Localization of sterically stabilized liposomes in Klebsiella pneumoniae-infected rat lung tissue: influence of liposome characteristics.

Sterically stabilized liposomes are able to localize at sites of infection and could serve as carriers of antimicrobial agents. For a rational optimization of liposome localization, the blood clearance kinetics and biodistribution of liposomes differing in poly(ethylene glycol) (PEG) density, particle size, bilayer fluidity or surface charge were studied in a rat model of a unilateral pneumonia caused by Klebsiella pneumoniae. It is shown that all liposome preparations studied localize preferentially in the infected lung compared to the contralateral non-infected lung. A reduction of the PEG density or rise in particle size resulted in a higher uptake by the mononuclear phagocyte system, lower blood circulation time and lower infected lung localization. Differences in bilayer fluidity did not affect blood clearance kinetics or localization in the infected lung. Increasing the amount of negatively charged phospholipids in the liposome bilayer did not affect blood clearance kinetics, but did reduce localization of this liposome preparation at the site of lung infection. In conclusion, the degree of localization at the infected site is remarkably independent of the physicochemical characteristics of the PEG liposomes. Substantial selective liposome localization can be achieved provided that certain criteria regarding PEG density, size and inclusion of charged phospholipids are met. These properties seem to be a direct consequence of the presence of the polymer coating operating as a repulsive steric barrier opposing interactions with biological components.     

Dispersion and bilayer interaction of single-walled carbon nanotubes modulated by covalent and noncovalent PEGylation

Single-walled carbon nanotubes (SWNTs) covalently functionalised with polyethylene glycol (PEG) or noncovalently coated with PEGylated lipids were simulated in water and in lipid bilayers at different PEG sizes and grafting densities using coarse-grained force fields. Starting with the random position of three SWNT–PEG complexes in water, larger PEGs at higher grafting densities more significantly inhibit the aggregation of SWNTs because of larger radii of gyration and hydrodynamic radii of the SWNT–PEG complex, which influence the thickness and the wrapping extent of PEG layer. In particular, PEG-functionalised SWNTs, where PEGs are evenly grafted along the SWNT, disperse, while PEG-coated SWNTs aggregate because SWNTs are less covered by randomly adsorbed PEGylated lipids. Simulations of SWNT–PEGs in lipid bilayers show that PEG (M_w = 550 and 2000)-functionalised SWNTs bind to the bilayer surface but do not insert into the bilayer, while PEG-coated SWNTs insert into the bilayer because PEGylated lipids detach from SWNTs and mix with bilayer lipids. These findings support recent experiments at the same PEG size and density, which suggested that PEG-coated SWNTs may form bundles and thus cannot be easily excreted through the renal route, while PEG-functionalised SWNTs may remain individual and thus show more renal excretion.     

Hydration of polyethylene glycol-grafted liposomes.

This study aimed to characterize the effect of polyethylene glycol of 2000 molecular weight (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodynamic stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodynamic and hydrational characterization. Using a differential scanning calorimetry technique we showed that each molecule of PEG2000 binds 136 +/- 4 molecules of water. For PEG2000 covalently attached to the lipid molecules organized in micelles, the water binding increases to 210 +/- 6 water molecules. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concentrations over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calculated from ultrasound velocity and density) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concentration up to approximately 7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodynamic stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concentrations, compressibility and partial volume of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five additional water molecules per lipid molecule for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.     

Interpretation of small angle X-ray measurements guided by molecular dynamics simulations of lipid bilayers

Reconstruction and interpretation of lipid bilayer structure from X-ray scattering often rely on assumptions regarding the molecular distributions across the bilayer. It is usually assumed that changes in head-head spacings across the bilayer, as measured from electron density profiles, equal the variations in hydrocarbon thicknesses. One can then determine the structure of a bilayer by comparison to the known structure of a lipid with the same headgroup. Here we examine this procedure using simulated electron density profiles for the benchmark lipids DMPC and DPPC. We compare simulation and experiment in both real and Fourier space to address two main aspects: (i) the measurement of head-head spacings from relative electron density profiles, and (ii) the determination of the absolute scale for these profiles. We find supporting evidence for the experimental procedure, thus explaining the robustness and consistency of experimental structural results derived from electron density profiles. However, we also expose potential pitfalls in the Fourier reconstruction that are due to the limited number of scattering peaks. Volumetric analysis of simulated bilayers allows us to propose an improved, yet simple method for scale determination. In this way we are able to remove some of the restrictions imposed by limited scattering data in constructing reliable electron density profiles.     

