Interrogating the role of liposome size in mediating the dynamics of a chromophore in the acyl chain region of a phospholipid bilayer

We have examined the temperature-dependent reorientation dynamics of perylene imbedded in bilayers of 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), where the bilayers exist in the form of unilamellar vesicles. Previous work using 100-nm diameter DMPC vesicles has shown that the phase transition from the gel phase to the fluid phase can be detected using the reorientation dynamics of perylene. In this work we explore the vesicle size dependence of the perylene reorientation dynamics in DMPC vesicles. The size of the vesicles is determined by extrusion and the reorientation dynamics of perylene are measured as a function of vesicle size between 100-nm and 5-microm diameter. We find that, while the phase transition for DMPC is seen in smaller vesicles, perylene becomes insensitive to the phase transition for vesicles larger than ca. 800-nm diameter. We also find a discontinuous change in perylene reorientation dynamics with increasing vesicle size, and we consider this result in the context of the location of perylene within the bilayer.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

Bis(monoacylglycero)phosphate and ganglioside GM1 spontaneously form small homogeneous vesicles at specific concentrations

The morphology and size of hydrated lipid dispersions of bis(monoacylglycero)phosphate (BMP) mixed with varying mole percentages of the ganglioside GM1 were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Electron paramagnetic resonance (EPR) spectroscopy of these same mixtures, doped at 0.5 mol% with doxyl labeled lipids, was used to investigate acyl-chain packing. Results show that for 20-30% GM1, hydrated BMP:GM1 mixtures spontaneously form small spherical vesicles with diameters ∼100 nm and a narrow size distribution profile. For other concentrations of GM1, hydrated dispersions with BMP have non-spherical shapes and heterogeneous size profiles, with average vesicle diameters >400 nm. All samples were prepared at pH 5.5 to mimic the lumen acidity of the late endosome where BMP is an essential component of intraendosomal vesicle budding, lipid sorting and trafficking. These findings indicate that GM1 and BMP under a limited concentration range spontaneously form small vesicles of homogeneous size in an energy independent manner without the need of protein templating. Because BMP is essential for intraendosomal vesicle formation, these results imply that lipid-lipid interactions may play a critical role in the endosomal process of lipid sorting and trafficking.     

A structural model of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata deduced from density measurements at different osmotic pressures

Density measurements made on cholinergic synaptic vesicles from the electric organs of Torpedo marmorata at different osmotic pressures are consistent with the following structural model of the vesicle. The particle behaves like a sphere 80-100 nm in diameter bounded by a semi-permeable membrane. The bulk of its soluble constituents are in true solution at physiological osmolalities. The limiting membrane is approximately 4-5 nm thick, suggesting that it contains large areas of phospholipid bilayer exposed to its bathing medium. The limiting membrane takes up about 26% (v/v) of the particle, a further 34% (v/v) of which is osmotically active water and 31% (v/v) hydrated core material at 800 mosmol/1. The buoyant density of the membrane is 1.132 g . cm-3. The density of the hydrated core material is approximately 1.05 g . cm-3. The membrane is selectively permeable to small molecules when subjected to hypo-osmotic stress. It is proposed that this occurs by the formation of small transient pores in the lipid bilayer of the membrane, which are induced by stretching caused by the osmotic pressure change.     

Extrusion of electroformed giant unilamellar vesicles through track-etched membranes

Unilamellar vesicle populations having a narrow size distribution and mean radius below 100 nm are preferred for drug delivery applications. In the present work, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used to prepare giant unilamellar vesicles (GUVs) by electroformation and multilamellar vesicles (MLVs) by thin film hydration. Our experiments show that in contrast to MLVs, a single-pass extrusion of GUVs through track-etched polycarbonate membranes at moderate pressure differences is sufficient to produce small liposomes having low polydispersity index. Moreover, we observe that the drug encapsulating potential of extruded liposomes obtained from GUVs is significantly higher compared to liposomes prepared by extrusion of MLVs. Furthermore, our experiments carried out for varying membrane pore diameters and extrusion pressures suggest that the size of extruded liposomes is a function of the velocity of GUV suspensions in the membrane pore.     

