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PubMed1994

Divalent cation-dependent interaction of sulfated polysaccharides with phosphatidylcholine and mixed phosphatidylcholine/phosphatidylglycerol liposomes.

The Ca(2+)-dependent interaction of various polyanionic polysaccharides (chondroitin sulfate, heparin, dextran sulfate, beta-cyclodextrin sulfate, hyaluronic acid and carboxymethyldextran) with multilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes was investigated by calorimetric and fluorescence spectroscopic measurements. It was found that an observed polysaccharide-induced phospholipid phase separation depends on the density of the sulfate groups along the polysaccharide chain independent of the presence of additional carboxyl groups. The phase separation resulting from the drastic dehydration of the covered membrane regions is monitored by the upward shift of the lipid phase transition and by the blue shift of the emission spectrum of a headgroup-dansylated phosphatidylethanolamine (DPE). This shift is only observable if the required polysaccharide chain length contains at least three glycosyl units. The Ca(2+)-mediated interaction of dextran sulfate with various phosphatidylcholines, differing in their compressibility, showed the maximal difference between the phase transition temperatures of the lipid phase covered by the polysaccharide and the uneffected lipid domains for dielaidinoyl phosphatidylcholine (DEPC), the most compressible phospholipid investigated here. Mixed negatively charged DMPC/dimyristoyl phosphatidylglycerol (DMPG) liposomes were found to compete with the likewise negatively charged dextran sulfate for the binding of Ca2+. At excess Ca2+ concentrations, the binding of the polysaccharide was strengthened, compared to pure DMPC liposomes. The monovalent cation sodium, was able to inhibit the interaction between the membrane surface and dextran sulfate. Various divalent cations were found to mediate the interaction, depending on their ionic radii and electron configuration. Within the second group of the periodic system Ca2+ is the most effective ion. However, within the horizontal forth period the ability to bind sulfated dextran to membrane surfaces decreases from Ca2+ to Ni2+, but then increases again if Cu2+ or Zn2+ was used as the mediating ion.

Calorimetry, Differential Scanning↗

PubMed2002

Effect of polyethylene glycol on the liquid-liquid phase transition in aqueous protein solutions.

We have studied the effect of polyethylene glycol (PEG) on the liquid-liquid phase separation (LLPS) of aqueous solutions of bovine gammaD-crystallin (gammaD), a protein in the eye lens. We observe that the phase separation temperature increases with both PEG concentration and PEG molecular weight. PEG partitioning, which is the difference between the PEG concentration in the two coexisting phases, has been measured experimentally and observed to increase with PEG molecular weight. The measurements of both LLPS temperature and PEG partitioning in the ternary gammaD-PEG-water systems are used to successfully predict the location of the liquid-liquid phase boundary of the binary gammaD-water system. We show that our LLPS measurements can be also used to estimate the protein solubility as a function of the concentration of crystallizing agents. Moreover, the slope of the tie-lines and the dependence of LLPS temperature on polymer concentration provide a powerful and sensitive check of the validity of excluded volume models. Finally, we show that the increase of the LLPS temperature with PEG concentration is due to attractive protein-protein interactions.

Animals↗

PubMed2005

Formation and spatio-temporal evolution of periodic structures in lipid bilayers.

Miscibility phase separation in lipid bilayers is widely implicated as an organizing principle in living cell membranes. However, the chemical and physical aspects of how membrane phase separation modulates protein activity remain obscure. Herein, we describe formation of ordered superstructures of coexisting liquid phases in bilayer membranes. Metastable stripe and hexagonal domain lattices are observed, as well as transitions between them. The high degree of order achieved by these methods facilitates statistical analysis of domain spatial distributions and enables measurement of domain interactions. Such long-range ordering principles may exist in more complicated membrane systems.

Biomimetic Materials↗

PubMed2004

Preparation of uniform-sized PLA microcapsules by combining Shirasu porous glass membrane emulsification technique and multiple emulsion-solvent evaporation method.

