dipalmitoyl-L-α-phosphatidylcholine Research Articles

Preparation of liposomes with phospholipids and study of their photo-interaction with a fluorophore-containing compound

Aim. To substantiate the optimal technology for obtaining two types of bio-related liposomes based on the composition of dipalmitoyl-L-α phosphatidylcholine (DPPC) or palmitoyl-sphingomyelin-N-hesadecanoyl-D erythrosphingosylphosphorylcholine (C16-SM) with cholesterol and study their interaction with a fluorophore-containing compound. Materials and methods. To prepare liposomal vesicles based on phospholipids – DPPC, C16-SM and cholesterol (1:1) the method of reverse-phase evaporation on a rotary vacuum evaporator Bϋchi-210 was used. Ultrasonic homogenization was performed using a Jeken (Codyson) PS-08A sonicator. A fluorescent compound R-203 (5’-isopropyl-3’- ((4-methyl-2-oxo-2H-chromen-7-yl) -carbamoyl) -2-oxospiro-[indoline-3,2’-pyrrolidine] -4’-carbon) with a 7-amino-coumarin (7-AMC) fluorophore was obtained by the synthesis previously described. Liposome particle size determination was measured using laser diffraction on a LA-960 Laser Particle Size Analyzer (Horiba Scientific). The measuring range of the device is 0.01 μm to 5000 μm; guaranteed accuracy is ± 0.6 %. Fluorescence spectra were recorded in the emission range of 365… 650 nm on a FluoroMax®-4 spectrofluorometer (Horiba Scientific) at an excitation wavelength of 350 nm. R-203 fluorescence spectra (λem = 412 nm) were measured in a 500 μl cuvette. Results and discussion. Target liposomes were obtained based on the composition of DPPC or C16-SM phospholipids with cholesterol in the ratio of phospholipids and cholesterol 1:1. The optimal size of liposomes of about 100 nm was achieved for the composition of C16-SM: cholesterol (1:1). The calculated packaging parameter (γ) allowed us to conclude that in the case of DPPC and C16-SM phospholipids, the parameter γ < 1, i.e. the molecules of these lipids had a cone shape and were packed in phase I type where the molecules could form spherical (vesicles) and / or cylindrical micelles. However, the C16-SM phospholipid has a larger polar area of the molecule (a0), in contrast to the DPPC molecule. Fluorescence spectra of R-203 interaction with DPPC : cholesterol and C16-SM : cholesterol (1:1) liposomes in phosphate-buffered saline pH 7.4 were studied. In the interaction of the fluorescent substance R-203, quenching of the fluorescence intensity was observed according to the Stern–Volmer equation; it was more intense for C16-SM: cholesterol liposomes than in the case of DPPC: cholesterol liposomes. Conclusions. The optimal technology for obtaining two types of bio-related liposomes based on the composition of DPPC or C16-SM with cholesterol (1: 1) has been proposed using the method of reverse phase evaporation with ultrasound treatment; the control parameters of their technology have been determined. Liposomes based on C16-SM interact more intensively with the 7-AMK fluorophore of the compound R-203 in the buffer.

Patterning of Magnetic Bimetallic Coordination Nanoparticles of Prussian Blue Derivatives by the Langmuir–Blodgett Technique

We report a novel method to prepare patterns of nanoparticles over large areas of the substrate. This method is based on the adsorption of the negatively charged nanoparticles dispersed in an aqueous subphase onto a monolayer of the phospholipid dipalmitoyl-l-α-phosphatidylcholine (DPPC) at the air-water interface. It has been used to prepare patterns of nanoparticles of Prussian blue analogues (PBA) of different size (K(0.25)Ni[Fe(CN)(6)](0.75) (NiFe), K(0.25)Ni[Cr(CN)(6)](0.75) (NiCr), K(0.25)Ni[Co(CN)(6)](0.75) (NiCo), Cs(0.4)Co[Cr(CN)(6)](0.8) (CsCoCr), and Cs(0.4)Co[Fe(CN)(6)](0.9) (CsCoFe)). The behavior of DPPC monolayer at the air-water interface in the presence of the subphase of PBA nanoparticles has been studied by the compression isotherms and Brewster angle microscopy (BAM) images. Atomic force microscopy (AFM) of the transferred films on mica substrates shows that patterns of the nanoparticles are observed for a 10(-4) M concentration of the subphase, based on the nanoparticle precursors, at surface pressures between 1 and 6 mN/m and transfer velocities from 10 to 80 mm/min. Vertical, horizontal, or tilted fringes of the nanoparticles with respect to the transfer direction can be obtained depending on the transfer velocity and surface pressure.