Structure of polymerizable lipid bilayers. I--1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, a tubule-forming phosphatidylcholine

This report presents the first X-ray diffraction data on diacetylenic phospholipids. The tubule-forming polymerizable lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), was studied by low angle X-ray diffraction from partially dehydrated oriented multibilayers in both polymerized and unpolymerized form. Bilayers of this material were found to be highly ordered, yielding as many as 16 orders of lamellar diffraction, in both the polymerized and unpolymerized states. The unit cell dimension was very small for a lipid of this size. In addition to the features usually observed in the electron density profile structure of phospholipid bilayers, the electron-dense diacetylenic portions of the fatty acyl chain produced electron density maxima at two well-defined levels on each side of the bilayer approximately 15 A and 9 A from the bilayer midplane. A model molecular conformation deduced from the one-dimensional electron density map features all-trans acyl chains tilted at approximately 28 degrees from the bilayer normal that are interdigitated with chains of the opposing monolayer by approximately two carbons at the bilayer center. The linear diacetylene moieties on beta- and gamma-chains appear at different positions along the bilayer normal axis and are roughly parallel to the bilayer surface. This model is discussed in terms of a polymerization mechanism.     

Phase behavior and aggregate structure in mixtures of dioleoylphosphatidylethanolamine and poly(ethylene glycol)-lipids

Cryo-transmission electron microscopy has been used to investigate the phase behavior and aggregate structure in dilute aqueous mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-phospholipids (PEG-lipids). It is shown that PEG-lipids (micelle-forming lipids) induce a lamellar phase in mixtures with DOPE (inverted hexagonal forming lipid). The amount of PEG-lipid that is needed to induce a pure dispersed lamellar phase, at physiological conditions, depends on the size of the PEG headgroup. In the transition region between the inverted hexagonal phase and the lamellar phase, particles with dense inner textures are formed. It is proposed that these aggregates constitute dispersed cubic phase particles. Above bilayer saturating concentration of PEG-lipid, small disks and spherical micelles are formed. The stability of DOPE/PEG-lipid liposomes, prepared at high pH, against a rapid drop of the pH was also investigated. It is shown that the density of PEG-lipid in the membrane, sufficient to prevent liposome aggregation and subsequent phase transition, depends on the size of the PEG headgroup. Below a certain density of PEG-lipid, aggregation and phase transition occurs, but the processes involved proceed relatively slow, over the time scale of weeks. This allows detailed studies of the aggregate structure during membrane fusion.     

Effects of physiologically relevant pressures of helium on the structure of cholesterol-containing lipid bilayers. A neutron diffraction study.

We have used neutron diffraction to study the effects of helium gas (1-210 atm) on the structure of a lipid bilayer model of neuronal plasma membranes. We have recorded diffraction patterns from hydrated multilayers of dimyristoyl lecithin and 40% (molar) cholesterol to a resolution of approximately 6.5 A and have calculated scattering amplitude density distributions as a function of pressure. We find that there are no significant changes in the scattering density profiles at 95% confidence over the range of pressures investigated, suggesting that the physiological effects of high helium pressure are unlikely to be a consequence of changes in the structures of the lipid bilayer portions of membranes.     

Molecular mechanism of polyethylene glycol mediated stabilization of protein

The effect of different molar ratios of polyethylene glycol (PEG) on the conformational stability of protein, bovine serum albumin (BSA), was studied. The binding of PEG with BSA was observed by fluorescence spectroscopy by measuring the fluorescence intensity after displacement of PEG with chromophore ANS and had further been confirmed by measuring the intrinsic fluorescence of tryptophan residues of BSA. Co-lyophilization of BSA with PEG at optimum BSA:PEG molar ratio led to the formation of the stable protein particles. Circular dichroism (CD) spectroscopy study suggested that a conformational change had occurred in the protein after PEG interaction and demonstrated the highest stability of protein at the optimum BSA:PEG molar ratio of 1:0.75. Additional differential scanning calorimetry (DSC) study suggested strong binding of PEG to protein leading to thermal stability at optimum molar ratio. Molecular mechanism operating behind the polyethylene glycol (PEG) mediated stabilization of the protein suggested that strong physical adsorption of PEG on the hydrophobic core of the protein (BSA) along with surface adsorption led to the stability of protein.     

