Deformability of multilamellar vesicles

Although a free unilamellar vesicle has zero or almost zero genuine surface tension, the multilamellar vesicle ("onion") exhibits a nonzero effective surface tension sigma(eff). The expression for sigma(eff) used in the literature is sigma(eff) approximately square root of kappaB/d(0), where B is the interaction modulus between the vesicle bilayers, d(0) the repeating distance between the bilayers in the droplet, and kappa their bending rigidity. In this paper we calculate the contributions to the effective surface tension of a lamellar droplet in the case when the layers interact with one another and when they are free. It is shown that the interaction contribution to the surface tension is small and sigma(eff) is determined mainly by kappa, the radius of the droplet R(0), and the number of the shape undulation modes l(max). A nonzero surface tension of the layers is also included in the calculation which is necessary when the vesicle membrane is stressed in the complex of other membranes.     

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.     

Crystallization of water in multilamellar vesicles

The two-step crystallization of water in multilamellar vesicles (MLVs) of phosphatidylcholines has been investigated. The main crystallization occurs near -15 degrees C and involves bulk water. Contrary to unilamellar vesicles, a sub-zero phase transition is observed for MLVs at -40 degrees C that corresponds to the crystallization of interstitial water, as proved by Fourier transform infrared absorption and differential scanning calorimetry (DSC) experiments. Furthermore, by means of the DSC method and, more specifically, using the enthalpy change values Delta H(sub) at the sub-zero transition, the number of water molecules per 1,2-dipalmitoylphosphatidylcholine (DPPC) molecule giving rise to this transition has been estimated for different H(2)O/DPPC molar ratios. The curve of the molecular fraction of water molecules involved in the sub-zero transition versus the H(2)O/DPPC molar ratio exhibits a maximum for H(2)O/DPPC equal to 27 (40% in mass of water) and tends towards zero for H(2)O/DPPC ratio values approaching that of the swelling limit of the membrane. A smaller enthalpy value of the sub-zero transition is found for 1-oleoyl-2-palmitoyl-3-phosphatidylcholine (OPPC) than for DPPC. This may be explained by the decrease of interstitial water's quantity when the lipid contains an unsaturated chain. When troxerutin, a hydrophilic drug, is added to the DPPC multilayers, the decrease of Delta H(sub) and melting enthalpy of bulk water is attributed to a decrease of the entropy of the liquid phase owing to the network of water molecules surrounding troxerutin molecules. In all cases, the experiments revealed that the sub-zero transition occurs only in the presence of excess water with respect to the swelling limit of membranes. This evidence could be, at least qualitatively, related to an increase of membrane pressure on interstitial water subsequent to bulk water crystallization.     

The captured volume of multilamellar vesicles

Characterization of classical 'hand-shaken' multilamellar lipid vesicles (MLVs) confirmed that these systems exclude solute during formation thus confounding previous captured volume measurements which typically have utilized solute as a merker of the occluded aqueous space. We used solvent rather than solute to determine the captured volume of these systems and obtained values at least twice those previously reported. We present here a captured volume and lamellarity profile of 'hand-shaken' MLVs and suggest that these parameters are dependent on the lipid concentration present during hydration.     

Generation of multilamellar and unilamellar phospholipid vesicles

Multilamellar and unilamellar vesicles can be generated by a variety of techniques which lead to systems with differing lamellarity, size, trapped volume and solute distribution. The straight-forward hydration of lipid to produce multilamellar vesicles (MLVs) results in systems which exhibit low trapped volumes and where solutes contained in the aqueous buffer are partially excluded from the MLV interior. Large trapped volumes and equilibrium solute distributions can be achieved by freeze-thawing or by ‘reverse phase’ procedures where the lipid is hydrated after being solubilized in organic solvent. Unilamellar vesicles can be produced directly from MLVs by extrusion or sonication or, alternatively, can be obtained by reverse phase or detergent removal procedures. The advantages and limitations of these techniques are discussed.     