Insertion properties of cholesterylcyclodextrins in phospholipid membranes: a molecular study

Amphiphilic cyclodextrins (CDs) are good candidates to functionalize natural membranes, as well as synthetic vesicles. In this paper, we fully describe and compare the insertion properties of the permethylated mono-cholesteryl α-CD (TASC) and its mono- and di-cholesteryl β-CD analogues (TBSC and TBdSC) in dipalmitoyl-L-α-phosphatidylcholine (DPPC) mono- and bi-layers as membrane models from the macroscopic to the molecular scale. By calculating the inverse compressibility moduli and free excess Gibbs energies from the Langmuir isotherms, the influence of the CD type, CD ratio and number of cholesteryl anchors on the membrane properties have been established. TBdSC, with its two cholesteryl residues, seems to be anchored best to the membrane compared to CD derivatives with only one anchor. Furthermore, TASC appears to be more firmly inserted into the membrane than TBSC. The in-plane structure is characterized by Brewster angle microscopy (BAM) at the air–water interface and atomic force microscopy (AFM) of the mono- and bi-layers deposited on mica. Depending on the compression, full miscibility of the cholesteryl CDs and the phospholipids is observed at low surface pressures and a clear demixing tendency is apparent during compression. CD-modified bilayers are stable and are subject to a gel–liquid phase transition upon heating. Due to their bulky CD moiety, the amphiphilic CDs exhibit a distinct fluidizing effect, shifting the DPPC's gel–liquid transition. The structure of the mixed TASC/DPPC mono- and bi-layers perpendicular to the surface is investigated with Angstrom resolution by neutron reflectivity. In this way a molecular model of the insertion has been established, which suggests that the CD cavities partly protrude into the subphase, which should leave them accessible for complex formation.

Structural characterization of liposomes made of diether archaeal lipids and dipalmitoyl-L-α-phosphatidylcholine

The physicochemical properties of binary lipid mixtures of diether C 25,25 lipids and dipalmitoyl-L-α-phosphatidylcholine (DPPC) were studied using photon correlation, fluorescence and electron paramagnetic resonance spectroscopy, and transmission electron microscopy. These two types of lipids can be mixed at all molar ratios to form unilamellar and multilamellar liposomes. Fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatrien in mixed liposomes indicates that the abrupt changes in order parameter in the hydrophobic part of bilayer membranes made of DPPC lipids disappears with increasing mol% C 25,25 lipids. Electron paramagnetic resonance spectroscopy shows that at temperatures below 50 °C, the interfacial regions of membrane bilayer of mixed liposomes is more fluid than for pure DPPC liposomes, while at higher temperatures, the impact of the long isoprenoid chains on the membrane stability becomes more pronounced. Photon correlation spectroscopy and transmission electron microscopy show that mixed liposomes do not fuse or aggregate, even after 41 days at 4 °C.

Amphiphilic Behavior and Membrane Solubility of a Dicholesteryl-Cyclodextrin

Amphiphilic cyclodextrins (CDs) are good candidates to functionalize natural membranes as well as synthetic vesicles. In this paper, we provide a full description of the interfacial behavior of pure 6I,6IV-(β-cholesteryl)succinylamido-6I,6IV-(6-deoxy-per-(2,3,6-O-methyl))cycloheptaose (TBdSC) and how it inserts in dipalmitoyl-l-α-phosphatidylcholine (DPPC) monolayers as a membrane model. Langmuir isotherms of pure TBdSC suggest a reorganization upon compression, which could be clarified using X-ray reflectivity. The CD head can adjust its conformation to the available area per molecule. A compatible model involving a rotation around a horizontal axis defined by the two selectively substituted glucose units is proposed. The in-plane structure is characterized at all scales by Brewster angle microscopy (BAM) on the water surface and atomic force microscopy (AFM) on monolayers deposited on solid substrates. The same tools are used for its mixtures with DPPC. We show in particular that TBdSC seems to be soluble in the liquid-expanded DPPC. However, phase segregation occurs at higher pressure, allowing for sequentially liquid-condensed DPPC and high-pressure conformation of TBdSC. This gives rise to a remarkable contrast inversion in both imaging methods.