Partitioning of glycomacropeptide in aqueous two-phase systems

The partition behavior of glycomacropeptide (GMP) was determined in polyethylene glycol (PEG) and sodium citrate aqueous two-phase systems (ATPS). It was found that the partitioning of GMP depends on PEG molar mass, tie line length, pH, NaCl concentration and temperature. The obtained data indicates that GMP is preferentially partitioned into the PEG phase without addition of NaCl at pH 8.0. Larger tie line lengths and higher temperatures favor GMP partition to the PEG phase. Furthermore, it was verified that PEG molar mass and concentration have a slight effect on GMP partition. The increase in the molar mass of PEG induces a reduction of the protein solubility in the top PEG rich phase, being shown that the use of PEG1500 is beneficial for the extraction of GMP. A protein recovery higher than 85% was obtained in the top phase of these systems, clearly demonstrating its suitability as a starting point for the separation of GMP.     

Affinity of lipid transfer protein for lipid and lipoprotein particles as influenced by lecithin-cholesterol acyltransferase

The effects of lecithin-cholesterol acyltransferase (LCAT) on the transfer of cholesterol esters mediated by lipid transfer protein (LTP) and its affinity for lipid and lipoprotein particles were investigated. When the single bilayer vesicle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apolipoprotein- (apo) A-I at the molar ratio of 90:30:1.2:0.18) or high density lipoprotein 3 (HDL3) were used as the cholesteryl ester donor and low density lipoproteins (LDL) as the acceptor, the transfer activity of LTP was enhanced by the addition of low concentrations of LCAT. In contrast, no enhancement of cholesteryl ester transfer was observed upon addition of LCAT to either the discoidal bilayer particle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apo-A-I at the molar ratio of 90:30:1.2:1.0) or high density lipoprotein 2 (HDL2). Although both apo-A-I and apo-A-II promoted the transfer of cholesteryl ester from vesicles to LDL, the additional enhancement of the transfer by LCAT was observed only with the vesicles containing apo-A-I. Gel permeation chromatography of LTP/vesicle and LTP/HDL3 mixtures in the presence and absence of LCAT showed that the affinity of LTP for both the vesicles and HDL3 increased upon addition of LCAT. In contrast, neither HDL2 nor discoidal bilayer particles showed any significant enhancement of LTP binding upon addition of LCAT. By using LCAT covalently bound to Sepharose 4B, a maximal interaction between LTP and bound LCAT was shown to occur at the ionic strength of 0.16. Deviation from this ionic strength reduced the extent of the interaction. At the ionic strength of 0.01 and 0.5, the elution volume of LTP was identical to that of bovine serum albumin.     

Structure of the dimyristoylphosphatidylcholine vesicle and the complex formed by its interaction with apolipoprotein C-III: X-ray small-angle scattering studies.

Single bilayer vesicles of dimyristoylphosphatidylcholine have been investigated by small-angle X-ray scattering at 28 degrees C. The results indicate that these vesicles are hollow spherical shell structures with an outer radius of approximately 12 nm and a molecular weight of (3.2 +/- 0.5) X 10(6). The shell was found to be 4.4 +/- 0.2 nm thick with a cross-sectional electron-density profile characteristic for a single phospholipid bilayer. Upon interaction of these vesicles with apolipoprotein C-III from human very low density lipoproteins at a protein/lipid ratio greater than 0.08 (g/g), a complex containing 0.25 g of protein/g of lipid, with molecular weight of (3.9 +/- 0.4) X 10(5), is formed. The shape analysis indicates a highly asymmetric particle with an internal partition of low and high electron density resembling that produced by a bilayer structure. Model calculations and curve-fitting procedures show good agreement between the experimental scattering curve and that computed for an oblate ellipsoidal structure with dimensions of 17 X 17 X 5 nm and a 1 nm thick shell of high electron density surrounding the core of low electron density.     

Osmotic stress induces a phase transition from interdigitated gel phase to bilayer gel phase in multilamellar vesicles of dihexadecylphosphatidylcholine

We have investigated the effects of poly(ethylene glycol) (PEG) on the structure and phase behavior of multilamellar vesicles of dihexadecylphosphatidylcholine (DHPC-MLVs) using an X-ray diffraction method. At low concentrations of PEG-6K (MW = 7500), DHPC-MLVs were in an interdigitated gel (L(beta)I) phase, a gel phase with interdigitated hydrocarbon chains. At around 24% (w/v) PEG 6K, a phase transition from the L(beta)I phase to a bilayer gel phase occurred in the DHPC-MLVs, and above this concentration, they were in a bilayer gel phase. On the other hand, ethylene glycol (EG), the monomer of PEG, did not induce this phase transition in the DHPC-MLVs. A mechanism of this phase transition is proposed and discussed; a decrease in the repulsive interaction between the head groups of the phospholipids in the bilayer gel phase with an increase in PEG concentration, which is due to a decrease in the cross-sectional area of the head group region by osmotic stress, may be the main reason for this phase transition.     

Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains

We demonstrate quantitative vibrational imaging of specific lipid molecules in single bilayers using laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with a lateral resolution of 0.25 mum. A lipid is spectrally separated from other molecules by using deuterated acyl chains that provide a large CARS signal from the symmetric CD(2) stretch vibration around 2100 cm(-1). Our temperature control experiments show that d62-DPPC has similar bilayer phase segregation property as DPPC when mixing with DOPC. By using epi-detection and optimizing excitation and detection conditions, we are able to generate a clear vibrational contrast from d62-DPPC of 10% molar fraction in a single bilayer of DPPC/d62-DPPC mixture. We have developed and experimentally verified an image analysis model that can derive the relative molecular concentration from the difference of the two CARS intensities measured at the peak and dip frequencies of a CARS band. With the above strategies, we have measured the molar density of d62-DPPC in the coexisting domains inside the DOPC/d62-DPPC (1:1) supported bilayers incorporated with 0-40% cholesterol. The observed interesting changes of phospholipid organization upon addition of cholesterol to the bilayer are discussed.     

Hexane dissolved in dioleoyllecithin bilayers has a partial molar volume of approximately zero

Neutron diffraction has been used to measure the amount and distribution of hexane incorporated from the vapor phase into oriented dioleoylphosphatidylcholine bilayers at 66% relative humidity. We reported earlier that hexane at low concentrations is located largely in a zone 10 A wide at the center of the bilayer [White, S. H., King, G. I., & Cain, J. E. (1981) Nature (London) 290, 161-163]. Extending these studies to high hexane concentrations, we find no readily apparent change in the volume of the hydrocarbon region of the bilayer even though more than one hexane molecule per lipid enters the region. The hexane partial molar volume in the bilayer hydrocarbon region is thus approximately zero. Within our statistical confidence limits, the partial molar volume is certainly no greater than one-third the molecular volume of the hexane. Further, analysis of the data suggests that the mass density of the bilayer is considerably less than 1 in the absence of hexane. These findings are in conflict with the assumptions usually made about lipid bilayers and their interaction with nonpolar hydrophobic molecules. In the course of these experiments, we found that standard methods of interpreting diffraction results were not suitable for our purposes. We thus developed several new methods which are summarized in the text and two appendixes. One of these methods allows us to define with precision the width of the hydrocarbon core of the bilayer. The other provides a means of calculating the effects of changes in the absolute scaling of the bilayers with changes in composition without placing the structures on an absolute scattering length density scale.     

Structure of magainin and alamethicin in model membranes studied by x-ray reflectivity

We have investigated the structure of lipid bilayers containing varied molar ratios of different lipids and the antimicrobial peptides magainin and alamethicin. For this structural study, we have used x-ray reflectivity on highly aligned solid-supported multilamellar lipid membranes. The reflectivity curves have been analyzed by semi-kinematical reflectivity theory modeling the bilayer density profile rho(z). Model simulations of the reflectivity curves cover a large range of vertical momentum transfer q(z), and yield excellent agreement between data and theory. The structural changes observed as a function of the molar peptide/lipid concentration P/L are discussed in a comparative way.     

An X-ray diffraction analysis of oriented lipid multilayers containing basic proteins

X-ray diffraction techniques have been used to study the structures of lipid bilayers containing basic proteins. Highly ordered multilayer specimens have been formed by using the Langmuir-Blodgett method in which a solid support is passed through a lipid monolayer held at constant surface pressure at an air/water interface. If the lipid monolayer contains acidic lipids then basic proteins in the aqueous subphase are transferred with the monolayer and incorporated into the multi-membrane stack. X-ray diffraction patterns have been recorded from multilayers of cerebroside sulphate and 40% (molar) cholesterol both with and without polylysine, cytochrome c and the basic protein from central nervous system myelin. Electron density profiles across the membranes have been derived at between 6 A and 12 A resolution. All of the membrane profiles have been placed on an absolute scale of electron density by the isomorphous exchange of cholesterol with a brominated cholesterol analog. The distributions and conformations of the various basic proteins incorporated within the cerebroside sulphate/cholesterol bilayer are very different. Polylysine attaches to the surface of the lipid bilayer as a fully extended chain while cytochrome c maintains its native structure and attaches to the bilayer surface with its short axis approximately perpendicular to the membrane plane. The myelin basic protein associates intimately with the lipid headgroups in the form of an extended molecule, yet its dimension perpendicular to the plane of the membrane of approx. 15 A is consistent with the considerable degree of secondary structure found in solution. In the membrane plane, the myelin basic protein extends to cover an area of about 2500 A2. There is no significant penetration of the protein into the hydrocarbon region of the bilayer or, indeed, beyond the position of the sulphate group of the cerebroside sulphate molecule.     