Relatively Uniform-sized biodegradable poly(lactide) (PLA) microcapsules were successfully prepared by combining a Shirasu Porous Glass (SPG) membrane emulsification technique and multiple emulsion-solvent evaporation method. An aqueous phase containing lysozyme was used as the internal water phase (w1), and PLA and Arlacel 83 were dissolved in a mixture solvent of dichloromethane (DCM) and toluene which was used as the oil phase (o). These two solutions were emulsified by a homogenizer to form a w1/o primary emulsion. The primary emulsion was permeated through the uniform pores (5.25 microm) of an SPG membrane into the external water phase by the pressure of nitrogen gas to form the uniform w1/o/w2 droplets. Then, the solid polymer microcapsules were obtained by simply evaporating the solvent. It is necessary to avoid the phase separation of primary emulsion during the SPG membrane emulsification. It was found that when the density difference of the internal water phase and oil phase was reduced to nearly zero and Arlacel 83 was used as the oil emulsifier, the phase separation was not observed within 24 h. The w1/o/w2 emulsion with uniform diameter was obtained only when Arlaecl 83 concentration was limited below 2.5 wt.% based on oil phase. The drug encapsulation efficiency was found to be related to several factors including PLA molecular weight, additive type and its concentration in the internal water phase, the emulsifier type and concentration in the oil phase, the NaCl concentration and the pH value in the external water phase. Comparing with the stirring method, it was found that the size was more uniform and the drug encapsulation efficiency was much higher when the microcapsules were prepared by SPG membrane emulsification technique and the highest drug encapsulation efficiency of 92.20% was obtained. This is the first study to prepare PLA microcapsules by combining an SPG membrane emulsification technique and multiple emulsion-solvent evaporation method.

Capsules↗

PubMed2006

Measurement of phosphatidylcholine hydroperoxides in solution and in intact membranes by the ferric-xylenol orange assay.

Formation of a colored complex between ferric iron and xylenol orange (XO) has been used for the determination of hydroperoxides (FOX method). Original or modified FOX methods were performed on aqueous or organic solutions consisting of a single phase. However, for lipid peroxides in heterogeneous samples, such as biological materials, much of the lipid is sequestered in a separate phase. Organic solvent extraction of these lipids is often incomplete and may result in additional peroxidation during the extraction procedure. In this study, we applied the FOX assay for measurement of the membrane phosphatidylcholine hydroperoxides (PC-OOH) in separated phases. The presence of membranous egg yolk phosphatidylcholine (EYPC) in 60% MeOH shifted the broad peak at 560 nm of Fe(3+)-XO complex to 610 nm with a sharp peak associated with the increased intensity of the absorbance. The shift of the peak is useful to measure the unknown amounts of Fe(3+) because the uncomplexed XO considerably contributed to the absorbance of the peak at 560 nm but did not affect the absorbance at 610 nm. EYPC was required to form the membranes to shift the peak because the shift occurred in 60% MeOH but did not by the treatment with detergents or in 90% MeOH in which EYPC did not form the membranes. The molar absorption coefficient (epsilon 610) was 32,700 M(-1) cm(-1), which was about twice the molar absorption co efficient (epsilon 560) reported. We applied this method to the assay of PC-OOH prepared from EYPC and obtained the molar absorption coefficients (epsilon 610), which were 79,100 and 115,700 M(-1) cm(-1) in the presence and absence of BHT, respectively. This finding allows the determination of PC-OOH concentration even in chemically complex systems.

Chemistry Techniques, Analytical↗

PubMed2005

Interpolymer complex between hydroxypropyl cellulose and maleic acid-styrene copolymer: phase behavior of semi-dilute solutions.

The phase behavior of a water/hydroxypropyl cellulose/maleic acid-styrene copolymer (H2O/HPC/MAc-S) system was investigated in the semi-dilute range by turbidimetry, rheology, and optical microscopy. The two polymers under investigation form interpolymer complexes via hydrogen bonding. In the case of a total polymer concentration of cpol = 5 mg . mL(-1) a second phase segregates upon heating the homogeneous ternary system. By applying a constant shear rate (gamma = 50 s(-1)) the phase separation temperature of the system is 10-15 degrees C lower than for an unsheared one. For cpol = 10 mg . mL(-1) phase separation has already occurred at room temperature when the two binary polymer solutions are mixed. The distribution of the partners among the coexisting phases was examined by FT-IR spectroscopy. The stoichiometry of the interpolymeric complex (IPC) was estimated to be HPC/MAc-S = 40:60 (w/w) independent of cpol.