Compressibilities and Volume Fluctuations of Archaeal Tetraether Liposomes

Bipolar tetraether lipids (BTLs) are abundant in crenarchaeota, which thrive in both thermophilic and nonthermophilic environments, with wide-ranging growth temperatures (4–108°C). BTL liposomes can serve as membrane models to explore the role of BTLs in the thermal stability of the plasma membrane of crenarchaeota. In this study, we focus on the liposomes made of the polar lipid fraction E (PLFE). PLFE is one of the main BTLs isolated from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using molecular acoustics (ultrasound velocimetry and densimetry), pressure perturbation calorimetry, and differential scanning calorimetry, we have determined partial specific adiabatic and isothermal compressibility, their respective compressibility coefficients, partial specific volume, and relative volume fluctuations of PLFE large unilamellar vesicles (LUVs) over a wide range of temperatures (20–85°C). The results are compared with those obtained from liposomes made of dipalmitoyl-L- α-phosphatidylcholine (DPPC), a conventional monopolar diester lipid. We found that, in the entire temperature range examined, compressibilities of PLFE LUVs are low, comparable to those found in gel state of DPPC. Relative volume fluctuations of PLFE LUVs at any given temperature examined are 1.6–2.2 times more damped than those found in DPPC LUVs. Both compressibilities and relative volume fluctuations in PLFE LUVs are much less temperature-sensitive than those in DPPC liposomes. The isothermal compressibility coefficient ( β T lipid) of PLFE LUVs changes from 3.59 × 10 −10 Pa −1 at 25°C to 4.08 × 10 −10 Pa −1 at 78°C. Volume fluctuations of PLFE LUVs change only 0.25% from 30°C to 80°C. The highly damped volume fluctuations and their low temperature sensitivity, echo that PLFE liposomes are rigid and tightly packed. To our knowledge, the data provide a deeper understanding of lipid packing in PLFE liposomes than has been previously reported, as well as a molecular explanation for the low solute permeation and limited membrane lateral motion. The obtained results may help to establish new strategies for rational design of stable BTL-based liposomes for drug/vaccine delivery.

Active Uptake of Drugs into Photosensitive Liposomes and Rapid Release on UV Photolysis ¶

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-l-α-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4′-n-butylphenyl)azo-4′-(γ-phenylbutyroyl))-glycero-3-phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30°C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photoinduced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.

Interaction of gadolinium with phospholipids bilayer membranes

The effects of concentration of gadolinium ions Gd3+ on dipalmitoyl L-α-phosphatidylcholine (DPPC) unilamellar vesicles in aqueous media were studied by photon correlation spectroscopy (PCS) and differential scanning calorimeter. The theoretical predictions of the colloidal stability of liposomes were followed using the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory. Changes in the size of liposomes were observed as Gd3+ concentration increases, suggesting that this cation induces the aggregation of vesicles. To determine the effect of Gd3+ on the transition temperature (Tc) and on the enthalpy (ΔHc) associated with the process differential scanning calorimetry (DSC) has been used. The addition of the metal ion provided DSC curves with different behavior to DPPC bilayer.

Real-Time Monitoring of the Kinetics and Gas-Phase Products of the Reaction of Ozone with an Unsaturated Phospholipid at the Air−Water Interface

While the kinetics and mechanisms of the reaction of O3 with alkenes in the gas and condensed phases are reasonably well understood, those with unsaturated organics in the intermediate regime at the air−water interface are not. Studies of the reaction of ozone at room temperature with the unsaturated phospholipid, 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) at the air−water interface, and, for comparison, the fully saturated dipalmitoyl-l-α-phosphatidylcholine (DPPC) were carried out. The phospholipids were exposed at varying surface areas per phosphocholine molecule on a water subphase to a flow of O3 in air (0.25−1 ppm), and atmospheric pressure ionization mass spectrometry (API-MS) was used to monitor the formation of gaseous products in real time. Nonanal was detected as a major gas-phase product of the reaction of ozone with OPPC; no volatile products were observed in the case of DPPC. The yield of nonanal, defined as the nonanal produced per phosphocholine molecule reacted at the air−wat...

