Free energy potential for aggregation of mixed phosphatidylcholine/phosphatidylserine lipid vesicles in glucose polymer (dextran) solutions.

The energetics of lipid vesicle-vesicle aggregation in dextran (36,000 mol wt) solutions have been studied with the use of micromechanical experiments. The affinities (free energy reduction per unit area of contact) for vesicle-vesicle aggregation were determined from measurements of the tension induced in an initially flaccid vesicle membrane as it adhered to another vesicle. The experiments involved controlled aggregation of single vesicles by the following procedure: two giant (approximately 20 micron diam) vesicles were selected from a chamber on the microscope stage that contained the vesicle suspension and transferred to a second chamber that contained a dextran (36,000 mol wt) salt solution (120 mM); the vesicles were then maneuvered into position for contact. One vesicle was aspirated with sufficient suction pressure to create a rigid sphere outside the pipette; the other vesicle was allowed to spread over the rigid vesicle surface. The aggregation potential (affinity) was derived from the membrane tension vs. contact area. Vesicles were formed from mixture of egg lecithin (PC) and phosphatidylserine (PS). For vesicles with a PC/PS ratio of 10:1, the affinity showed a linear increase with concentration of dextran; the values were on the order of 10(-1) ergs/cm2 at 10% by weight in grams. Similarly, pure PC vesicle aggregation was characterized by an affinity value of 1.5 X 10(-1) ergs/cm2 in 10% dextran by weight in grams. In 10% by weight in grams solutions of dextran, the free energy potential for vesicle aggregation decreased as the surface charge (PS) was increased; the affinity extrapolated to zero at a PC/PS ratio of 2:1. When adherent vesicle pairs were transferred into a dextran-free buffer, the vesicles did not spontaneously separate. They maintained adhesive contact until forceably separated, after which they would not read here. Thus, it appears that dextran forms a "cross-bridge" between the vesicle surfaces.     

Experimental and theoretical studies of DNA separations by capillary electrophoresis in entangled polymer solutions.

Studies of electrophoretic separations of DNA restriction fragments by capillary electrophoresis in solutions of (hydroxyethyl) cellulose (HEC) were performed. Rheological studies were used to confirm that the entanglement threshold (phi*) for the HEC solutions used is approximately 0.004 g/mL, in good agreement with theoretical predictions. A mesh size an order of magnitude smaller than that found in agarose gels (on a per weight basis) was calculated using polymer-entanglement theory and was confirmed by electrophoretic measurements. Electrophoretic migration was shown to follow the Ogston regime under most conditions. An approach for obtaining smaller mesh sizes is presented.     

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.     

Aggregation behavior of lipid IVA in aqueous solutions at physiological pH. 1: Simple buffer solutions.

We have investigated the aggregation behaviour of lipid IVA (a bioactive precursor of lipid A and the lipid anchor of lipopolysaccharide) in aqueous solutions in the physiological pH range using dynamic light scattering, nuclear magnetic resonance, fluorescence, surface pressure, electron microscopy and force field simulation studies. The sonication of lipid IVA in PBS, Tris and Hepes produces vesicles which are stable in the concentration range of 10(-3) - 10(-7) M, possibly even at lower concentrations. The vesicle size is not sensitive to the nature of the buffer, only to the pH and to some extent to the ionic strength. The long time stability of the small unilamellar vesicles as well as the structureless 1H-NMR spectra might be attributed to a rigid surface structure. This structure is also supported by the simulation studies. We have tentatively proposed a coexistence of micelles and/or other aggregates with the bilayered vesicles at higher lipid concentrations in order to explain some of the experimental observations.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Hybrid polymer/lipid vesicles: Influence of polymer architecture and molar mass on line tension

Hybrid polymer/lipid vesicles are self-assembled structures that have been the subject of an increasing number of studies in recent years. They are particularly promising tools in the development of cell membrane models because they offer the possibility to fine-tune their membrane structure by adjusting the distribution of components (presence or absence of “raft-like” lipid domains), which is of prime importance to control their membrane properties. Line tension in multiphase membranes is known to be a key parameter on membrane structuration, but remains unexplored, either experimentally or by computer modeling for hybrid polymer/lipid vesicles. In this study, we were able to measure the line tension on different budded hybrid vesicles, using a micropipette aspiration technique, and show the influence of the molar mass and the architecture of block copolymers on line tension and its consequences for membrane structuration.     

Ice nucleation and supercooling behavior of polymer aqueous solutions

We determined the homogeneous nucleation temperature depression, ΔT_f,hom, the equilibrium melting point depression, ΔT_m, and the value λ, which can be obtained from the linear relationship ΔT_f,hom = λΔT_m, for aqueous solutions of PEG (200-20,000 g mol⁻¹), PVP (10,000, 35,000, 40,000 g mol⁻¹), and dextran (10,000 g mol⁻¹) in the concentration range 0-40 wt% using the emulsion method. The molecular weight dependence of T_f,hom, T_m, and λ in PEG aqueous solutions was found to change in the vicinity of Mw 600-1540 at all concentrations. In addition, it was confirmed that for all of the polymers studied, there was a good linear relationship between λ and the logarithmic value of the self-diffusion coefficient D₀ of the solute molecule. These results indicate that the parameters that describe non-equilibrium freezing, such as T_f,hom and λ, are dependent on solution properties such as viscosity and self-diffusion of solute molecules.     