山莨菪碱与膜相互作用的小角X射线衍射研究

应用小角X射线衍射和差示扫描量热法研究了中药提取物山莨菪碱与DPPC多层膜的相互作用.结果表明,山莨菪碱不仅能降低DPPC多层膜的相变温度,而且能使多层膜发生分相现象,根据衍射结果并采用推广傅里叶合成去卷积法计算了脂双分子层膜的电子密度剖面图.从中我们估计山莨菪碱分子可能分布于脂双分子层极性头部的内侧     

Biotinylated lipid bilayer disks as model membranes for biosensor analyses

The aim of this study was to investigate the potential of polyethylene glycol (PEG)-stabilized lipid bilayer disks as model membranes for surface plasmon resonance (SPR)-based biosensor analyses. Nanosized bilayer disks that included 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)₂₀₀₀] (DSPE-PEG₂₀₀₀-biotin) were prepared and structurally characterized by cryo-transmission electron microscopy (cryo-TEM) imaging. The biotinylated disks were immobilized via streptavidin to three different types of sensor chips (CM3, CM4, and CM5) varying in their degree of carboxymethylation and thickness of the dextran matrix. The bilayer disks were found to interact with and bind stably to the streptavidin-coated sensor surfaces. As a first step toward the use of these bilayer disks as model membranes in SPR-based studies of membrane proteins, initial investigations were carried out with cyclooxygenases 1 and 2 (COX 1 and COX 2). Bilayer disks were preincubated with the respective protein and thereafter allowed to interact with the sensor surface. The signal resulting from the interaction was, in both cases, significantly enhanced as compared with the signal obtained when disks alone were injected over the surface. The results of the study suggest that bilayer disks constitute a new and promising type of model membranes for SPR-based biosensor studies.     

Determination of the depth of bromine atoms in bilayers formed from bromolipid probes

X-ray diffraction analysis has been performed on a series of 1-palmitoyl-2-dibromostearoyl-phosphatidylcholines (BRPCs) with bromine atoms at the 6, 7-, the 11, 12-, or the 15, 16-positions on the sn-2 acyl chains. The diffraction patterns indicate that, when hydrated, each of these lipids forms liquid-crystalline bilayers at 20 degrees C. For each lipid, electron density profiles and continuous Fourier transforms were calculated by the use of swelling experiments. In the electron profiles, high-density peaks, due to the bromine atoms, are observed. The separation between these bromine peaks in the profile decreases as the bromine atoms are moved toward the terminal methyl of the acyl chain. For the 6, 7- and 11, 12-bromolipids, experimental Fourier transforms can be approximated by the sum of the transform of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and the transform of two symmetrically placed peaks of electron density (the bromines). For the case of the 15, 16-bromolipids, a better fit is obtained for the transforms of a model bilayer where the thickness of the methylene chain region of the bilayer is 3 A greater than that of POPC. Our analysis indicates the following: for each of these bromolipids, the bromines are well localized in the bilayer; the distance of the bromines from the head-group-hydrocarbon boundary are 3.5, 8.0, and 14 A, for 6, 7-, 11, 12-, and 15, 16-BRPC, respectively; the bilayer thickness and perturbation to bilayer hydrocarbon chain packing caused by the bromine atoms depend on the position of the bromines on the hydrocarbon chain.     

The structure of Staphylococcus aureus alpha-toxin-induced ionic channel

Polyethylene glycols (PEG) with molecular weight less than or equal to 3000 were shown to effectively protect human erythrocytes from osmotic lysis induced by alpha-staphylotoxin (ST). PEG with MW less than 3000 do not change the conductivity of ion channels induced by ST in bilayer lipid membranes (BLM). Changing the bilayer from a pure phosphatidylcholine (PC) to a negatively charged phosphatidylserine (PS) film results in an asymmetry of the current-voltage characteristics. This is evidenced by the asymmetrical position of the ST-channel pore in bilayer membranes. The results obtained allow to conclude that the ST-channel is an interprotein pore filled with water (with an inner diameter of 2.5-3 nm and a length of approximately 10 nm). It is composed of six molecules of alpha-toxin from Staphylococcus aureus. The ST-channel incorporates into a membrane with only one mouth in contact with the polar lipid heads and the other one protruding 4.5-5 nm from the bilayer plane in water solution.