Vesicle-micelle transition of phosphatidylcholine and octyl glucoside elucidated by cryo-transmission electron microscopy.

Vesicle-micelle transition structures of egg phosphatidylcholine (PC) and octyl glucoside (OG) mixtures were observed in the vitrified hydrated state by cryo-transmission electron microscopy (cryo-TEM) and correlated with the macroscopic and molecular changes previously associated with micellization monitored by 90 degrees light scattering and resonance energy transfer between fluorescent lipid probes. Several distinct structural changes occurred as OG was added to the PC vesicles. First, the average vesicle size decreased from 160 nm to less than 66 nm with no apparent change or decrease in optical density (OD). Then, associated with a small rise in OD, samples with open vesicles were observed coexisting with pieces of lamellae and long cylindrical micelles; more micelles were seen at higher [OG]. This mixture of vesicles and cylindrical micelles occurred in the region of the phase diagram previously attributed to vesicle opening, and possibly vesicle size increase. At higher [OG], small spheroidal micelles coexisting with cylindrical micelles correlated with a decrease in OD and changes in the fluorescence signal. At high [OG] when the solution appeared clear, spheroidal micelles were the dominant structure. By using cryo-TEM, a technique which preserves the original microstructure of fluid systems and provides direct images at 1 nm resolution, we have elucidated the vesicle-micelle transition and identified intermediates not known previously in the PC/OG system.     

Size analysis and stability study of lipid vesicles by high-performance gel exclusion chromatography, turbidity, and dynamic light scattering

Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 micron or by filtration through 0.8-micron cellulosic or 0.22-micron polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 micron, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF "tortuous" ones being less efficient than "straight bores" polycarbonate ones; (ii) mixed EPC/EPA REVs sequentially extruded down through 0.2-micron polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC/EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC/EPA (mole ratio: 91/9) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.     

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.     

Comparative electrophysiological study of reconstituted giant vesicle preparations of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were reconstituted into two types of giant vesicles: (1) Giant proteoliposomes prepared by freeze-thawing of a mixture of SR vesicles and sonicated phospholipid vesicles without the use of detergent. (2) Giant SR vesicles prepared by fusion of SR vesicles using poly(ethylene glycol) (PEG) as a fusogen and without the addition of exogenous lipid. These giant vesicles were patch-clamped and properties of the single voltage-dependent potassium channel in the excised patch were studied. Single-channel conductance in a symmetrical solution of 0.1 M KCl and 1 mM CaCl2 was 140.0 +/- 10 pS (n = 5) for freeze-thawed vesicles and 136.4 +/- 15 pS (n = 7) for PEG vesicles. Both types of vesicles exhibited a sub-conductance state having 55% of the fully open state conductance. The voltage-dependence of open-channel probability could be expressed in terms of thermodynamic parameters of delta Gi = 0.95 kcal/mol and z = -0.77 for freeze-thawed vesicles and delta Gi = 0.92 kcal/mol and z = -0.87 for PEG vesicles. These values correlated well with previous data obtained by fusion of native SR vesicles with a planar lipid membrane. Channel orientation was found to be conserved in both types of vesicles used in the present study.     

Prolonged oxygen-carrying ability of hemoglobin vesicles by coencapsulation of catalase in vivo

Hemoglobin (Hb) vesicles (particle diameter, ca. 250 nm) have been developed as Hb-based oxygen carriers in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The oxidation of Hb to nonfunctional ferric Hb (metHb) was caused by reactive oxygen species, especially hydrogen peroxide (H(2)O(2)), in vivo in addition to autoxidation. We focused on the enzymatic elimination of H(2)O(2) to suppress the metHb formation in the Hb vesicles. In this study, we coencapsulated catalase with Hb within vesicles and studied the rate of metHb formation in vivo. The Hb vesicles containing 5.6 x 10(4) unit mL(-1) catalase decreased the rate of metHb formation by half in comparison with Hb vesicles without catalase. We succeeded in prolonging the oxygen-carrying ability of the Hb vesicle in vivo by the coencapsulation of catalase.     

