Unilamellar Dimyristoylphosphatidylcholine Research Articles

Reduction of nanoscopic dynamics in the zwitterionic membrane by salt

The effect of addition of lithium chloride on the lateral diffusivity and internal motion of lipid in unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles was investigated using elastic incoherent neutron scattering, quasielastic incoherent neutron scattering, and differential scanning calorimetry. A shift of the main phase transition to a higher temperature indicated a broader temperature range of the thermodynamic stability of the ordered phase resulting from the salt addition. A universal stiffening effect exerted by lithium chloride on the lipid membrane manifested itself in (1) lower mean-square displacement and (2) decreasing diffusivities, both lateral and localized, in both the ordered gel and the disordered fluid phase state. A strong reduction in the lateral lipid diffusivity was likely primarily due to the strong interaction of Li+ ions with the head group of the zwitterionic DMPC lipid and was consistent with the previous reports of the effect of a salt addition on the self-diffusivity of zwitterionic lipids in a membrane. Besides the whole lipid long-range diffusivity, quasielastic neutron scattering allowed measurements of the local internal motion of the lipid. Despite the weak interaction between the ions and the uncharged lipid tails, in both the ordered and the disordered phase states, the local diffusivity of the lipid tails decreased by a factor of 2.2 in the presence of the salt, which closely correlates with the decrease in the diffusivity (that is, an increase in viscosity) of water in the LiCl salt solution by a factor of 2.4. Regardless of the phase state of the membrane, the decrease in the local diffusivity of the lipid seems to mimic the decrease in the diffusivity (which is inverse of the increase of viscosity) of the solvents.

The THE INFLUENCE OF CHLORPROMAZINE HYDROCHLORIDE ON THE SIZE OF SMALL UNILAMELLAR DIMYROSTYLPHOSPHATIDYLCHOLINE LIPOSOMES AS REVEALED BY LIGHT SCATTERING PHOTOMETRY

Objectives: This study aims to investigate the possible influence of the model, cationic, surface-active solute chlorpromazine hydrochloride (CPZ-HCl) on the size of small unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes as a function of temperature and CPZ-HCl concentration, below and above the critical micelle concentration (CMC).
 Methods: Small unilamellar DMPC liposomes were prepared by dissolving DMPC in chloroform and the solvent was rota-evaporated in a water bath adjusted at 40 °C. The lipid film was then dispersed in 0.1 M KCl solution adjusted at pH 6.2 to form large multilamellar liposomes which are then sonicated and fractionated via Sepharose 2B-Cl gel. The elution profile was followed spectrophotometrically at λ 260 nm. Combined fractions from the trailing edge of the included peak which is due to small unilamellar liposomes, were used as a source throughout this study. The SOFICA light scattering photometer (Model 42000) was used to determine the weight average liposomes weight (Lw) of small unilamellar DMPC liposomes. The Lw was determined in the absence and presence of CPZ-HCl both above and below the CMC over the temperature range of 25 °C to 40 °C.
 Results: The Lw was observed to increase linearly in the absence and presence of CPZ-HCl.
 The Lw was observed to increase linearly in the absence of CPZ-HCl, from 1.88×106+0.02 g/mol at 25 °C to 3.25×106+0.03 g/mol at 40 °C. Similarly, the Lw was observed to increase linearly in the present of CPZ-HCl, for example at 18 mmol drug concentration, the Lw increases from 11×106+0.04 g/mol at 25 °C to 13.75×106+0.03 g/mol at 40 °C. When the data are presented as a function of CPZ-HCl concentration, a gradual increase in Lw was observed below the CMC. Little increase in Lw however, was observed at post-micellar concentrations of 14 mmol and 18 mmol.

Investigation of the structure of unilamellar dimyristoylphosphatidylcholine vesicles in aqueous sucrose solutions by small-angle neutron and X-ray scattering

The structure of a polydispersed population of unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles in sucrose solutions has been investigated by small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). Calculations within the model of separated form factors (SFF) show that the structure of the vesicle system depends strongly on the sucrose concentration.

