Adhering Vesicles Research Articles

PH responsive adhesion of phospholipid vesicle on poly(acrylic acid) cushion grafted to poly(ethylene terephthalate) surface

Polymer-supported lipid bilayer is a key enabling technology for the design and fabrication of novel biomimetic devices. To date, the physical driving force underlying the formation of polymer-supported lipid bilayer remains to be determined. In this study, the interaction between dipalmitoylphosphocholine (DPPC) vesicle and poly(ethylene terephthalate) [PET] surface with or without grafted poly(acrylic acid) [PAA] layer is examined with several biophysical techniques. First, vesicle deformation analysis shows that the geometry of adherent vesicle on either plain PET or PAA-grafted PET surface is best described by a truncated sphere model. At neutral pH, the degree of deformation and adhesion energy are unaltered by the grafted polymerization of acrylic acid on PET surface. Interestingly, the average magnitude of adhesion energy is increased by 185% and −43% on PAA-grated PET and plain PET surface, respectively, towards an increase of pH at room temperature. Our results demonstrate the possibility of tuning the adhesive interaction between vesicle and polymer cushion through the control of polyelectrolyte ionization on the solid support.

Temperature dependence of vesicle adhesion

The influence of thermal fluctuations on the adhesion behavior of fluid vesicles is investigated with the help of Monte Carlo simulations. The adhesion area A(ad) of a fluid vesicle adhering to a smooth attractive substrate is studied systematically for different values of temperature, adhesion strength, and potential range. For low temperatures T , the ratio A(ad) /A between the adhesion area and the total area A of the vesicle is a linear function of T/kappa , where kappa is the bending rigidity. Linear fits of the simulation data allow an extrapolation to T=0 which corresponds well with data obtained from a simplified analytic model. A new ansatz for A(ad) (T) which is based on the eigenmodes of the adhering vesicle explains the linear behavior of A(ad) (T) for low T and helps to define a fit function which reproduces the linear behavior of the obtained simulation data. This fit function may be used in order to determine the bending rigidity and the adhesion strength from the observed adhesion geometry.

Membrane composition determines the fate of aggregated vesicles

Vesicles incorporating a fluorescent metal-chelating lipid can be linked together by addition of copper(II) and poly-L-histidine, but the stability of adhering vesicles towards fusion depends upon membrane composition.

Patterned Supported Lipid Bilayers and Monolayers on Poly(dimethylsiloxane)

A simple and practical method for patterning supported lipid bilayers on poly(dimethylsiloxane) is presented. By using electron microscopy grids to laterally control the extent of plasma oxidation, the substrate is partitioned into regions of different hydrophilicities. Addition of vesicles then results in the spontaneous formation of lipid bilayers and monolayers side-by-side on the surface, separated by regions that contain no lipid and/or a region with adhering vesicles. By using millimeter-sized plastic masks we are able to control the formation of these lipid structures on macroscopic patches by simply varying the plasma-cleaning time. For the first time, we are able to influence, in a controlled fashion, the chemical composition of a substrate in such a way that it supports fluid lipid monolayers, rejects lipid adhesion, adsorbs intact lipid vesicles, or supports fluid bilayers.

Surface-Dependent Transitions during Self-Assembly of Phospholipid Membranes on Mica, Silica, and Glass

Formation of supported membranes by exposure of solid surfaces to phospholipid vesicles is a much-used technique in membrane research. Freshly cleaved mica, because of its superior flatness, is a preferred support, and we used ellipsometry to study membrane formation kinetics on mica. Neutral dioleoyl-phosphatidylcholine (DOPC) and negatively charged dioleoyl-phosphatidylserine/dioleoyl-phosphatidylcholine (20% DOPS/80% DOPC) vesicles were prepared by sonication. Results were compared with membrane formation on silica and glass, and the influence of stirring, buffer, and calcium was assessed. Without calcium, DOPC vesicles had a low affinity (Kd approximately 30 microM) for mica, and DOPS/DOPC vesicles hardly adsorbed. Addition of calcium promptly caused condensation of the adhering vesicles, with either loss of excess lipid or rapid additional lipid adsorption up to full surface coverage. Vesicle-mica interactions dominate the adsorption process, but vesicle-vesicle interactions also seem to be required for the condensation process. Membranes on mica proved unstable in Tris-HCl buffer. For glass, transport-limited adsorption of DOPC and DOPS/DOPC vesicles with immediate condensation into bilayers was observed, with and without calcium. For silica, vesicle adsorption was also rapid, even in the absence of calcium, but the transition to condensed layers required a critical surface coverage of about 50% of bilayer mass, indicating vesicle-vesicle interaction. For all three surfaces, additional adsorption of DOPC (but not DOPS/DOPC) vesicles to condensed membranes was observed. DOPC membranes on mica were rapidly degraded by phospholipase A2 (PLA2), which pleads against the role of membrane defects as initial PLA2 targets. During degradation, layer thickness remained unchanged while layer density decreased, in accordance with recent atomic force microscopy measurements of gel-phase phospholipid degradation by PLA2.

