Vesicle Radius Research Articles

Osmotic shrinkage and reswelling of giant vesicles composed of dioleoylphosphatidylglycerol and cholesterol

The osmotic shrinkage of giant unilamellar dioleoylphosphatidylglycerol (DOPG) vesicles in a hypertonic osmotic solution is investigated. The volume reduction for given membrane area leads to a vesiculation of the bilayer into the interior of the giant. The size of the daughter vesicles that appear inside the giant is uniform and an increasing function of the cholesterol content, but independent of the osmotic gradient applied. The radius of the daughter vesicles increases from 0.2 μm to 3.0 μm when the cholesterol content is changed from 0 to 40%. It is argued that the size of the daughter vesicles is regulated by the membrane persistence length, which is an exponential function of the mean bending modulus. From the kinetics of shrinkage it follows that approximately 14% of the daughter vesicles remain attached to the mother giant. This is in reasonable agreement with osmotic swelling experiments which show that approximately 11% of the daughter vesicles is available for area expansion.

Membrane Fusogenic Activity of the Alzheimer's Peptide Aβ(1–42) Demonstrated by Small-Angle Neutron Scattering

Amyloid-β peptide (Aβ) is considered a triggering agent of Alzheimer's disease. In relation to a therapeutic treatment of the disease, the interaction of Aβ with the cell membrane has to be elucidated at the molecular level to understand its mechanism of action. In previous works, we had ascertained by neutron diffraction on stacked lipid multilayers that a toxic fragment of Aβ is able to penetrate and perturb the lipid bilayer. Here, the influence of Aβ(1–42), the most abundant Aβ form in senile plaques, on unilamellar lipid vesicles of phospholipids is investigated by small-angle neutron scattering. We have used the recently proposed separated form factor method to fit the data and to obtain information about the vesicle diameter and structure of the lipid bilayer and its change upon peptide administration. The lipid membrane parameters were obtained with different models of the bilayer profile. As a result, we obtained an increase in the vesicle radii, indicating vesicle fusion. This effect was particularly enhanced at pH 7.0 and at a high peptide/lipid ratio. At the same time, a thinning of the lipid bilayer occurred. A fusogenic activity of the peptide may have very important consequences and may contribute to cytotoxicity by destabilizing the cell membrane. The perturbation of the bilayer structure suggests a strong interaction and/or insertion of the peptide into the membrane, although its localization remains beyond the limit of the experimental resolution.

Microscopic structure and collapse of depletion-induced gels in vesicle-polymer mixtures

We present the behavior of depletion-induced gels for vesicle-polymer mixtures when the ratio of the polymer radius of gyration to the mean vesicle radius is 0.09 and 0.27. As the polymer concentration increases, density gradients build up and an interface is developed between a highly turbid vesicle-rich phase and a polymer-rich phase. Increasing the polymer concentration further forms a gel (CP=0.3 and 0.1 wt% for Rg/a approximately 0.09 and 0.27, respectively), which subsequently collapses. This collapse is characterized by a slow initial rising for a finite delay time, a rapid collapse, and a slow final compaction to an equilibrium height. However, we observe a remarkably different polymer concentration dependence on the collapse rate. Unlike other colloidal gels, we find that the delay time for the vesicle collapse decreases with increasing polymer concentration. We show that this behavior can be accounted for by considering the permeability for solvent backflow, which is directly related to the characteristic pore area of the gel obtained using confocal microscopy.

Water distribution function across the curved lipid bilayer: SANS study

The neutron scattering length density across the membrane is simulated on the basis of fluctuated model of lipid bilayer. The use of a separated form factors method has been applied for the identification of the structural features of the polydispersed unilamellar DMPC vesicle system. The hydration of vesicle is described by sigmoid distribution function of the water molecules. The application of the model to the obtained SANS spectra allow the determination of the main parameters of the system, such as the average vesicle radius (and its polydispersity), the membrane thickness, the thickness of hydrocarbon chain region, the number of water molecules located per lipid molecule, and the phospholipid surface area. Moreover the approach allow the calculation of some relevant parameters connected with the water distribution function across the bilayer system. The main features of the obtained results furnish an explanation of why lipid membrane is easily penetrated by the water molecules of the solution.

Shrinkage Dynamics of a Vesicle Induced by Chemical Reactions

We study the shrinkage dynamics of a vesicle out of equilibrium. In our previous paper, we introduced a kinetic theory of a vesicle interacting with surfactant molecules. In the present paper, we extend the theory to cover more variety of chemical reactions. It is shown that the chemical kinetics change drastically the relation between the waiting time of pore formation and the radius of vesicle. Numerical simulations are carried out to confirm our analytical results.

