each PC bilayer showed a linear dependence on the acyl chain length. The binding of Mac1.1 exhibited a biphasic dependency between the amount of bound Mac1.1 and bilayer thickness, whereby the mass of bound peptide increased from C14 to C16 and then decreased from C16 to C22. Significant perturbation of 31P chemical shift anisotropy (CSA) values was only observed for DOPC (C18) and DEPC (C22), respectively. In the case of DEPC, the greater range in CSA indicated different headgroup conformations or environments in the presence of Mac1.1. Overall, the results indicated that there is a significant change in the bilayer order upon binding of Mac1.1 and this change occurred in a co-operative manner at higher concentrations of Mac1.1 with increasing bilayer thickness and order. Overall, an optimum bilayer thickness and lipid order may be required for effective membrane perturbation by Mac1.1 and increasing the bilayer thickness and order may counteract the activity of Mac1.1 and play a role in antimicrobial resistance to AMPs.     

Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles

The thickness of the lipid bilayer in vesicles made of pure phosphatidylcholines, with acyl chain lengths ranging from 10 to 24 carbons, has been determined by analysis of continuous X-ray scattering data from vesicle pellets at temperatures above the lipid phase transition temperature. Bilayer thickness was found to vary linearly with the number of carbons per acyl chain. The lines for saturated and monounsaturated acyl chains were slightly displaced but had similar slopes. For the saturated species di-12:0, di-14:0, di-16:0, and di-18:0 phosphatidylcholine the surface areas per molecule were all 65.7 to 66.5 A2, while the monounsaturated species and di-10:0 phosphatidylcholine all occupied 67.7 to 70.1 A2 per molecule.     

Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying.

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze-drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry egg PtdCho by more than 20 degrees C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.     

Secondary structure of a mitochondrial signal peptide in lipid bilayer membranes

The secondary structure of the synthetic signal peptide of cytochrome c oxidase subunit IV (coxIV-25) has been measured by circular dichroism spectroscopy in different lipid environments. CoxIV-25 is polymorphic in membranes. It forms an amphiphilic alpha-helix both in negatively charged lipid bilayers (up to 49% helix) and in detergent micelles (up to 42% helix). In association with bilayers of the zwitterionic lipid phosphatidylcholine, coxIV-25 takes an aperiodic, unidentified structure. CoxIV-25 is also partially alpha-helical in bilayers of cardiolipin, mitochondrial lipid extracts and mixtures of synthetic phosphatidylcholine and phosphatidylglycerol.     

Preparation of passive bilayer liposomes

In studies of in-membrane molecular interactions, need may arise for a matrix that cannot itself interact, except hydrophobically, with the reactants. Such a bilayer matrix should, ideally, consist of only a hydrophobic zone without ionic outer layers and without hydrogen belts (the membrane strata containing CO and OH groups). However, because of the necessity of anchoring the bilayer to its aqueous surroundings, there must be polar substituents. Hydrophilic ether groups in the form of polyoxyethylenes can provide nearly sufficient anchoring and yet not confer unwanted reactivity to the membrane since they are only very weak H-bond acceptors. The stability of the bilayer is ensured by the presence of a few percent of an amphiphile (which may be the substrate to be studied, e.g. a phospholipid) or by a free polyethylene hydroxy group far remote from the original hydrogen belt region. Our most impermeable liposomes consisted of O-methylcholesterol/O-methoxyethoxyethoxyethylcholesterol; the most readily prepared liposomes were made from O-methylcholesterol and hydroxy(ethoxy)4dodecane (Brij 30) or Triton.     

Mitochondria transfection by oligonucleotides containing a signal peptide and vectorized by cationic liposomes.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.     

