1-palmitoyl-2-oleoylglycero-3-phosphoglycerol and 1-2-dipalmitoylphosphatidylglycerol

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30 mN m(-1)) and the HIV-1 FP (35 mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (GE). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive GE values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP-lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.

Topics: Biomimetic Materials; Erythrocytes; GB virus C; Hemolysis; HIV-1; Humans; Kinetics; Lipid Bilayers; Membranes, Artificial; Phosphatidylglycerols; Thermodynamics; Viral Envelope Proteins; Viral Fusion Proteins

Layer-by-layer (LbL) films have been exploited in drug delivery systems that may be used in the form of patches, but the encapsulation of poor water soluble drugs and their release with a controlled rate are still major challenges to be faced. In this paper, we demonstrate the controlled release of aloin (barbaloin), an important component of the widely used Aloe vera, encapsulated into liposomes and immobilized in LbL films with a polyelectrolyte. With a systematic study using fluorescence spectroscopy of aloin release from solutions and from LbL films with different phospholipid liposomes, we inferred that optimized release was achieved with aloin incorporated into palmitoyl oleyl phosphatidyl glycerol (POPG) or dipalmitoyl phosphatidyl glycerol (DPPG) liposomes immobilized in LbL films. Significantly, with this optimized system aloin was almost completely released within 30 h, with a small release rate at the end, which followed a sharp release in the first 5h. Upon comparing the rates of the distinct systems, we conclude that the main factors controlling the release are the electrostatic interactions involving the negatively charged phospholipids. Because these interactions can be tuned in LbL films, the approach used here opens the way for new drug delivery systems to be developed with fine control of the drug release.

Topics: Absorption; Administration, Cutaneous; Chemistry, Pharmaceutical; Drug Delivery Systems; Emodin; Liposomes; Phosphatidylglycerols; Solutions; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

We studied the conformation of β2-human glycoprotein (β2GPI) in solution and bound to the anionic lipids palmitoyl oleoyl phosphatidylglycerol (POPG), dimiristoyl phosphatidylglycerol (DMPG) and dipalmitoyl phosphatidylglycerol (DPPG) as a function of the temperature. We used the infrared amide I' band to study the protein conformation, and the position of the antisymmetric stretching band of the methylene groups in the lipid hydrocarbon chains to study the lipid order. Lipid-protein complexes were studied in media of low and high ionic strengths. In solution, β2GPI displayed a conformational pre-transition in the range 47-50°C, characterized by a shift in the band of β secondary structure, previous to the main unfolding at 64°C. When the protein was bound to the anionic lipid membranes at 25°C, a similar shift as in the pre-transition in solution was observed, together with an increase in the band corresponding to α-helix secondary structure. Lipid-protein complexes formed large aggregates within the temperature range 10≅60°C. At temperatures above the protein unfolding, the complexes were disrupted to yield vesicles with bound protein. This finding indicated that the native fold was required for the formation of the lipid-protein aggregates. Cycles of heating and cooling showed hysteresis in the formation of aggregates.

Topics: Calorimetry; Glycoproteins; Light; Lipids; Nephelometry and Turbidimetry; Phosphatidylglycerols; Protein Conformation; Protein Denaturation; Protein Structure, Secondary; Scattering, Radiation; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Temperature

Oritavancin, a lipoglycopeptide with marked bactericidal activity against vancomycin-resistant Staphylococcus aureus and enterococci, induces calcein release from CL:POPE and POPG:POPE liposomes, an effect enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG (Domenech et al., Biochim Biophys Acta 2009; 1788:1832-40). Using vesicles prepared from lipids extracted from S. aureus, we showed that oritavancin induces holes, erosion of the edges, and decrease of the thickness of the supported lipid bilayers (atomic force microscopy; AFM). Oritavancin also induced an increase of membrane permeability (calcein release) on a time- and dose-dependent manner. These effects were probably related to the ability of the drug to bind to lipid bilayers as shown by 8-anilino-1- naphthalene sulfonic acid (ANS) assay. Interaction of oritavancin with phospholipids at the level of their glycerol backbone and hydrophobic domain was studied by monitoring changes of Laurdan excitation generalized polarization (GP(ex)) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy upon temperature increase. Oritavancin increased GP(ex) values and the transition temperature, indicating a more ordered structure at the level of the glycerol backbone. Oritavancin slightly decreased DPH fluorescence depolarization intensities, suggesting an increase in fluidity at the level of acyl chains. Together, our data confirm the interaction of oritavancin with lipids and the potential role of a rigidifying effect at the level of glycerol backbone for membrane permeabilization. This work shows how AFM and biophysical methods may help in characterizing drug-membrane interactions, and sheds further light on the mode of action of oritavancin.