Bending rigidity of mixed phospholipid bilayers and the equilibrium radius of corresponding vesicles

In spite of the large mean bending moduli observed for phospholipid bilayers, stable vesicle phases were recently observed for dilute solutions of charged phospholipids. A correspondingly large negative Gaussian bending modulus associated with charged membranes results in an overall curvature energy that is so low that entropic stabilization is possible. The mean bending modulus determines the membrane persistence length and therefore it is reasonable that there is a correlation between the membrane rigidity and the size of the lipid vesicles. Here we show that in mixtures of the anionic phospholipid dioleoylphosphatidylglycerol and the zwitterionic phospholipid dioleoylphosphatidylcholine the radius of vesicles produced by repetitive freeze-thaw cycles is considerably smaller than expected from the rigidities of the corresponding pure lipid bilayers. Self-consistent field calculations indicate that the changes in the equilibrium radius of mixed bilayers can be attributed to the dependences of the mean bending modulus k(c) on lipid mixing and the average surface charge density.

Defect Scars on Flexible Surfaces with Crystalline Order

The crystallography of two-dimensional particle packings on flexible surfaces of spherical topology is investigated. Examples are viral capsids and crystalline vesicles. Computer simulations of dynamically triangulated surfaces are employed to study the shape and structure of lattice defects as a function of the Föppl-von Kármán number gamma. We find that grain-boundary scars become much more fuzzy with increasing temperature, that the size of grain-boundary scars saturates with increasing vesicle radius, and that the buckling transition shifts to higher values of gamma due to the presence of scars.

Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4

We show that cryptdin-4 (Crp4), an antimicrobial peptide found in mice, induces the aggregation and hemi-fusion of charged phospholipid vesicles constructed of the anionic lipid POPG and the zwitterionic lipid POPC. Hemi-fusion is confirmed with positive total lipid-mixing assay results, negative inner monolayer lipid-mixing assay results, and negative results from contents-mixing assays. Aggregation, as quantified by absorbance and dynamic light scattering, is self-limiting, creating finite-sized vesicle assemblies. The rate limiting step in the formation process is the mixing of juxtaposed membrane leaflets, which is regulated by bound peptide concentration as well as vesicle radius (with larger vesicles less prone to hemi-fusion). Bound peptide concentration is readily controlled by total peptide concentration and the fraction of anionic lipid in the vesicles. As little as 1% PEGylated lipid significantly reduces aggregate size by providing a steric barrier for membrane apposition. Finally, as stable hemi-fusion is a rare occurrence, we compare properties of Crp4 to those of many peptides known to induce complete fusion and lend insight into conditions necessary for this unusual type of membrane merger.

Entropic Stabilization and Equilibrium Size of Lipid Vesicles

We have studied the phase behavior of zwitterionic phospholipid dioleoylphosphatidylcholine (DOPC) vesicles (membranes) and interpreted our results using scaling arguments in combination with molecular realistic self-consistent field (SCF) calculations. DOPC membranes acquire a partial negative charge per lipid molecule at intermediate NaBr concentrations. As a result of this, dilute DOPC solutions form stable unilamellar vesicles. Both at low and high salt concentrations phase separation into a lamellar and a vesicular phase is observed. The vesicle radius decreases as a power law with decreasing lipid concentration. This power-law concentration dependence indicates that the vesicle phase is entropically stabilized; the size of the DOPC vesicles result from a competition between the bending energy and translation and undulation entropy. This scaling behavior breaks down for very small vesicles. This appears to be consistent with SCF predictions that point to the fact that in this regime the mean bending modulus kc increases with curvature. The SCF theory predicts that, at low ionic strength, the membrane stability improves when there is more charge on the lipids. Upon a decrease of the ionic strength, lipids with a full negative charge form vesicles that grow exponentially in size because the mean bending modulus increases with decreasing ionic strength. At the same time the Gaussian bending modulus becomes increasingly negative such that the overall bending energy tends to zero. This indicates that small micelles become the dominant species. The SCF theory thus predicts a catastrophic break down of giant vesicles in favor of small micelles at sufficiently low ionic strength and high charge density on the lipids.