Interaction of fluoxetine with phosphatidylcholine liposomes

Fluoxetine (Prozac) is one of the latest of a new generation of antidepressants, approved by FDA in 2002. The interactions of fluoxetine with multilamellar liposomes of pure phosphatidylcholine (PC) or containing cholesterol 10% molar were studied as a function of the lipid chain lengths, using differential scanning calorimetry and spin labelling EPR techniques. The DSC profiles of the gel-to-fluid state transition of liposomes of DMPC (C14:0) are broadened and shifted towards lower temperatures at increasing dopant concentrations and, with less than 10% fluoxetine, any detectable transition is destroyed. The broadened profiles and the lowered transition temperatures demonstrate that both the size and the packing of the cooperative units undergoing the transition are modified by fluoxetine, leading to a looser and more flexible bilayer. No phase separation was observed. The effects of fluoxetine on the thermotropic phase behaviour of DPPC (C16:0) and, even more, of DSPC (C18:0) are different from that of DMPC. In fact, in the former cases, two peaks appeared at increasing dopant concentrations, suggesting the occurrence of a phase separation phenomenon, which is a sign of a binding of fluoxetine in the phosphate region. In cholesterol containing membranes, fluoxetine, even at low concentrations, leads to a general corruption of the membrane, both in terms of packing and cooperativity, and the formation of any new phase is no longer observable. EPR spectra reflect the disordered motion of acyl chains in the bilayer. It was found that fluoxetine lowers the order of the lipid chains mainly in correspondence of the fifth carbon position of SASL, indicating a possible accumulation near the interfacial region.     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

Effect of phosphatidylcholine liposomes on the mitogen-stimulated lymphocyte activation.

The effect of phosphatidylcholine liposomes on the mitogen-stimulated lymphocyte activation was examined $\text{in vitro}$ in an attempt to determine whether liposomes influence the cell growth. Phosphatidylcholine liposomes reduced the cellular cholesterol level and effectively inhibited lymphocyte activation. On the other hand, phosphatidylcholine-cholesterol liposomes (molar ratio 1:1) increased the cellular cholesterol level and was relatively ineffective in the inhibition. After phosphatidylcholine treatment, the addition of high-density lipoprotein to the medium reversed the inhibition of lymphocyte activation. It is concluded that the inhibition was related to the attraction and association of cellular cholesterol with liposomes. This is consistent with the notion that cholesterol is required for successful blast transformation.     

Permeability of dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-crystalline phases.

The passive permeation of glucose and a small zwitterionic molecule, methyl-phosphoethanolamine, across two-component phospholipid bilayers (dimyristoyl phosphatidylcholine (DMPC)/dipalmitoyl phosphatidylcholine (DPPC) mixtures) exhibit a maximum when gel domains and fluid domains coexist. The permeability data of the two-phase bilayers cannot be fitted to single-rate kinetics, but are consistent with a Gaussian distribution of rate constants. In pure DMPC and DPPC as well as in their mixtures, at the temperature of the maximum excess heat capacity, the logarithm of the average permeability rate constants are linearly correlated with the mole fraction of DPPC in the total system. In addition, in the 50:50 mixture, the excess heat capacity values as well as the apparent fractions of interfacial lipid correlate with the logarithm of the excess permeabilities in the two-phase region. These results suggest that small polar molecules can cross the membrane at the interface between gel and fluid domains at a much faster rate than through the homogeneous phases; the acyl chains located at the domain interface experience lateral density fluctuations that are inversely proportional to their average length, and large enough to allow rapid transmembrane diffusion of the solute molecules. The distribution of the permeability rate constants may reflect temporal and spatial fluctuations of the lipid composition at the phase boundaries.     

Bile salt damage of egg phosphatidylcholine liposomes

Physiochemical damage of egg phosphatidylcholine liposomes, caused by the salts of three bile acids, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid, has been investigated. Of the three bile salts, that of chenodeoxycholic acid was the most destructive, and the effect of the damage was examined by monitoring the induced 6-carboxyfluorescein release from the liposomes. For all three of the bile salts and under the experimental conditions, the minimum (effective) concentrations causing the 6-carboxyfluorescein release were below their critical micelle concentrations. In the case of the salt of chenodeoxycholic acid, the presence of cholesterol in the liposomal bilayers did not show any significant effect on the induced 6-carboxyfluorescein release, while, for the salts of ursodeoxycholic acid and cholic acid, the presence of cholesterol tended to depress the release. Permeation of bile salts into the membranes of liposomal bilayers made these membranes more fluid, and this fluidity was monitored by measuring the change in fluorescence polarization using 1,6-diphenylhexatriene entrapped in the liposomes. Coating the liposomes with polysaccharides, to make them more hydrophobic, led to their easier lysis by the bile salts.     