Bilayer curvature and certain amphipaths promote poly(ethylene glycol)-induced fusion of dipalmitoylphosphatidylcholine unilamellar vesicles.

Unilamellar vesicles of varying and reasonably uniform size were prepared from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) by the extrusion procedure and sonication. Quasi-elastic light scattering was used to show that different vesicle preparations had mean (Z-averaged) diameters of 1340, 900, 770, 630, and 358 A (sonicated). Bilayer-phase behavior as detected by differential scanning calorimetry was consistent with the existence of essentially uniform vesicle populations of different sizes. The response of these different vesicles to treatment with poly(ethylene glycol) (PEG) was monitored using fluorescence assays for lipid transfer, contents leakage, and contents mixing, as well as quasi-elastic light scattering. No fusion, as judged by vesicle contents mixing and change in vesicle size, was detected for vesicles of diameter greater than 770 A. The diameters of smaller vesicles increased dramatically when treated with high concentrations of PEG, although mixing of their contents could not be detected both because of their small trapped volumes and because of the extensive leakage induced in small vesicles by high concentrations of PEG. Lipid transfer was detected between vesicles of all sizes. We conclude the high bilayer curvature does encourage fusion of closely juxtaposed membrane bilayers but that highly curved vesicles appear also to rupture and form larger structures when diluted from high PEG concentration, a process that can be confused with fusion. Despite the failure of PEG to induce fusion of large, uncurved vesicles composed of a single phosphatidylcholine, these vesicles can be induced to fuse when they contain small amounts of certain amphiphathic compounds thought to play a role in cellular fusion processes. Thus, vesicles which contained 0.5 mol % L-alpha-lysopalmitoylphosphatidylcholine, 5 mol % platelet activating factor, or 0.5 mol % palmitic acid fused in the presence of 30%, 25%, and 20% (w/w) PEG, respectively. However, vesicles containing 1,2-dipalmitoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, 1-oleoyl-2-acetyl-sn-glycerol, or monooleoyl-rac-glycerol at surface concentrations up to 5 mol % did not fuse in the presence or absence of PEG. There was no correlation between the abilities of these amphipaths to induce phase separation or nonlamellar phases and their abilities to support fusion of pure DPPC unilamellar vesicles in the presence of high concentrations of PEG. The results are discussed in terms of the type of disrupted lipid packing that could be expected to favor PEG-mediated fusion.     

Preparation and characterization of monodisperse unilamellar phospholipid vesicles with selected diameters of from 300 to 600 nm

A method has been developed for making large unilamellar vesicles (LUV) with low polydispersity. The LUV, constituted of dioleoylphosphatidic acid (DOPA), 300 nm in diameter are made by a modification of the pH adjustment technique (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683-1687). This size is 10 times that (30 nm) of vesicles prepared by prolonged sonication. Vesicle size is increased stepwise by adding cholesterol (to a maximum of 40 mol% cholesterol) to form vesicles in 0.15 M KCl with up to 600 nm diameter. The vesicle size is measured by photon correlation spectroscopy, electron microscopy, and by measurement of the internal volume with cyanocobalamin while calculating the number of DOPA molecules per vesicle. Vesicles are stable for at least three weeks. Sepharose 4B column chromatography of the preparation yields a peak of fractions with the same polydispersity as the original sample and shows that 30 to 40% of the original lipid in a sample is recovered as LUV. Less than 2% of the sample forms small unilamellar vesicles (SUV) (diameter = 30 nm), which emerge from the column in a separate peak. Since the remaining lipid is not suspended in the buffer during vesicle formation, for most purposes the vesicles may be used immediately after titration so that they can be prepared in less than 40 min.     