Pigment containing lipid vesicles. I. Preparation and characterization of chlorophyll a-lecithin vesicles.

Vesicles obtained by sonication of chlorophyll a-lecithin mixtures dispersed in anaqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophyll a. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophyll a molecule per 55 lecithins can be incorporated into membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102 +/- 5 A and independent of the chloropyll a content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Location of valinomycin in lipid vesicles

The location of the cyclododecadepsipeptide, valinomycin in vesicles formed from two synthetic lipids is studied by differential scanning calorimetry, spin-label partitioning electron paramagnetic resonance and [¹H]-nuclear magnetic resonance. The results show that valinomycin is located near the head group region of dipalmitoyl phosphatidyl choline vesicles and in the hydrophobic core of the dimyristoyl phosphatidyl choline vesicles in the liquid crystalline phase.     

Affinity purification of lipid vesicles

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.     

Electric breakdown of lipid vesicles

Theoretical expression for free energy F of spherical lipid vesicle containing through pore in the presence of diffusional potential difference is derived. It is assumed that the pore radius is small in comparison with vesicle size. According to estimation the variation of elastic energy of vesicle membrane with pore radius is small. Therefore electrical breakdown becomes reversible for reasonable region of r values. Conditions of equilibrium and dynamic modes of breakdown are analyzed. Random oscillation mode of intravesicular label discharge is shown for some region of vesicle parameters.     

Auto-oxidation-induced fusion of lipid vesicles

Spontaneous size changes of small unilamellar vesicles with initial mean diameters of 25 nm measured by quasi-elastic light scattering (QELS) and electron microscopy are reported. After the size conversion the vesicles have mean diameters of about 70 nm and are of the unilamellar and multilamellar type. The fact that auto-oxidation initiates this process is established by the comparison of the results for vesicles which differ only in the degree of auto-oxidation. The role of phosphatidylcholine hydroperoxides as fusogens is discussed.     

Inclusion-induced boundary layers in lipid vesicles

The equilibrium shapes of lipid vesicles are perturbed by rigid inclusions. In a two-dimensional vesicle, that may also model a cylindrically elongated tubule, the shape modifications can be determined analytically, and turn out to be significant even far from the inclusion. On the contrary, previous numerical work has given evidence that in the three-dimensional case the shape perturbations decay quite rapidly and are negligible a few inclusion radii away. In this paper, we use the tools of asymptotic analysis to derive analytically the shape of the boundary layer induced by the inclusion. As a result, we are able to determine the dominant part of the free-energy perturbation that, in turn, allows to identify the vesicle points where the inclusion prefers to sit.     

Light scattering and turbidity measurements on lipid vesicles.

The dynamic behaviour of model membranes in the form of sonicated liposomes in excess water was studied by means of 90 degrees C light scattering and turbidity measurements. Computer calculations based on the Rayleigh-Gans theory of light scattering were used to estimate the average size of lipid vesicles dispersed in water, taking into account the various structures of the vesicles. Normal reversible changes in the scattered light intensity and turbidity with temperature could be accounted for mainly by the changes in the refractive index of the lipid and irreversible anomalous changes were explained on the basis of fusion of smaller aggregated vesicles.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

Lipid packing and transbilayer asymmetries of mixed lipid vesicles.

Predictions, based on a previously developed theory, of the radii and asymmetric lipid distribution of mixed phosphatidylcholine/lysophosphatidylcholine and phosphatidylcholine/cholesterol vesicles of variable composition are presented. The results compare well with available experimental data, except for cis-unsaturated phosphatidylcholine/cholesterol vesicles at high concentrations of cholesterol. It is concluded that specific lipid-lipid interactions need not be invoked for saturated and trans-unsaturated phosphatidylcholine mixed with lysophosphatidylcholine or cholesterol. A discussion of the effect of packing stresses on induced flip-flop and non-spherical vesicles is also presented.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Preparation and characteristics of lipid vesicles

Summary As determined by electron microscopy, lipid sonicated in buffer initially forms large vesicles which may be multilamellar. Prolonged sonication results in a population of vesicles of smaller, but not uniform diameters. These vesicles are bounded by only one bilayer. The lipid suspension can be partially fractionated according to size by column chromatography. A fraction of the eluate has been selected for further study. The weight-average vesicle weight and average radius of gyration are obtained by lightscattering measurements. The volume of buffer enclosed by the vesicles is determined using¹⁴C- or³H-labelled sugars as a marker. These values are in reasonable agreement with the corresponding values calculated from the size distribution of the vesicle fraction obtained by electron microscopy.