Topics: Anilino Naphthalenesulfonates; Anti-Bacterial Agents; Diphenylhexatriene; Fluorescence Polarization; Glycopeptides; Lipid Bilayers; Lipids; Lipoglycopeptides; Microscopy, Atomic Force; Permeability; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Staphylococcus aureus; Time Factors; Unilamellar Liposomes

Field induced droplet ionization mass spectrometry (FIDI-MS) comprises a soft ionization method to sample ions from the surface of microliter droplets. A pulsed electric field stretches neutral droplets until they develop dual Taylor cones, emitting streams of positively and negatively charged submicrometer droplets in opposite directions, with the desired polarity being directed into a mass spectrometer for analysis. This methodology is employed to study the heterogeneous ozonolysis of 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (POPG) at the air-liquid interface in negative ion mode using FIDI mass spectrometry. Our results demonstrate unique characteristics of the heterogeneous reactions at the air-liquid interface. We observe the hydroxyhydroperoxide and the secondary ozonide as major products of POPG ozonolysis in the FIDI-MS spectra. These products are metastable and difficult to observe in the bulk phase, using standard electrospray ionization (ESI) for mass spectrometric analysis. We also present studies of the heterogeneous ozonolysis of a mixture of saturated and unsaturated phospholipids at the air-liquid interface. A mixture of the saturated phospholipid 1,2-dipalmitoyl-sn-phosphatidylglycerol (DPPG) and unsaturated POPG is investigated in negative ion mode using FIDI-MS while a mixture of 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) and 1-stearoyl-2-oleoyl-sn-phosphatidylcholine (SOPC) surfactant is studied in positive ion mode. In both cases FIDI-MS shows the saturated and unsaturated pulmonary surfactants form a mixed interfacial layer. Only the unsaturated phospholipid reacts with ozone, forming products that are more hydrophilic than the saturated phospholipid. With extensive ozonolysis only the saturated phospholipid remains at the droplet surface. Combining these experimental observations with the results of computational analysis provides an improved understanding of the interfacial structure and chemistry of a surfactant layer system when subject to oxidative stress.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Air; Oxidation-Reduction; Ozone; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids; Pulmonary Surfactants; Spectrometry, Mass, Electrospray Ionization; Surface-Active Agents; Time Factors

We have investigated the effect of cholesterol concentration on the properties of lipid monolayers at air/water interfaces at low surface tensions. This is of interest for understanding the properties and function of lung surfactant monolayers. Lung surfactant lines the gas exchange interface in the lungs and dramatically reduces the surface tension, thereby preventing lung collapse and decreasing the work associated with breathing. Changes in the lipid composition of lung surfactant, particularly an increase in cholesterol concentration, can result in inhibition of its function, as in the case of acute respiratory distress syndrome. We have used molecular dynamics simulations with both atomistic and coarse-grained force fields to study lipid monolayers containing DPPC, POPG and cholesterol in molecular ratios of 8:2:1 and 4:1:4 at surface tensions of 40, 20 and 0 mN m(-1) at 310 K. These mixtures model the lipid component of lung surfactant at normal (approximately 9%) and elevated (approximately 44%) cholesterol concentration. We have characterised the structural and dynamic properties of these monolayers and calculated the free energy for transfer of each lipid from its equilibrium position in the monolayer into water and into air (vacuum). The results show that at low surface tensions an increase in cholesterol concentrations leads to formation of a liquid-condensed phase with low area compressibility, which is in agreement with experimental findings.

Topics: Cholesterol; Computer Simulation; Membranes, Artificial; Phosphatidylglycerols; Pulmonary Surfactants; Surface Tension

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L(alpha) or the gel phase L(beta) lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (>or=44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R(P)=0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.

Topics: Cryoelectron Microscopy; Fluorescent Dyes; Lipid Bilayers; Liposomes; Membranes, Artificial; Peptides; Phosphatidylglycerols; Polylysine; Static Electricity

Novel peptidomimetic backbone designs with stability towards proteases are of interest for several pharmaceutical applications including intracellular delivery. The present study concerns the cellular uptake and membrane-destabilising effects of various cationic chimeras comprised of alternating N-alkylated beta-alanine and alpha-amino acid residues. For comparison, homomeric peptides displaying octacationic functionalities as well as the Tat(47-57) sequence were included as reference compounds. Cellular uptake studies with fluorescently labelled compounds showed that guanidinylated chimeras were taken up four times more efficiently than Tat(47-57). After internalisation, the chimeras were localised primarily in vesicular compartments and diffusively in the cytoplasm. In murine NIH3T3 fibroblasts, the chimeras showed immediate plasma membrane permeabilising properties, which proved highly dependent on the chimera chain length, and were remarkably different from the effects induced by Tat(47-57). Finally, biophysical studies on model membranes showed that the chimeras in general increase the permeability of fluid phase and gel phase phosphatidylcholine (PC) vesicles without affecting membrane acyl chain packing, which suggests that they restrict lateral diffusion of the membrane lipids by interaction with phospholipid head groups. The alpha-peptide/beta-peptoid chimeras described herein exhibit promising cellular uptake properties, and thus represent proteolytically stable alternatives to currently known cell-penetrating peptides.