Cellulose↗

PubMed2003

Calcium modulates the mechanical properties of anionic phospholipid membranes.

Using micropipette aspiration and fluorescence techniques, we have studied the material properties of charged lipid vesicles in calcium solutions. Vesicles were composed of phosphatidylglycerol (PG)/phosphatidylcholine (PC) or phosphatidic acid (PA)/PC mixtures. For the case of PG/PC membranes, we measure no effect of anionic lipid fraction on elasticity but a monotonic decrease up to 20% for tension required to induce membrane failure. Both of these observations are rationalized by a model we have developed to describe membrane electrostatic interactions in a two-component salt solution and the resulting changes in membrane properties. Critical tensions measured for PA/PC membranes, on the other hand, did not depend on anionic lipid fraction and were uniformly approximately 35% lower than PG/PC vesicles. This is likely due to a lateral phase separation in the membrane. By combining mechanical properties with fluorescence observations we propose that the PA-rich phase separates into small unconnected domains.

Anions↗

PubMed2001

Structure formation by polysaccharides in concentrated solution.

Molecular structure determination in concentrated solutions is generally considered unfeasible. This conclusion, drawn from experience with linear flexible chains, relates to the complete interpenetration of coils and their ensuing entanglement. Such deep interpenetration is prevented by the obstacles of branching units in branched macromolecules. The present study with branched (and one linear cellulose) polysaccharides demonstrates that different architectures can be distinguished via their different repulsive interactions. The procedure displayed in this article permits the estimation of true molar mass and true radius of gyration at finite concentration c. It furthermore allows, within upper and lower limits, an estimation of the growing size, when association takes place. Results from starches, glycogen, dextran, and cellulose, in aqueous media are discussed. The cellulose was dissolved in a recently developed aqueous cadmium complex. Association can lead to gel formation and/or to phase separation. The former is characterized by repulsive interactions which only slightly decrease when the gel point is approached. Phase separation, on the other hand, results from a decrease of the repulsive interaction induced, for instance, by another polymer. The effect is demonstrated with starches containing different amounts of amylose, where pure amylose forms unstable solutions and precipitates in time as a semicrystalline solid.

Cellulose↗

PubMed2005

Molecular dynamics simulation study of the role of evenly spaced poly(ethylene oxide) tethers on the aggregation of C60 fullerenes in water.

The aggregation behavior of C60 fullerenes and C60 fullerenes with six symmetrically tethered poly(ethylene oxide) oligomers [(PEO)-6-C60] in aqueous solutions has been studied using implicit solvent molecular dynamics simulations. Our simulations reveal that while the attraction between two (PEO)-6-C60 fullerenes in aqueous solution is stronger and longer range than that between two bare C60 fullerenes, the (PEO)-6-C60 fullerenes do not phase-separate in water but rather aggregate in chain-like clusters at concentrations where unmodified fullerenes completely phase-separate.

Letter↗

PubMed2004

Design, synthesis and properties of a degradable polyurethane scaffold for meniscus regeneration.

Longitudinal lesions in menisci are among the most frequent orthopedic problems of the knee. Repair by simple techniques is only limited to the vascular part of the meniscus. For repair of the avascular part of the meniscus a scaffold, which will assist the body in the formation of new meniscus cell tissue, might be applicable. In this study a biomedical segmented polyurethane with poly(epsilon-caprolactone) as soft segment and 1,4-butanediisocyanate and 1,4-butanediol as uniform hard segments has been synthesised. The material has a micro phase separated morphology and excellent mechanical properties. A porous scaffold was prepared via a combination of liquid-liquid phase separation and salt leaching. The foams prepared combined a very high interconnectivity and porosity with the desired compression modulus. After six months of implantation in the knees of beagles full ingrowth with cells was obtained and it was found that meniscus like tissue had been formed in the scaffold. Moreover, compression behaviour appeared to be comparable to native meniscus tissue.