Curvature Effect on the Structure of Phospholipid Bilayers

High-resolution small-angle X-ray scattering (SAXS), complemented by small-angle neutron scattering (SANS) and dynamic light scattering (DLS) experiments, was used to study the effect of curvature on the bilayer structure of dioleoyl-phosphatidylcholine (DOPC) and dioleoyl-phosphatidylserine (DOPS) unilamellar vesicles (ULVs). Bilayer curvature, as a result of finite vesicle size, was varied as a function of vesicle radius and determined by DLS and SANS measurements. Unilamellarity of large DOPC ULVs was achieved by the addition of small amounts (up to 4 mol %) of the charged lipid, DOPS. A comparison of SANS data over the range of 0.02 < q <0.2 A-1 indicated no change in the overall bilayer thickness as a function of ULV diameter (620 to 1840 A). SANS data were corroborated by high-resolution (0.06 < q <0.6 A-1) SAXS data for the same diameter ULVs and data obtained from planar samples of aligned bilayers. Both the inner and outer leaflets of the bilayer were found to be indistinguishable. This observation agrees well with simple geometric models describing the effect of vesicle curvature. However, 1220-A-diameter pure DOPS ULVs form asymmetric bilayers whose structure can most likely be rationalized in terms of geometrical constraints coupled with electrostatic interactions, rather than curvature alone.

Transition from long- to short-lived transient pores in giant vesicles in an aqueous medium

We have observed large pores in the membrane of giant vesicles in an aqueous medium. The lifetime of the pores can reach 2 min and their size (a few micrometers) enables their visualization by fluorescence microscopy. These pores are obtained thanks to a destabilization of the membrane due to the synergistic action of a cone-shaped and nitrobenzodiazole (NBD) labeled phospholipid illuminated in the presence of dithionite. The opening of the pore occurs immediately after illumination starts so that it can be accurately triggered. A concomitant decrease of the vesicle radius is observed; we interpret it as a solubilization of the membrane. Depending on the rate of this solubilization, long- or short-lived pores were observed. At the transition between both regimes for a 30 microm vesicle, the solubilization rate was about 1/300 s{-1} . In order to interpret these observations, we have revisited the current model of pore opening to take into account this solubilization. This proposed model along with simulations enables us to prove that solubilization explains why the large long-lived pores are observed even in an aqueous medium. The model also predicts the solubilization rate at the transition between a single long-lived pore and a cascade of short-lived pores.

Self-Assembly in a Catanionic Mixture with an Aminoacid-Derived Surfactant: From Mixed Micelles to Spontaneous Vesicles

The aqueous self-assembly of a novel lysine-derived surfactant with a gemini-like architecture, designated here as 12-Lys-12, has been experimentally investigated for the amphiphile alone in water and in a mixture with dodecyltrimethylammonium bromide (DTAB). The neat surfactant forms interesting micrometer-sized rigid tubules in the dilute region, resulting in very viscous solutions. For the catanionic mixture with DTAB, various single and multiphase regions were identified (up to a total surfactant concentration of 1.5 wt %) by means of combined polarizing light microscopy, cryo-TEM, and NMR. In the DTAB-rich side, for a mixing molar ratio in the range 2 < DTAB/12-Lys-12 < 4, a region of stable, unilamellar vesicles can be found. Furthermore, it was found that upon addition of 12-Lys-12 to pure DTAB solutions, the mixed micelles grow and beyond a given mixing ratio, vesicles assemble and coexist with small micelles. The transition is not continuous, since there is a narrow mixing range where phase separation occurs. Self-diffusion measurements and cryo-TEM imaging show that the average vesicle radius is on the order of 30-40 nm.

Curved single-bilayers in the region of the anomalous swelling: Effect of curvature and chain length

We have carried out density and DSC measurements on large unilamellar vesicles (LUVs) of different phosphocholines in the temperature range corresponding to the anomalous swelling regime of multibilayer systems, adjacent to the chain melting transition region. Experiments were performed for DPPC, DMPC and DC 13PC, as a function of aggregate size. The steep drop in density occurring around the main transition gets broader as the vesicle radius decreases and a two-step process is clearly observed for DMPC and DC 13PC vesicles smaller than 200 nm. No similar effect is observed for DPPC vesicles, whatever the aggregate dimension. DSC measurements corroborate density results, showing a splitting of the main peak that becomes more evident as the chain length decreases and as the curvature of the aggregate increases.