Three classes of spiny-(Clathrin-) coated vesicles in rodent liver based on size distributions

Summary Clathrin-coated vesicles in rodent (rat and mouse) liver distribute into three distinct populations based on measurements of vesicle diameter. The first population consists of 60–80 nm vesicles found almost exclusively within the Golgi apparatus region. The second population is of 100–160 nm coated vesicles located within 100–500 nm of the cell surface. A third population of coated vesicles of intermediate diameter (ca. 90 nm) is present both at the Golgi apparatus and at the cell surface. We speculate that clathrin and clathrin-coated vesicles within the region of the Golgi apparatus and of the cell surface exist in two recycling populations. The third population of vesicles of intermediate diameter could represent a shuttle to link the two major compartments.     

Characterization of Large Unilamellar Vesicles as Models for Studies of Lipid Peroxidation Initiated by Azocompounds

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.     

Osmotic properties of large unilamellar vesicles prepared by extrusion.

We have examined the morphology and osmotic properties of large unilamellar vesicles (LUVs) prepared by extrusion. Contrary to expectations, we observe by cryo-electron microscopy that such vesicles, under isoosmotic conditions, are non-spherical. This morphology appears to be a consequence of vesicle passage through the filter pores during preparation. As a result when such LUVs are placed in a hypoosmotic medium they are able to compensate, at least partially, for the resulting influx of water by "rounding up" and thereby increasing their volume with no change in surface area. The increase in vesicle trapped volume associated with these morphological changes was determined using the slowly membrane-permeable solute [3H]-glucose. This allowed calculation of the actual osmotic gradient experienced by the vesicle membrane for a given applied differential. When LUVs were exposed to osmotic differentials of sufficient magnitude lysis occurred with the extent of solute release being dependent on the size of the osmotic gradient. Surprisingly, lysis was not an all-or-nothing event, but instead a residual osmotic differential remained after lysis. This differential value was comparable in magnitude to the minimum osmotic differential required to trigger lysis. Further, by comparing the release of solutes of differing molecular weights (glucose and dextran) a lower limit of about 12 nm diameter can be set for the bilayer defect created during lysis. Finally, the maximum residual osmotic differentials were compared for LUVs varying in mean diameter from 90 to 340 nm. This comparison confirmed that these systems obey Laplace's Law relating vesicle diameter and lysis pressure. This analysis also yielded a value for the membrane tension at lysis of 40 dyn cm-1 at 23 degrees C, which is in reasonable agreement with previously published values for giant unilamellar vesicles.     

The size and curvature of anionic covesicle substrate affects the catalytic action of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) is normally located in the cytosol, but in response to cellular activation the enzyme binds to the membrane at the lipid/water interface where it catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. Synthetic phospholipid vesicle systems have been used in kinetic and mechanistic analyses of cPLA2, but these systems result in a rapid loss of enzyme activity. In the present research, covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing 相似文献   

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.     

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.     

Effect of Hb-encapsulation with vesicles on H2O2 reaction and lipid peroxidation

We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H(2)O(2)) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H(2)O(2), metHb (Fe(III)) and ferrylHb (Fe(IV)=O) are produced, and H(2)O(2) is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H(2)O(2) that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H(2)O(2). Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H(2)O(2), the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H(2)O(2), whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.     

Vesicle size distributions measured by flow field-flow fractionation coupled with multiangle light scattering.

The separation method, flow field-flow fractionation (flow FFF), is coupled on-line with multiangle laser light scattering (MALLS) for simultaneous measurement of the size and concentration of vesicles eluting continuously from the fractionator. These size and concentration data, gathered as a function of elution time, may be used to construct both number- and mass-weighted vesicle size distributions. Unlike most competing, noninvasive methods, this flow FFF/MALLS technique enables measurement of vesicle size distributions without a separate refractive index detector, calibration using particle size standards, or prior assumptions about the shape of the size distribution. Experimentally measured size distributions of vesicles formed by extrusion and detergent removal are non-Gaussian and are fit well by the Weibull distribution. Flow FFF/MALLS reveals that both the extrusion and detergent dialysis vesicle formation methods can yield nearly size monodisperse populations with standard deviations of approximately 8% about the mean diameter. In contrast to the rather low resolution of dynamic light scattering in analyzing bimodal systems, flow FFF/MALLS is shown to resolve vesicle subpopulations that differ by much less than a factor of two in mean size.