Microelectrophoresis studies of the binding of glycosaminoglycans to phosphatidylcholine liposomes.

The binding of the glycosaminoglycans (GAG) chondroitin sulfate and heparin and the homologous molecule dextran sulfate to multilamellar dimyristoyl phosphatidylcholine (DMPC), dilaureyl phosphatidylcholine (DLPC) and egg lecithin liposomes was investigated by microelectrophoresis measurements. Drastic changes of the zeta potential of the liposomes to negative values indicate the binding of the highly anionic macromolecules. Binding depends strongly on Ca2+ and NaCl concentrations in the medium and does not occur in the absence of Ca2+. The adsorption is saturated at concentrations of about 0.1 mg/ml chondroitin sulfate and heparin and 0.01 mg/ml dextran sulfate. In the gel state of the phospholipid bilayer more GAG can associate with the surface compared to the fluid state.     

Location of terminal methyl groups of acyl chains in the bilayer of unilamellar phosphatidylcholine liposomes: a 13C-NMR study

The spin-lattice 13C-NMR relaxation time T1 of carbons in egg yolk phosphatidylcholine (EYPC) unilamellar liposomes was measured at 15 MHz, 25 MHz and 75 MHz in the presence of diamagnetic La3+ and paramagnetic Gd3+ ions. Supposing formation of ML2 complexes (where M is the metal ion and L the lipid) and using a simplified Solomon-Bloembergen-Morgan equation, a value of less than r-3IS greater than 2 = 0.1880 +/- 0.0005 nm-6 was obtained for C omega carbon of lipid chains, where rIS is the distance of Gd3+ unpaired electron and C omega nucleus, and angle brackets denote a weighted average. This value may serve as the basis for testing the application of statistical mechanics to lipid chain conformation and chain terminal group distribution in lipid bilayers.     

Small-angle neutron scattering study of the n-decane effect on the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine liposomes

Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.     

Interactions of liposomes with planar bilayer membranes

Summary The adhesion to horizontal, planar lipid membranes of lipid vesicles containing calcein in the aqueous compartment or fluorescent phospholipids in the membranes has been examined by phase contrast, differential interference contrast and fluorescence microscopy. With water-immersion lenses, it was possible to study the interactions of vesicles with planar bilayers at magnifications up to the useful limit of light microscopy. In the presence of 15 mM calcium chloride, vesicles composed of phosphatidylserine and either phosphatidylethanolamine or soybean lipids adhere to the torus, bilayer and lenses of planar bilayers of the same composition. Lenses of solvent appear, at the site where vesicles attach to decane-based bilayers and lipid fluorophores move from the vesicles to the lenses. Because the calcein contained in such vesicles is not released, we interpret this as indicating fusion of only the outer monolayer (hemifusion) of the vesicles with the decane lenses. In the case of squalene-based black lipid membranes (BLMs), in contrast, vesicles do not nucleate lenses but they apparently do fuse with the torus at the bilayer boundary. Interactions leading to hemifusions between vesicles and planar membranes thus occur predominantly in regions where hydrocarbon solvent is present. Osmotic water flow, induced by addition of urea to the compartment containing vesicles, causes coalescence of lenses in decane-based, BLMs as well as coalescence of the aqueous spaces of the vesicles that have undergone hemifusion with the lenses. We did not observe transfer of the aqueous phase of vesicles to therans side of either decane-or squalene-based planar membranes; however, we cannot rule out the possibility particularly in the latter case, that rupture of the planar membrane may have been an immediate result of vesicle fusion and thus precluded its detection.