Topics: Animals; Cell Membrane; Cell Membrane Permeability; Cytoplasmic Vesicles; Flow Cytometry; Fluoresceins; Gene Products, tat; Guanidine; HeLa Cells; Humans; Membranes, Artificial; Mice; Microscopy, Confocal; NIH 3T3 Cells; Peptides; Peptoids; Phase Transition; Phosphatidylcholines; Phosphatidylglycerols; Temperature

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I' band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 degrees C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 degrees C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 degrees C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.

Topics: Animals; Calorimetry, Differential Scanning; Carrier Proteins; Chickens; Lipid Bilayers; Liver; Membrane Glycoproteins; Phase Transition; Phosphatidylglycerols; Protein Conformation; Protein Folding; Spectroscopy, Fourier Transform Infrared; Static Electricity; Temperature

We have observed a discontinuous unbinding transition of lipid bilayer stacks composed of phosphatidylethanolamine and phosphatidylglycerol using x-ray diffraction. The unbinding is reversible and coincides with the main (L(beta)-->L(alpha)) transition of the lipid mixture. Interbilayer interaction potentials deduced from the diffraction data reveal that the bilayers in the L(beta) phase are only weakly bound. The unbinding transition appears to be driven by an abrupt increase in steric repulsion resulting from increased thermal undulations of the bilayers upon entering the fluid L(alpha) phase.

Topics: Lipid Bilayers; Liposomes; Models, Chemical; Phosphatidylethanolamines; Phosphatidylglycerols; X-Ray Diffraction

Cell membranes and their lipids play critical roles in calcification. Specific membrane phospholipids promote the formation of calcium phosphate and become a part of the organic matrix of growing calcification. We propose that membrane lipids also promote the formation of calcium oxalate (CaOx) and calcium phosphate (CaP) containing kidney stones, and become a part of their stone matrix.. Human urine, crystals of CaOx and CaP produced in the urine of healthy individuals, and urinary stones containing struvite, uric acid, CaOx and CaP crystals for the presence of membrane lipids were analyzed. Crystallization of CaOx monohydrate at Langmuir monolayers of dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG) and dimyristoylphosphatidylglycerol (DMPG) was investigated to directly demonstrate that phospholipid assemblies can catalyze CaOx nucleation.. Urine as well as CaOx and CaP crystals made in the urine and various types of urinary stones investigated contained some lipids. Urine of both CaOx and uric acid stone formers contained significantly more cholesterol, cholesterol ester and triglycerides than urine of healthy subjects. However, urine of CaOx stone formers contained more acidic phospholipids. The organic matrix of calcific stones contained significantly more acidic and complexed phospholipids than uric acid and struvite stones. For each Langmuir monolayer precipitation was heterogeneous and selective with respect to the orientation and morphology of the CaOx crystals. Crystals were predominantly monohydrate, and most often grew singly with the calcium rich (10-1) face toward the monolayer. The number of crystals/mm2 decreased in the order DPPG> DPPC and was inversely proportional to surface pressure and mean molecular area/molecule.. Stone forming conditions in the kidneys greatly impact their epithelial cells producing significant differences in the urinary lipids between healthy and stone forming individuals. Altered membrane lipids promote face selective nucleation and retention of calcium oxalate crystals, and in the process become a part of the growing crystals and stones.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adult; Aged; Aged, 80 and over; Calcium Oxalate; Crystallization; Female; Humans; Kidney Calculi; Lipids; Male; Membrane Fluidity; Middle Aged; Phosphatidylglycerols; Phosphatidylserines

Langmuir isotherms, fluorescence microscopy, and atomic force microscopy were used to study lung surfactant specific proteins SP-B and SP-C in monolayers of dipalmitoylphosphatidylglycerol (DPPG) and palmitoyloleoylphosphatidylglycerol (POPG), which are representative of the anionic lipids in native and replacement lung surfactants. Both SP-B and SP-C eliminate squeeze-out of POPG from mixed DPPG/POPG monolayers by inducing a two- to three-dimensional transformation of the fluid-phase fraction of the monolayer. SP-B induces a reversible folding transition at monolayer collapse, allowing all components of surfactant to remain at the interface during respreading. The folds remain attached to the monolayer, are identical in composition and morphology to the unfolded monolayer, and are reincorporated reversibly into the monolayer upon expansion. In the absence of SP-B or SP-C, the unsaturated lipids are irreversibly lost at high surface pressures. These morphological transitions are identical to those in other lipid mixtures and hence appear to be independent of the detailed lipid composition of the monolayer. Instead they depend on the more general phenomena of coexistence between a liquid-expanded and liquid-condensed phase. These three-dimensional monolayer transitions reconcile how lung surfactant can achieve both low surface tensions upon compression and rapid respreading upon expansion and may have important implications toward the optimal design of replacement surfactants. The overlap of function between SP-B and SP-C helps explain why replacement surfactants lacking in one or the other proteins often have beneficial effects.

Topics: Amino Acid Sequence; Anions; Humans; Lung; Membranes, Artificial; Microscopy, Atomic Force; Microscopy, Fluorescence; Molecular Sequence Data; Mutation; Phosphatidylglycerols; Phospholipids; Protein Binding; Proteolipids; Pulmonary Surfactants; Surface Tension; Temperature