Interaction of trypsin with multilamellar vesicles of soybean lipids

The pH dependence of complex formation of trypsin with multilamellar vesicles (MLV) of soybean lipids has been investigated. The lipids were characterized by the same phospholipid composition, but the content of other lipids differed. Decrease of pH or introduction of negatively charged components into the lipid samples increased trypsin content in the protein-lipid complexes. This suggests electrostatic interaction between the protein and soybean lipids. The dependence of trypsin activity in the complexes with MLV on their concentration and on the presence of an ionic detergent was studied. Trypsin-MLV interaction did not result in complete inactivation of the protein molecules. Moreover, the effects of dilution and addition of ionic detergent on trypsin activity were additive. Using a fluorescence technique, complex formation with MLV was found to stabilize trypsin molecules, preventing their autolysis.     

In vitro and in vivo stability of new multilamellar vesicles

Factors affecting multilamellar vesicles transport to the blood compartment after oral administration to rats were evaluated first in vitro. A high entrapment of protein A was obtained when the vesicles were prepared by shearing a lyotropic lamellar phase composed of soybean phosphatidylcholine, cholesterol and polyoxyethylene alcohol (C12H25(OCH2CH2)4OH) as neutral detergent. In vitro tests showed that these vesicles (spherulites) were stabled in 50% of fetal calf serum, in acidic (pH 3) or basic (pH 10) buffers, in pancreatin but are partially lysed in 20mM bile salts. Oral administration of spherulites entrapping 111In-NTA in fasting rats showed a increase of radioacticivity in blood. This could be explained by passage of some spherulites in the enterocytes.     

Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles

It has recently been observed (Gruner, Lenk, Janoff and Ostro (1985) Biochemistry, in the press) that mechanical dispersion of dry lipid in an aqueous buffer to form multilamellar vesicle (MLV) systems does not result in equilibrium trans-membrane distributions of solute. In particular, the entrapped buffer exhibits reduced solute concentrations. Here we demonstrate that egg phosphatidylcholine MLV systems dispersed in the presence of Mn2+ also exhibit non-equilibrium solute distributions, and that repetitive freeze-thawing cycles can remove such solute heterogeneity. Further, the resulting freeze-thawed MLVs exhibit dramatically enhanced trapped volumes and trapping efficiencies. At 400 mg phospholipid per ml, for example, the trapping efficiencies can be as high as 90%. This is associated with a remarkable change in MLV morphology where large inter-bilayer separations are commonly observed.     

Thermal acoustic radiation from multilamellar vesicles in lipid phase transition

A new acoustical method for the investigation of lipid phase transition is introduced based on the measurement of the thermal acoustic radiation (TAR) inherent in lipids. The TAR of multilamellar vesicles from dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) was measured in the megahertz range and the variations in the radiation intensity during the lipid phase transition were recorded. Two types of variations are possible: if the temperature of the vesicles decreases (in the process of transition from the liquid crystalline state to the gel state) then the TAR intensity increases, and if the temperature increases (in the reverse transition) then the TAR intensity decreases. These effects are connected with an increase in the ultrasonic absorption in the vesicles under lipid phase transition. Basing on the results of the TAR investigation, a new theoretical estimate has been developed of the variation in the absorption coefficient during the lipid phase transition. In this estimate, the variation is equated to the ratio of the phase transition entropy to the gas constant.     

Slow relaxation of the sub-main transition in multilamellar phosphatidylcholine vesicles.

The influence of ionic strength and equilibration time on the appearance of the sub-main transition in fully hydrated multilamellar vesicles composed of phosphatidylcholines has been investigated by means of calorimetry and densitometry. The heat capacity measurements show that the transition enthalpy of the sub-main transition is affected by both salt concentration (KCl) and equilibration time. The small heat capacity peak appearing in vesicles made in pure water is significantly increased upon addition of salt. Furthermore, equilibration of the multilamellar vesicles at low temperatures for several weeks results in a pronounced enhancement of the transition enthalpy of the sub-main transition. Neither salt concentration nor equilibration time affected the transition temperature of the sub-main transition. In the densitometry measurements a small volume change is detectable for high salt concentrations. In order to gain further insight into the physical mechanisms involved in the sub-main transition, a Monte Carlo computer simulation study has been carried out using a microscopic model. The combined experimental and simulation results suggest that the sub-main transition involves an acyl chain disordering of phospholipids in lipid bilayer regions that are characterized by a locally decreased lateral pressure most likely caused by a curvature stress.     