The effect of intercalants on the host liposome

When phospholipids are vigorously dispersed in water, liposomes are formed. In the present study, we have explored the effect of intercalant concentration on various properties of unilamellar liposomes. Liposomes were sonically intercalated with vitamin E acetate (VitEAc) and hypericin (Hy) until no difference in light transmission was observed, which reflects the formation of liposomes of minimal diameter. Our studies indicate that the intercalant structure and concentration have an influence on the liposome diameter, which could be directly measured by cryogenic transmittance electronic microscopy. Thus, intercalated VitEAc substantially decreased the diameter of unilamellar dimyristoylphosphatidylcholine liposomes, whereas Hy did not. In addition, we followed peak intensities in the absorbance and fluorescence spectra of Hy as a function of intercalant concentration in the liposomal solution. Initially, the fluorescence intensity increased linearly with concentration; however, the curve then arched asymptotically, followed by a decrease in fluorescence at yet higher concentrations. Because the Hy monomer is the only species that emits fluorescence, we believe that the decrease of fluorescence intensity is the result of Hy aggregation.

Structure and phospholipase function of peroxiredoxin 6: identification of the catalytic triad and its role in phospholipid substrate binding

Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A(2) (PLA(2)) activities, and it alone among mammalian peroxiredoxins can hydrolyze phospholipids. After identifying a potential catalytic triad (S32, H26, D140) from the crystal structure, site-specific mutations were used to evaluate the role of these residues in protein structure and function. The S32A mutation increased Prdx6 alpha-helical content, whereas secondary structure was unchanged by mutation to H26A and D140A. Lipid binding by wild-type Prdx6 to negatively charged unilamellar liposomes showed an apparent rate constant of 11.2 x 10(6) M(-1) s(-1) and a dissociation constant of 0.36 microM. Both binding and PLA(2) activity were abolished in S32A and H26A; in D140A, activity was abolished but binding was unaffected. Overoxidation of the peroxidatic C47 had no effect on lipid binding or PLA(2) activity. Fluorescence resonance energy transfer from endogenous tryptophanyls to lipid probes showed binding of the phospholipid polar head in close proximity to S32. Thus, H26 is a site for interfacial binding to the liposomal surface, S32 has a key role in maintaining Prdx6 structure and for phospholipid substrate binding, and D140 is involved in catalysis. This putative catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate to optimize the protein-substrate complex for hydrolysis.

Receptor-mediated biological responses are prolonged using hydrophobized ligands

Hormone–receptor interactions occur following three-dimensional diffusion of the ligand to the membrane-embedded receptor. However, prior hydrophobization of the ligand might restrict its movement to two dimensions along the membrane surface, and the biological response might therefore be modulated. This idea was tested using the C-terminal nonapeptide, CCK 9, of the satiating hormone, cholecystokinin (CCK). The hormone was lipidated by linking it covalently to distearoylphosphatidylethanolamine via a poly(ethylene glycol) (PEG) spacer. The desired conjugate was isolated by thin-layer chromatography and incorporated into preformed small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. The hormone-bearing vesicles were injected intraperitoneally into Wistar rats and food intake monitored. Compared to the biological effect elicited by the same amount of soluble non-derivatized CCK 9, food intake reduction showed a delayed onset, but lasted for a significantly longer time. We believe this prolonged effect was due to the transfer of the derivatized CCK 9 from the vesicles to the natural membrane containing the hormone receptor. Ultimately, this event may result in sustained receptor occupation and, thus, food intake reduction. The underlying mechanism for the physiological effects observed may be of relevance in interpreting results obtained using artificial measuring devices; for example, the signal produced by biosensors may be drastically affected by the hydrophobicity of the ligand.

On the origin of the dynamic Stokes shift of Laurdan molecules in lipid membranes

Frequency-resolved time correlated single photon counting experiments have been performed to investigate the time scales and molecular mechanisms for solvation dynamics of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) embedded in liquid-crystalline bilayers of unilamellar dimyristoyl-phosphatidylcholine (DMPC) vesicles. The reconstructed fluorescence spectra as a function of time exhibit a pronounced double-exponential Stokes shift, which is strongly correlated with the rotational diffusion dynamics of the chromophore as evidenced by experiments on transient fluorescence depolarization.