Probing Lipid Vesicles by Bimolecular Association and Dissociation Trajectories of Single Molecules

Vesicles prepared by DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid molecules having sizes smaller than the diffraction-limited focused laser beam have been used to confine single molecules in the laser focus. The confinement of single molecules in a volume smaller than the focused laser beam leads to a Gaussian distribution of single molecule fluorescence intensity. The interactions of single Nile Red molecules with DMPC and SOPC lipid bilayers were studied by single molecule fluorescence confocal microscopy. Nile Red molecules were observed to associate with and dissociate from individual DMPC and SOPC vesicles adsorbed on a glass surface, generating on-and-off fluctuations in a fluorescence signal representing a very low noise two-state trajectory. Off-time statistics were used to investigate the mean radius of the vesicles and the size distribution functions. The means of the on-time distributions of Nile Red in DMPC and SOPC vesicles were significantly different. The association and dissociation reactions of single Nile Red molecules with a vesicle have been studied. Features of the bimolecular interaction between the probe Nile Red and the vesicle were evaluated from the uncorrelated mean on-time and vesicle radius distributions, and the linear Nile Red concentration dependence of the mean off-time. Nile Red is shown to be a useful probe of the structural fluctuations and heterogeneity of these membrane structures, and it is a useful model with which to directly study a diffusion-influenced reversible bimolecular reaction.

Membrane Curvature Change Induced by an Antimicrobial Peptide Detected by 31P Exchange NMR

The influence of an antimicrobial peptide, protegrin-1 (PG-1), on the curvature and lateral diffusion coefficient (D(L)) of phosphocholine bilayers is investigated using one- (1D) and two-dimensional (2D) (31)P exchange NMR. The experiments utilize the fact that lipid lateral diffusion over the curved surface of vesicles changes the molecular orientation and thus the (31)P chemical shift anisotropy. This reorientation is manifested in 2D spectra as off-diagonal intensities and in 1D stimulated-echo experiments as reduced echo heights. The 2D spectra give information on the reorientation-angle distribution while the decay of the stimulated-echo intensity, which closely tracks the second-order correlation function in our experiments, yields the correlation times of the reorientation. The relationships among the 2D exchange spectra, stimulated-echo intensities, the correlation function, and reorientation-angle distributions are analyzed in detail. In the absence of PG-1, both dilaurylphosphotidylcholine (DLPC) and palmitoyloleoylphosphatidylcholine (POPC) vesicles show biexponential decays of the stimulated-echo intensities to equilibrium values of 0.20-0.25, suggesting that the curvature of the lipid vesicles has a bimodal distribution. The addition of PG-1 to DLPC vesicles increased the decay time constants, indicating that D(L) decreases due to peptide binding. In contrast, the addition of PG-1 to POPC vesicles decreased the decay constants by three to fivefold, indicating that the POPC vesicles are fragmented into smaller vesicles. On the basis of the changes in D(L) and the decay constants, we estimate that the radius of the POPC vesicles decreases by threefold due to PG-1 binding. Simulations of the 2D exchange spectra yielded quantitative reorientation-angle distributions that are consistent with the bimodal distributions of the vesicle curvature and the effects of the peptide on the two types of lipid bilayers. Thus, (31)P exchange NMR provides useful insights into the membrane morphological changes induced by this antimicrobial peptide.

Influence of phytosphingosine-type ceramides on the structure of DMPC membrane

The present paper describes the influence of the ceramides with phytosphingosine base, N-stearoylphytosphingosine (Cer[NP]) and α-hydroxy- N-stearoylphytosphingosine (Cer[AP]), on the structure and properties of multilamellar (MLVs) and unilamellar vesicles (ULVs) of dimyristoylphosphatidylcholine (DMPC). The lamellar repeat distance, D, has been measured at various temperatures using small angle X-ray diffraction. The incorporation of ceramides into the DMPC membrane causes larger D compared to pure DMPC membrane. For both ceramide types, at 32 °C, there is a linear relationship between the D value and the ceramide concentration. However, there is no such dependence at 13 or 60 °C. Unlike Cer[AP], Cer[NP] induces a new phase with a repeat distance of 38.5 Å. The membrane thickness and the vesicle radius of ULVs in water and in sucrose solution were calculated from small angle neutron scattering curves. Phytosphingosine ceramides increase both the membrane thickness and the radius in comparison to pure DMPC ULVs. The stability of ULVs in time was studied by dynamic light scattering. Both ceramides induce an aggregation of the ULVs into micrometer sized non-multilamellar structures in pure water. Presence of sucrose in the environment averts the vesicle aggregation.

CTP:Phosphocholine Cytidylyltransferase Binds Anionic Phospholipid Vesicles in a Cross-Bridging Mode

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.