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles

Summary The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3–10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986.Methods Enzymol. 127:400–416).In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986.Methods Enzymol. 127:522–538).In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state.Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage.The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer.The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.     

The microsecond rotational motions of eosin-labelled fatty acids in multilamellar vesicles

The rotational properties of two eosin-labelled fatty acids of different alkyl chain length have been studied in large multilamellar dimyristoylphosphatidylcholine vesicles. The location of the probes at the surface region were ascertained by quenching experiments using a hydrophilic divalent cation solubilized in the aqueous phase (Cu2+) and a hydrophobic aromatic aniline (N,N-dimethylaniline) associated with the lipid. Phosphorescence anisotropy measurements reveal that above the phospholipid phase transition the polarization of eosin luminescence decays monoexponentially in the micro-to-millisecond time range, while below the phase transition a biexponential decay is observed. A model is proposed which attributes the time constants to two separate motions, discrete jumps or 'flipping' of the eosin moiety within restricted boundaries and long-axis rotation. The value of the time-independent term changes with probe position and temperature and reflects orientational constraints imposed by lipid-chromophore interactions. The implications of these results for the study of protein rotations in membranes are discussed.     

The ultrastructure of multilamellar bodies and surfactant in the human lung

Summary Normal tissues from human lungs were dehydrated through Epon 812 resin to retain many of the lipids and carbohydrates in thin section. The three-dimensional structure of the multilamellar body was determined. The paired layer of phospholipid heads (PH) is 36Å thick; the layer of fatty-acid tails (FA) is 31Å, the same as reported previously for non-human primates and rodents. The human multilamellar body is apparently unique: the lamellae of the major focus divide into two or three lamellae; the matrix material of the core is without vesicular bodies and a projection core is present. When compared with those of the rat, human tissues contain a greater number of lamellar foci and fewer lamellae per focus. The presence of a peripheral layer of lamellae, an ever-present external limiting membrane, and the fusion of multilamellar bodies are also characteristic. Tubular myelin surfactant has the same appearance as in other mammals.Multilamellar bodies were observed in direct communication with Golgi vesicles. Their origin from multivesicular bodies and their maturation through secretion and exocytosis were demonstrated.Untransformed multilamellar bodies in the alveolar space demonstrated three periodicities (P): (1) compact regular lamellae, PH = 36Å, FA = 31Å, P = 66Å; (2) compact broad lamellae, PH = 72Å, FA = 22Å, P = 94Å; (3) loose lamellae, PH = 36Å, FA = 36Å, FA = 31Å with a variable interlamellar space.Appreciation is expressed to Nuket Olson and Phil Offenhauser for their technical assistance. Supported by a grant from the American Lung Association     

Preparation of multilamellar vesicles of defined size-distribution by solvent-spherule evaporation

A novel method of preparing multilamellar vesicles is described. The process involves dispersing in aqueous solutions small spherules of volatile hydrophobic solvents in which amphipathic lipids are dissolved. The lipids form vesicles when the solvents are evaporated in the proper manner. The resulting vesicles have been characterized morphologically with microscopy and electron microscopy. The method yields multilamellar vesicles with a defined size distribution which can be adjusted by varying the duration of mechanical agitation of the spherules and by varying the concentration of amphipathic lipids in the solvents. This is the first fundamentally new method of multilamellar vesicle preparation since Bangham's report in 1965 (Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252).     

Drug entrapment in surfactant vesicles.

Surfactant vesicles, formed from dioctadecyldimethylammonium chloride, entrapped 8-azaguanine, methotrexate and 6-mercaptopurine in amounts greater than liposomes. Changes in pH and addition of cholesterol influence drug entrapment and release.     

Modification of anomalous swelling in multilamellar vesicles induced by alkali halide salts

By use of small-angle X-ray scattering it is shown that addition of alkali halide salts in small amounts (0-200 mM) shifts the repeat spacing in multilamellar DC13PC vesicles and alters the anomalous swelling behaviour close to the main transition. Both effects follow the Hofmeister series of the ions. We suggest that the shift of repeat spacing can be explained by ion effects on the van der Waals attractive forces between the membranes and on the decay length of the repulsive hydration force. The anomalous swelling is explained in terms of a critical unbinding of the membranes. The proximity of the critical temperature of the unbinding to the main transition temperature can be tuned by varying the concentration and type of salt in the sample.     

Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles

The preparation of a new kind of multilayered liposome, called a stable plurilamellar vesicle (SPLV), is described. Although SPLVs and classical multilamellar vesicles (MLVs) are made of the same materials and appear overtly similar in the electron microscope, the two types of vesicles differ as determined by stability, entrapment efficiency, electron spin resonance (ESR), NMR, X-ray diffraction, and biological effects. It is demonstrated that, contrary to what has been assumed, classical MLVs exclude solutes during their formation and, thus, are under a state of osmotic compression. By contrast, the SPLV process produces liposomes that are not compressed. The effects of osmotic compression are discussed. It is suggested that the state of osmotic stress is an important variable that distinguishes various types of liposomes and that has significant physical and biological consequences.     

NMR structure of lung surfactant peptide SP-B(11-25)

Surfactant protein B (SP-B) is a 79-residue essential component of lung surfactant, the film of lipid and protein lining the alveoli, and is the subject of great interest for its role in lung surfactant replacement therapies. Here we report circular dichroism results and the solution NMR structure of SP-B(11-25) (CRALIKRIQAMIPKG) dissolved in CD(3)OH at 5 degrees C. This is the first report of NMR data related to the protein SP-B, whose structure promises to help elucidate the mechanism of its function. Sequence-specific resonance assignments were made for all observable (1)H NMR signals on the basis of standard 2D NMR methods. Structures were determined by the simulated annealing method using restraints derived from 2D NOESY data. The calculations yielded 17 energy-minimized structures, three of which were subjected to 0.95 ns of restrained dynamics to assess the relevance of the static structures to more realistic dynamic behavior. Our CD and NMR data confirm that this segment is an amphiphilic alpha helix from approximately residue L14 through M21. The backbone heavy-atom RMSD for residues L14 through M21 is 0.09 +/- 0.12 A, and the backbone heavy-atom RMSD for the whole peptide is 0.96 +/- 2.45 A, the difference reflecting fraying at the termini. Aside from the disordered termini, the minimized structures represent dynamic structures well. Structural similarity to the homologous regions of related saposin-like proteins and the importance of the distribution of polar residues about the helix axis are discussed.     

Effects of cholesterol on acyl chain dynamics in multilamellar vesicles of various phosphatidylcholines

Phase modulation fluorescence spectroscopy was used to investigate the influence of cholesterol (0 to 50 mol%) on acyl chain dynamics in multilamellar vesicles of phosphatidylcholine. Four different phosphatidylcholines (DPPC, DOPC, POPC, and egg PC) and six different fluorescent probes (diphenylhexatriene and five anthroyloxy fatty acids) were employed. We found that: (1) Increased cholesterol content had only slight effects on fluorescence lifetimes of the six probes. (2) Increased cholesterol content increased the steady-state fluorescence anisotropy (r) of all the probes except 16-anthroyloxy palmitate (16-AP) in each of the four phosphatidylcholines. (3) Added cholesterol tended to limit the extent of probe rotation (as reflected by r_∞, the infinite-time anisotropy) to a much greater extent than it altered the rate of probe rotation. (4) The tendency for cholesterol to order the structure of the bilayer was greatest in the proximal half of the acyl chains and diminished toward the center of the bilayer. (5) In some phosphatidylcholines the rotation rates of probes located near the bilayer center (diphenylhexatriene and 16-AP) were apparently increased by increasing levels of cholesterol. (6) In several respects dipalmitoylphosphatidylcholine (DPPC) vesicles responded differently to increased cholesterol than vesicles of the other three phosphatidylcholines. (7) A single second-order equation described the relationship between r_∞and r for the five anthroyloxy fatty acid probes in the four different phosphatidylcholines over a wide range of cholesterol content. The data for diphenylhexatriene in the different phosphatidylcholines could not be fit by a single equation.