Investigation of thin films formed from liposome suspensions on quartz substrate

The stability of thin wetting films formed from 0.15 M NaCl solutions containing small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles of different concentrations on quartz surface has been investigated by the microinterferometric method. The intensity of monochromatic light reflected from both film surfaces has been recorded as a function of the time of film thinning. Two temperatures were used in the experiments (20 and 35°C). Films containing 10 −3, 5×10 −3 and 10 −2 mg/ml DMPC were unstable and ruptured, while films with 10 −1 and 1 mg/ml DMPC were stable. Film stability was explained on the basis of hydrophobic interactions. Film thickness dependence on time was calculated. The kinetics of film thinning did not obey Reynolds equation and a linearization was observed in co-ordinates ln( h) as a function of time. This phenomenon was explained by a non-homogeneous thinning process, which might be due to the existence of some areas of different structure of the DMPC adsorption layers.

A Thermodynamic Study of the Effects of Cholesterol on the Interaction between Liposomes and Ethanol

The association of ethanol with unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes of varying cholesterol content has been investigated by isothermal titration calorimetry over a wide temperature range (8–45°C). The calorimetric data show that the interaction of ethanol with the lipid membranes is endothermic and strongly dependent on the phase behavior of the mixed lipid bilayer, specifically whether the lipid bilayer is in the solid ordered (so), liquid disordered (ld), or liquid ordered (lo) phase. In the low concentration regime (<10 mol%), cholesterol enhances the affinity of ethanol for the lipid bilayer compared to pure DMPC bilayers, whereas higher levels of cholesterol (>10 mol%) reduce affinity of ethanol for the lipid bilayer. Moreover, the experimental data reveal that the affinity of ethanol for the DMPC bilayers containing small amounts of cholesterol is enhanced in the region around the main phase transition. The results suggest the existence of a close relationship between the physical structure of the lipid bilayer and the association of ethanol with the bilayer. In particular, the existence of dynamically coexisting domains of gel and fluid lipids in the transition temperature region may play an important role for association of ethanol with the lipid bilayers. Finally, the relation between cholesterol content and the affinity of ethanol for the lipid bilayer provides some support for the in vivo observation that cholesterol acts as a natural antagonist against alcohol intoxication.

Solvation Dynamics of DCM in Lipid

Solvation dynamics of 4-(dicyanomethylene)-2-methyl-6(p-dimethylaminostyryl)-4H-pyran (DCM) in dimyristoylphosphatidylcholine (DMPC) vesicles in water is studied by using the picosecond time-resolved Stokes shift technique. In unilamellar DMPC vesicles, DCM exhibits wavelength-dependent fluorescence decays with a fast decay at the blue end and a slow decay preceded by a distinct growth at the red end of the emission spectrum. The solvation dynamics of DCM in DMPC vesicles is found to be biexponential with two components 230 ps (40%) and 1.6 ns (60%) and is attributed to the relaxation of the water molecules inside the water pool of DMPC vesicles.

Polymer-Cushioned Bilayers. II. An Investigation of Interaction Forces and Fusion Using the Surface Forces Apparatus

We have created phospholipid bilayers supported on soft polymer “cushions” which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to “solid-supported” membranes, such “soft-supported” membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature ( T m ≈ 24°C). Above T m, hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T m (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions.

A Calorimetric Investigation of the Interaction of Short Chain Alcohols with Unilamellar DMPC Liposomes

The molecular mechanisms by which ethanol and other short-chain alcohols perturb biological systems have been suggested to involve specific interactions with proteins and/or nonspecific interactions with the lipid bilayer part of biological membranes. To gain insight into the influence of alcohols on well-defined lipid bilayers, isothermal titration calorimetry (ITC) is used to determine the excess partial molar enthalpy of alcohol in liposome suspensions as well as the excess partial molar enthalpy of liposomes in dilute alcohol−water mixtures at temperatures above the main phase transition of dimyristoyl phosphatidylcholine (DMPC). The alcohols investigated were ethanol, 1-propanol, and 1-butanol. The calorimetric data were used to evaluate molecular interactions and based on a simple partitioning model the standard molar enthalpy of transferring alcohol from water into unilamellar DMPC liposomes was determined. The results reveal that the interaction of short-chain alcohols with the DMPC lipid bilayer ...