Pulling tethers from adhered vesicles.

The competition between adhesion and tether formation in bound vesicles is investigated. A theoretical model is developed in which tethers are induced by the application of a pulling force to the top of a strongly adhered vesicle. A critical onset force is identified where the tether spontaneously appears as part of a first order shape transition. Further growth of the tether initiates a detachment process that culminates in a continuous unbinding of the vesicle at a finite detachment force. Both critical forces, as well as all shape parameters, are calculated as a function of the reduced volume and the strength of adhesive potential.

A quantitative contour analysis of axisymmetric vesicles spontaneously adhering onto a substrate

The determination of membrane-substrate profile for adherent vesicle using confocal reflectance interference contrast microscopy (C-RICM) has pushed for the need of advanced mechanics model for interpreting adhesion mechanisms. In this work, a model for vesicles or cells adhesion is established, the governing equation is derived from the variation of the potential energy at the cohesive zone. A closed-form solution is found for vesicle spontaneously adheres to a substrate when its shear modulus, μ vanishes. Based on the model and C-RICM experiments the magnitude of the adhesion force is calculated for a lipid vesicle adheres to a glass substrate.

Effects of carbon chain difference and lipid composition on the contact mechanics of two-component vesicle

The composition of phospholipid mixture is known to influence the lateral organization of the resulting biomembrane. In this study, the substrate-induced adhesion of two component vesicles composed of dimyristoyl-phosphatidylcholine/distearoyl-phosphatidylcholine (DMPC/DSPC) is probed by confocal reflectance interference microscopy (C-RICM), cross-polarized light microscopy and contact mechanics modeling. First, our contact mechanics model of adherent vesicle is validated for the two-component DMPC/DSPC vesicles. The incorporation of 10 mol% of DSPC in DMPC bilayer enhances both the degree of vesicle deformation and adhesion energy of the resulting two-component vesicle in comparison with those of one-component DMPC or DSPC vesicles. It is further illustrated that the compositional variation of the binary DMPC/DSPC mixture does not affect the contact mechanics parameters of adherent vesicles when the mole fraction of DSPC increases from 0 to 70%. Interestingly, the difference in the acyl chain length between the two lipids within the phospholipid mixture is directly correlated with the degree of vesicle deformation and adhesion energy. Overall, the biophysical responses of adherent vesicles are shown to be tunable by the bio-membrane composition and the carbon chain difference.

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T m. In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T m and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 μm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.

The effect of electrostatics on the contact mechanics of adherent phospholipid vesicles

Adhesion of cells on biomaterial surface is resulted from the complex interplay of specific recognitions and colloidal interactions. Thus understanding the role of electrostatic interactions in bioadhesion may help to elucidate the physiochemical basis of cell signaling pathway on therapeutic devices. In this report, high-resolution reflection interference contrast microscopy, cross-polarized light microscopy and contact mechanics modeling are applied to probe the equilibrium adhesion of giant phospholipid vesicles on 3-amino-propyl-triethoxy-silane coated glass. Simultaneously, the effects of vesicle wall thickness, pH, osmotic stress and surface chemistry on the electrostatic interactions at the membrane–substrate interface are evaluated. The results show that both unilamellar vesicles (ULV) and multilamellar vesicles (MLV) strongly adhere on the cationic substrates at neutral pH. In the presence of electrostatic interactions, ULV is slightly deformed on the substrate as the dimension of its adhesive–cohesive zone is only 6–10% higher than the theoretical value of a rigid sphere with the same mid-plane diameter. The variances of contact angle and capillary length at different locations surrounding MLV are ten times higher than those of ULV. The adhesion energy of ULV with mid-plane diameter of 45 and 20 μm is determined as 3.8×10 −12 and 8.6×10 −12 J/m 2, respectively, from the truncated sphere model. Moreover, the increase of osmotic stress induces irregular pattern in ULV's adhesion disc and raises the adhesion energy by 10-fold. Finally, the reduction of pH further enhances the electrostatic attractions/repulsions between vesicle surface and cationic or anionic substrates and leads to an increase of adhesion strength.