Photochemical Generation of Gold Nanoparticles in Langmuir−Blodgett Films

Gold nanoparticles were generated by ultraviolet irradiation of Langmuir−Blodgett (LB) films of octadecylamine (ODA), 4-hexadecylaniline (HDA), and benzyldimethylstearylammonium chloride monohydrate (BDSAC) deposited from aqueous HAuCl4 subphases. In contrast, no gold crystals were observed in irradiated LB films prepared from monolayers of dipalmitoyl-dl-α-phosphatidyl-l-serine (DPPS) and dipalmitoyl-l-α-phosphatidylcholine (DPPC). X-Ray photoelectron spectroscopy, UV−visible absorption spectroscopy, atomic force microscopy, and transmission electron microscopy measurements indicated the marked influence of the surfactants used to prepare the LB matrix on the shape of the gold particles. Particles formed in ODA and BDSAC LB films were grown with well-defined crystal faces, while particles generated in HDA LB films were irregular in shape.

Plasmodium falciparum: Effects of Membrane Modulating Agents on Direct Binding of Rhoptry Proteins to Human Erythrocytes

We studied the effects of membrane modulation on the interaction of Plasmodium falciparum rhoptry proteins of 140/130/110 kDa (Rhop-H) with human and mouse erythrocytes. Cells treated with 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylcydopropyl)octanoate, myristoleyl alcohol, and proteins extracted with sublytic concentrations of membrane solubilizing detergents were used in erythrocyte binding assays. Protein binding was evaluated by immunoblotting using Rhop-H- and SERA-specific antisera, 1B9, K15, and 5E3, respectively. Protein binding to liposomes prepared with dipalmitoyl-L-α-phosphatidylcholine (DPPC) or dilauroyl-L-α-phosphatidylcholine (DLPC) was also examined. Our results show that erythrocyte membrane modulation markedly enhanced direct Rhop-H binding to intact human erythrocytes. Binding of SERA to intact human erythrocytes appeared unaffected. Both DPPC and DLPC liposomes had similar Rhop-H and SERA protein binding activities. However, binding to DLPC liposomes was reduced. Rhop-H and SERA extracted with the detergents octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, sodium deoxycholate, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate bound directly to intact human erythrocytes, probably by partitioning hydrophobically into the membranes. Sodium carbonate treatment demonstrated a nonintegral association of Rhop-H with the erythrocyte membrane during invasion. Membrane modulation may expose cryptic phospholipid binding sites in the bilayer.

Interaction of heparin with amphiphile assemblies and biocompatibility of the heparin complexes.

Addition of heparin to preformed vesicles of a cationic surface-active compound, dioctadecyldimethylammonium bromide (DODAB), resulted in fast precipitation of the heparin/DODAB complex. On the other hand, heparin stabilized the vesicle composed of dipalmitoyl L-alpha-phosphatidylcholine (DPPC) which is a neutral lipid. However, DPPC precipitated with heparin by the addition of a small amount of stearylamine (SA), which is a cationic substance. Differential scanning calorimetry of the heparin/amphiphile mixtures showed that the heparin/DODAB complex possess a structure different from that of the original DODAB vesicle. The structure of the DPPC vesicle was unaffected by the addition of heparin, while the structure of the heparin/DPPC-SA complex was found to be different from that of the DPPC-SA vesicle. The interaction of heparin with vesicles depended on the nature of amphiphiles. Heparin was solubilized in organic solvent when complexed with DODAB or DPPC-SA vesicles. Polyurethaneurea membranes blended with heparin/DODAB or heparin/DPPC-SA complexes were highly nonthrombogenic, but somewhat cytotoxic.