Structural Studies of Polymer-Cushioned Lipid Bilayers

The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.

Solvation Dynamics of Coumarin 480 in Vesicles

Solvation dynamics of Coumarin 480 (C-480) in dimyristoylphosphatidylcholine (DMPC) vesicles in water is studied using picosecond time-resolved Stokes shift. In sonicated unilamellar DMPC vesicles C-480 exhibits wavelength dependent fluorescence decays. At short wavelengths a fast decay is observed while at the long wavelengths a growth in the nanosecond time scale precedes the decay. The solvation dynamics of C-480 in DMPC vesicles is found to be bimodal with two components of 0.6 and 11 ns, which is similar to the solvation dynamics of C-480 in the large water pools of AOT/n-heptane/water (AOT = aerosol OT) microemulsions.

Novel Features of the Main Transition of Dimyristoylphosphocholine Bilayers Revealed by Fluorescence Spectroscopy

Lateral heterogeneity is evidenced in large unilamellar dimyristoylphosphatidylcholine vesicles by the transient peak at T* ≈ 22 °C (approximately two degrees below the main transition temperature Tm) in intermolecular excimer formation by the pyrene-labeled phospholipid probe 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC). Using three different fluorescence quenchers, cholesteryl 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bis(nor-5-cholen-3β-ol) (NBD-chol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresceinthiocarbamoyl) (DPPF), and 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproylphosphocholine (NBD-PC) partitioning preferentially into the (i) phase boundary, (ii) gel phase, and (iii) fluid phase, respectively, we could demonstrate PPDPC at T* to become weakly enriched into the interfacial boundary separating “fluid” domains from the bulk gel phase. Unexpectedly, at Tm there was a minimum in the colocalization of all three quenchers with PPDPC excimers. Our data are thus incompatible with the coexistence of fluid and gel state domains at Tm. Accordingly, the nature of the fluctuating entities underlying the heat capacity maximum at Tm must be reconsidered.1 Phospholipid main transition is suggested to involve two transitions, from the gel state to an intermediate and from this intermediate state to the fluid state. Our data indicate the possibility that the intermediate phase in the vicinity of Tm is a strongly fluctuating “pseudocrystalline” superlattice of fluid (“excited”) and gel (“ground”) state lipids, with augmented short-range positional order of embedded impurities.

Time-Resolved Infrared ATR Measurements of Liposome Transport Kinetics in Human Keratinocyte Cultures and Skin Reveals a Dependence on Liposome Size and Phase State

A novel in vitro method for studying the permeation kinetics of superficially applied liposomes or vesicles through layers of human skin or keratinocytes on a solid support is presented, employing attenuated total reflection infrared spectroscopy. The method is applied to investigate transport kinetics of unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) through cultured human keratinocyte layers and through human skin. We find a strong resemblance of the qualitative features of the permeation kinetics of small unilamellar DMPC vesicles for skin and keratinocytes. Detailed studies of the vesicles transport through keratinocyte layers show that DMPC vesicles with an average diameter of 55 nm can readily permeate through the layer at 37 degrees C with a diffusion constant of D = (4.0 +/- 0.8) x 10(-15) m2/second, whereas larger vesicles of twice that diameter do not permeate at all. In contrast, liposomes containing a chemical permeation enhancer permeate through the layer significantly faster [D = (7.0 +/- 0.5) x 10(-15) m2/second] than the small DMPC vesicles despite their five-times-larger diameter. Moreover, the transport of the DMPC vesicles depends drastically on their phase state. No permeation was observed for small DMPC vesicles at a temperature of 10 degrees C when the lipid is in the crystalline phase state. Our results indicate that keratinocyte culture layers can pose a significant permeation barrier for vesicles. The permeation mechanism can be explained by diffusion of the vesicles through small pores and gaps in the layer, presumably driven by transdermal osmotic gradients.