Absorbable Implants↗

PubMed2002

Phase behaviour of premixed 0.25% isoflurane in 50% nitrous oxide and 50% oxygen.

BACKGROUND: Isoflurane (0.25%) in premixed nitrous oxide and oxygen 50/50, v/v, (IN(2)O), has been suggested for pain relief in labour. METHODS: Possible phase separation of this mixture was studied by analysis of samples from pre-filled cylinders as they were cooled. RESULTS: Condensation of isoflurane was found at 3.1 degrees C in a cylinder, which held 8.7 MPa at 15 degrees C. In a cylinder holding 13.8 MPa, which is the standard filling pressure stipulated by the National Health Service, the condensation temperature was -2.3 degrees C. At the highest cylinder filling pressure investigated (14.15 MPa) the separation temperature was even less, -3.0 degrees C. After exposure of cylinders to -40 degrees C and complete phase separation of the mixture, complete mixing was achieved by 24 h storage in the horizontal position at room temperature and, either three complete inversions of the cylinder or mechanical rolling at 30 r.p.m. CONCLUSIONS: These findings should assist the use and storage of IN(2)O.

Anesthesiology↗

PubMed2002

Osmotic pressure of charged colloidal suspensions: a unified approach to linearized Poisson-Boltzmann theory.

We study theoretically the equation of state of a fluid suspension of charged objects (e.g., colloids, polyelectrolytes, clay platelets, etc.) dialyzed against an electrolyte solution using the cell model and linear Poisson-Boltzmann (PB) theory. From the volume derivative of the grand potential functional of linear theory we obtain two expressions for the osmotic pressure in terms of the potential or ion profiles, neither of which coincides with the expression known from nonlinear PB theory, namely, the density of microions at the cell boundary. We show that the range of validity of linearization depends strongly on the linearization point and prove that expansion about the self-consistently determined average potential is optimal in several respects. For instance, screening inside the suspension is automatically described by the actual ionic strength, resulting in the correct asymptotics at high colloid concentration. Together with the analytical solution of the linear PB equation for cell models of arbitrary dimension and electrolyte composition, explicit and very general formulas for the osmotic pressure ensue. A comparison with nonlinear PB theory is provided. Our analysis also shows that whether or not linear theory predicts a phase separation depends crucially on the precise definition of the pressure, showing that depending on the choice, an artificial phase separation in systems as important as DNA in physiological salt solution may result.

Biophysical Phenomena↗

PubMed1989

Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study.

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1982

Permeability of bilayers composed of mixtures of saturated phospholipids.

Liposomes composed of an equimolar binary mixture of phospholipids were from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Mixtures were chosen such that the two phospholipids differed either in terms of head group alone, chain length alone, or both head group and chain length. Cation effluxes, both with and without ionophores (nigericin and valinomycin) were measured over a range of temperatures that encompassed the regions of phase separation for these different lipid mixtures. There was a good correlation between the temperatures at which permeability maxima and phase separation occur. For phospholipid mixtures with the same acyl chain but different head groups (PC vs. PE), the PC component 'controls' permeability. For mixtures of PCs differing in chain length, the short chain lipid dominates the permeability pattern particularly if the chain length are sufficiently different. Lipids differing in both head group and chain length give rise to more complex permeability patterns. The results of the present study are interpreted in terms of a model in which one of the lipid components of the mixture may specifically congregate at defects between co-existing phases and thus 'regulate' permeability.

Liposomes↗

PubMed2002

Anisotropic ordering in sheared binary fluids with viscous asymmetry: experiment and computer simulation.