Particle Size Distribution in DMPC Vesicles Solutions Undergoing Different Sonication Times

Size distribution of dimyristoylphosphatidylcholine liposome suspensions was investigated by dynamic-light scattering (DLS) as a function of the sonication time (t(s)). Cumulant expansion (second- and third-order) and regularized Laplace inversion (CONTIN) of dynamic single-angle laser light-scattering data were performed. With both methods, the intensity-weighted mean hydrodynamic radius r(I) depended on the investigated lengthscale. The number-weighted mean hydrodynamic radius (r(N)), obtained from CONTIN by modeling dimyristoylphosphatidylcholine vesicles as thin-walled hollow spheres, resulted as independent on the lengthscale. However, the r(N) value obtained from cumulant expansions remained lengthscale-dependent. Therefore, the number-weighted radius distribution function is highly asymmetric. The number-weighted mean radius, the standard deviation, and the number-weighted radius at the peak (r(N)(peak)) all decreased to a plateau when increasing sonication time. At t(s) longer than 1 h, the r(N)(peak) compares well with the radius of unilamellar vesicles in equilibrium with monomers predicted on a thermodynamic basis. The reliability of our analysis is proved by the comparison of experimental Rayleigh ratios with simulated ones, using the normalized number-weighted radius distribution function p(N)(r) determined by DLS data. A perfect agreement was obtained at longer sonication times, and the average aggregation number was determined. At lower t(s) values, simulations did not match experimental data, and this discrepancy was ascribed to the presence of large and floppy unilamellar vesicles with ellipsoidal shapes. Our investigation shows that, from single-angle DLS data, the radius distribution function of the vesicles can only be obtained if p(N)(r) is known.

Controlled Initiation of Enzymatic Reactions in Micrometer-Sized Biomimetic Compartments

We present a technique to initiate chemical reactions involving few reactants inside micrometer-scale biomimetic vesicles (10(-12) to 10(-15) L) integral to three-dimensional surfactant networks. The shape of these networks is under dynamic control, allowing for transfer and mixing of two or several reactants at will. Specifically, two nanotube-connected vesicles were filled with reactants (substrate and enzyme, respectively) by microinjection. Initially, the vesicles are far apart and any diffusive mixing (on relevant experimental time scales) between the contents of the separated vesicles is hindered because of the narrow diameter and long axial extension of the nanotube. To initiate a reaction, the vesicles were brought close together, the nanotube was consumed by the vesicles and at a critical distance, the nanotube-vesicle junctions were dilated leading to formation of one spherical reactor, and hence mixing of the contents. We demonstrate the concept using a model enzymatic reaction, which yields a fluorescent product (two-step hydrolysis of fluorescein diphosphate by alkaline phosphatase), where product formation was measured as a function of time using a FRAP fluorescence microscopy protocol. By comparing the enzymatic activity with bulk measurements, the enzyme concentration inside the vesicle could be determined. Reactions could be followed for systems having as few as approximately 15 enzyme molecules confined to a reactor vesicle. To describe the experiments we use a simple diffusion-controlled reaction model and solve it using a survival probability approach. The agreement with experiment is qualitative, but the model describes the trends well. It is shown that the model correctly predicts (i) single-exponential decay after a few seconds, and (ii) that the substrate decay constant depends on the number of enzymes and geometry of reaction container. The numerical correction factor Lambda is introduced in order to ensure semiquantitative agreement between experiment and theory. It was shown that this numerical factor depends weakly on vesicle radius and number of enzymes, thus it is sufficient to determine this factor only once in a single calibration measurement.

Fluid Mixing in Growing Microscale Vesicles Conjugated by Surfactant Nanotubes

This work addresses novel means for controlled mixing and reaction initiation in biomimetic confined compartments having volume elements in the range of 10(-12) to 10(-15) L. The method is based on mixing fluids using a two-site injection scheme into growing surfactant vesicles. A solid-state injection needle is inserted into a micrometer-sized vesicle (radius 5-25 microm), and by pulling on the needle, we create a nanoscale surfactant channel connecting injection needle and the vesicle. Injection of a solvent A from the needle into the nanotube results in the formation of a growing daughter vesicle at the tip of the needle in which mixing takes place. The growth of the daughter vesicle requires a flow of surfactants in the nanotube that generates a flow of solvent B inside the nanotube which is counterdirectional to the pressure-injected solvent. The volume ratio psi between solvent A and B inside the mixing vesicle was analyzed and found to depend only on geometrical quantities. The majority of fluid injected to the growing daughter vesicle comes from the pressure-based injection, and for a micrometer-sized vesicle it dominates. For the formation of one daughter vesicle (conjugated with a 100-nm radius tube) expanded from 1 to 200 microm in radius, the mixing ratios cover almost 3 orders of magnitude. We show that the system can be expanded to linear strings of nanotube-conjugated vesicles that display exponential dilution. Mixing ratios spanning 6 orders of magnitude were obtained in strings of three nanotube-conjugated micrometer-sized daughter vesicles.