NMR study of phosphatidylcholine hydration in relation to its state of aggregation in CDCl 3

Hydration of dipalmitoyl-L- α - phosphatidylcholine (DPPC) was studied in relation to its aggregation in CDCl 3] by 1H and 31P NMR. Proton chemical shifts of DPPC segments were studied as a function of DPPC concentration in CDCl 3 to reveal the segments which could serve as aggregation sensitive internal probes. The most aggregation-sensitive are the fragments closes to the phosphatydic group, namely POCH 2chol and Ch 2OP gly. Fragments not sensitive to aggregation are the hydrocarbon tail groups (CH 2)n and CH 3. Water equimolar to DPPC serves as a very sensitive external probe of DPPC aggregation. Contrary to the aggregated DPPC the monomers of DPPC in CDCl 3 do not appear to be hydrated. Evidence for this are the 1H chemical shift and spin-lattice relaxation time of water and the 31P chemical shift of the phosphatidyl group of DPPC.

PHOTOSENSITIZED ELECTRON TRANSPORT IN THE ASYMMETRICAL VESICLES INCORPORATING A SURFACE-ACTIVE ZINC PORPHYRIN AND AN ELECTRON MEDIATOR

Abstract Photosensitized electron transport from ethylenediaminetetraacetic acid disodium salt (EDTA·2Na) or sodium ascorbate (Asc·Na) to methylviologen (MV++) across vesicle membranes of dipalmitoyl-l-α-phosphatidylcholine (DPPC) which incorporate 5,10,15-tris(1-methylpyridinium-4-yl)-20-[4-(octadecyloxy)phenyl]porphinatozinc(II) trichloride (Zn–P) asymmetrically (only on the outer side of the membrane) is facilitated by an electron mediator such as ubiquinone Q10 (UQ10) or tetraphenylporphine (TPP). Zn–P plays an important role in the redox reaction at the interface of the vesicles and the efficiency can be correlated with the redox potentials of the mediators.

Orientational order in the choline headgroup of sphingomyelin: A 14N-NMR study

An aqueous dispersion of fully hydrated bovine sphingomyelin was studied using 14N-NMR spectroscopy. Spectra were obtained as a function of temperature over the range 15–80°C, in both the liquid crystal and gel phases. In the liquid crystal phase, powder pattern lineshapes were obtained, whose quadrupolar splitting slowly decreases with increasing temperature. The spectra are increasingly broadened as the temperature is lowered through the phase transition into the gel phase. The linewidths and the second moments of these spectra indicate that the onset of a broad phase transition occurs at approx. 35°C, in agreement with previous calorimetric and 31P-NMR measurements. There is no evidence from the lineshapes for an hexagonal phase in this system, and this conclusion is supported by X-ray diffraction measurements carried out on aqueous dispersions of sphingomyelin in both phases. Assuming that the static nitrogen quadrupole coupling constant is the same for both sphingomyelin and dipalmitoyl- l-α-phosphatidylcholine (DPPC), the decrease observed in the quadrupolar splitting of sphingomyelin compared to that of DPPC indicates that the orientational order of the choline headgroup in liquid crystalline sphingomyelin is not the same as that of its counterpart in DPPC. Preliminary relaxation time measurements of T 1 and T 2 are presented which suggest that there are also dynamic differences between sphingomyelin and DPPC in the choline headgroup.

ELECTRON TRANSPORT ACROSS LIPID MEMBRANES PHOTOSENSITIZED BY AN AMPHIPHILIC ZINC PORPHYRIN

Abstract Electron transport reaction from EDTA·2Na to methylviologen (MV2+) across the membrane of single-lamella vesicles of dipalmitoyl-l-α-phosphatidylcholine (DPPC) is photosensitized by an amphiphilic zinc porphyrinato complex, 5,10,15-tris-(l-methylpyridinium-4-yl)-20-(4-stearoxyphenyl)porphyrinatozinc(II) trichloride (Zn–P). This porphyrin complex can be incorporated into the vesicles symmetrically (on both sides of the membrane) or asymmetrically (only on the outer side). The symmetrical system facilitates the electron transport even in the absence of electron mediators.