Optical measurements of the structure and morphology of phase-separating polymer blend under simple shear flow have been performed and the results are compared with computer simulations of sheared phase-separating binary mixtures with viscous asymmetry in the fluid components. Information about the structure is obtained from the two-point composition correlation function. Both experiment and simulation suggest subtle differences in the shear response depending on whether the more viscous phase is dispersed or continuous. Measurements of the string width along the neutral direction suggest power-law decay in the shear rate with an exponent of 1/3 when the more viscous phase is dispersed. The simulations suggest that the mean string width, measured along the velocity-gradient direction in the two-dimensional model calculation, exhibits power-law decay in the shear rate with an exponent of 1/3 independent of which phase is dispersed.

Journal Article↗

PubMed1996

Determination of choline in pharmaceutical formulations by reversed-phase high-performance liquid chromatography and postcolumn suppression conductivity detection.

Choline is a primary degradation product of succinylcholine chloride. Determination of low concentration choline in succinylcholine chloride bulk drug and formulation is a challenge, due to the lack of sensitive detection methods. A reversed-phase separation method with postcolumn suppression conductivity detection is described for the determination of choline. Hexanesulfonic acid is employed as an ion-pair reagent in the mobile phase, which allows the accomplishment of both reversed-phase separation and a sensitive conductivity detection. Detection sensitivity is significantly enhanced by passing the mobile phase through a postcolumn cation suppressor, where hexanesulfonic acid is removed and the background conductance is reduced. This method is simple and sensitive. No sample derivatization procedure is required. The detection limit for choline is about 10 pmol.

Choline↗

PubMed2005

Sedimentation and phase transitions of colloidal gibbsite platelets.

We study the competition between sedimentation, gelation, and liquid crystal formation in suspensions of colloidal gibbsite platelets of five different sizes at three ionic strengths. For large particles (with diameters of 350, 420, and 570 nm) sedimentation is initially the most important factor determining the macroscopic behavior. Only after the main part of the sample has sedimented in an amorphous phase, phase separation takes place. For the smallest particles (diameter 210 and 270 nm), it is the other way around: fast (within one week) phase separation or gelation takes place, after which sedimentation determines the final macroscopic appearance. We distinguish six different scenarios within this two-fold scheme and interpret these on the basis of the previously obtained phase diagram of colloidal gibbsite platelets (van der Beek, D.; Lekkerkerker, H. N. W. Langmuir 2004, 20, 8582).

Journal Article↗

PubMed1984

Modulation of neuraminidase activity by the physical state of phospholipid bilayers containing gangliosides Gd1a and Gt1b.

The thermotropic behavior of large unilamellar dipalmitoylphosphatidylcholine vesicles containing the disialoganglioside Gd1a and the trisialoganglioside Gt1b on their outer surface has been studied as a function of the ganglioside molar fraction and Ca2+ concentration by using high-sensitivity differential scanning calorimetry and steady-state fluorescence spectroscopy. These studies indicate that both gangliosides have an ordering effect on the hydrocarbon region of the bilayer and that this effect is enhanced by the presence of Ca2+ ions. The calorimetric experiments also indicate that ganglioside Gt1b has an intrinsic tendency to phase separate into compositional-rich ganglioside domains even in the absence of Ca2+. Ganglioside Gd1a, on the other hand, only phase separates at Ca2+ concentrations equal to or higher than 10 mM. These studies have allowed us to identify and evaluate the factors affecting the rates of hydrolysis of gangliosides by the soluble neuraminidase from Clostridium perfringens. The data presented in this paper indicate that the rates of hydrolysis of membrane-bound gangliosides are correlated to the physical state of the membrane and the state of aggregation of the ganglioside molecules within the lipid bilayer. For membrane-bound gangliosides, maximal activation energies were found at temperatures slightly below the lipid phase transition temperature. The rates of hydrolysis of the soluble substrate sialyllactose or that of the micellar ganglioside is independent of Ca2+ concentration, whereas the rates of hydrolysis of membrane-bound ganglioside are inhibited by Ca2+ especially under conditions in which the clustering effect of Ca2+ is maximal. These studies suggest that the soluble neuraminidases from Clostridium perfringens prefer ganglioside substrates that are dispersed within the membrane and not forming part of largely aggregated clusters.

Calcium↗