A fluorine-19 NMR study of interactions between the ATP-citrate lyase inhibitor SB-200517 and model membranes

Fluorine-19 NMR ( 19F-NMR) has been used to study the properties of SB-200517 (I), an inhibitor of the hepatic enzyme ATP-citrate lyase (ACL), in small sonicated unilamellar dimyristoyl phosphatidylcholine (DMPC) vesicles, SUVs. Two relatively sharp signals are observed in this environment, only the lower field of which is broadened by the paramagnetic relaxation-enhancing probe Mn 2+. This suggests that the splitting observed in SUVs is due to the slightly different environments experienced by fluorine atoms of inhibitor molecules localised to either the inner (higher field signal) or the outer (lower field signal) leaflet of the vesicles. The presence of fluorine in the compound thus provides a quick and convenient method of studying its partitioning into non-aqueous environments; such partitioning can have significant implications for bio-availabilty.

Atomic force microscopy of dimyristoylphosphatidylcholine membranes.

We have observed the surface images of dimyristoylphosphatidylcholine (DMPC) monolayer and DMPC vesicles with an atomic force microscope (AFM). It was confirmed that the lattice structure of polar head groups of DMPC molecules in a Langmuir-Blodgett monolayer is similar to those of large unilamellar DMPC vesicles. AFM images of small unilamellar vesicles of DMPC showed larger lattice structures than those of the monolayer, reflecting differences of molecular packing in these membranes.

Direct measurement of interaction forces in free thin liquid films stabilized with phosphatidylcholine

Microscopic foam films from suspensions of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles have been obtained. The film formation is facilitated at temperatures above the gel-liquid crystalline transition of DMPC. A detailed study of the dependence of equilibrium thickness of DMPC foam films on electrolyte concentration at constant capillary pressure and a direct measurement of the disjoining pressure isotherm has been carried out. Formation of thick equilibrium horizontal microscopic films stabilized with DMPC at low external pressures and low electrolyte concentrations was found and interpreted as being due to the existence of long-range electrostatic interactions in these films. A diffuse electric layer potential of 36 mV has been calculated. The DMPC films have been compared to films obtained from non-ionic surfactant solutions where the long range electrostatic repulsion is explained as being due to specific adsorption of OH− at the film interfaces. However, unlike the results obtained for surfactant films, in this study formation of common black films and thinning of the DMPC Newton film with pressure have been observed.

Comparison of the photochromism of a spirobenzopyran derivative in unilamellar surfactant vesicles and solvent-cast surfactant films

The thermal decolouration of the photoinduced merocyanine form of 1′-hexadecyl-3′,3′-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2′-indoline](SP-16) has been examined in a number of different aqueous unilamellar surfactant vesicle solutions and solvent-cast surfactant film matrices. The thermal decolouration of SP-16 in solvent cast dihexadecylphosphate (DHP) films and unilamellar dimyristoyl phosphatidylcholine (DMPC) vesicles exhibits single first-order kinetics. In DHP vesicles and solvent-cast DMPC films the thermal decolouration deviates from single first-order behaviour. The thermal decolouration in both these systems is well described by the super-position of two first-order rate processes. Dual first-order kinetics in the vesicular DHP media has been attributed to the merocyanine moieties of SP-16 partitioning between two residential sites within the vesicles, probably the surfactant/water interfacial region and the hydrocarbon chain domain. Dual first-order kinetics in the DMPC films has been ascribed to part of the merocyanine population being complexed with phosphatidylcholine headgroups and the other part of the population being located in hydrocarbon chain domains. In solvent-cast poly(diallyl dimethylammonium) dihexadecylphosphate (DHPP) films the thermal decolouration of SP-16 deviates from single first-order behaviour. The thermal decolouration in DHPP films is inadequately described as a dual first-order process but is well described as a dispersive process.