Colloidal adhesion of phospholipid vesicles: high-resolution reflection interference contrast microscopy and theory

High-resolution reflection interference contrast microscopy (HR-RICM) was developed for probing the deformation and adhesion of phospholipid vesicles induced by colloidal forces on solid surfaces. The new technique raised the upper limit of the measured membrane–substrate separation from 1 to 4.5 μm and improved the spatial resolution of the heterogeneous contact zones. It was applied to elucidate the effects of wall thickness, pH and osmotic stress on the non-specific adhesion of giant unilamellar vesicles (ULV) and multilamellar vesicles (MLV) on fused silica substrates. By simultaneous cross-polarization light microscopy and HR-RICM measurements, it was observed that ULV with the wall thickness of a single bilayer would be significantly deformed in its equilibrium state on the substrate as the dimension of its adhesive–cohesive zone was 29% higher than the theoretical value of a rigid sphere with the same diameter. Besides, electrostatic interaction was shown as a significant driving force for vesicle adhesions since the reduction in pH significantly increased the degree of deformation of adhering ULV and heterogeneity of the adhesion discs. The degree of MLV deformation on the solid surfaces was significantly less than that of ULV. When the wall thickness of vesicle increased, the dimension of contact zone was reduced dramatically due to the increase of membrane bending modulus. Most important, the adhesion strength of colloidal adhesion approached that of specific adhesion. Finally, the increase of osmotic stress led to the collapse of adhering vesicles on the non-deformable substrate and raised the area of adhesive contact zone. To interpret these results better, the equilibrium deformation of adhering vesicle was modeled as a truncated sphere and the adhesion energy was calculated with a new theory.

Binding of Lipid Vesicles to Protein-Coated Solid Polymer Surfaces: A Model for Cell Adhesion to Artificial Biocompatible Materials

The adhesion of lipid vesicles (liposomes) having controlled chemical and physical structure to polymer supported human serum albumin (HSA) thin layers was investigated by a spectrofluorimetric technique. The vesicle lipid bilayer was labeled with a small amount of an apolar fluorescent probe (diphenylexathriene) and the vesicle suspension was set in contact with the protein film. After washing and drying, the adhering vesicles containing sample was dissolved in chloroform and the homogeneous solution was analyzed by standard spectrofluorimetric techniques. Different parameters of the lipid bilayer, suspending solution, and protein film were varied and their influence on the liposome binding was investigated. Concerning the lipid bilayer, we studied the effect of liposome surface charge by using different mixtures of neutral (dipalmitoyl-phosphatidylcholine) and charged (dipalmitoyl-phosphatidic acid) phospholipids and the fluid or gel nature of the lipid bilayer (switched on and off by temperature variation). Variations of the local environment involve Ca2+ and H+ changes in the millimolar range as well as different hydrodynamical flows (in the range 0.1–10 cm/s). Preliminary measurements using different protein layers were also performed. Results show: (a) negligible adhesion without the protein layer, (b) the presence of a maximum for the liposome adhesion vs ion concentration (depending on the liposome composition and kind of the adsorbed ions), (c) a much stronger adhesion for vesicles in the fluid phase (overcoming the entropy-driven desorption increase with temperature), and (d) a dramatic lowering of the adhesion capability under hydrodynamic flow. Points a–c have been interpreted on the basis of a simple mechanoelectrical model.

Measuring Ligand−Receptor Unbinding Forces with Magnetic Beads: Molecular Leverage

We describe a new method for the quantitative measurement of weak forces between adhesion domains formed by receptor−ligand pairs. The method relies on the microinterferometric analysis of adhering vesicles subjected to an external force produced by a magnetic bead adhered to the membrane. The forces exerted on pinning centers can be deduced using classical elasticity theory. The method is applied to measure the binding strengths between solubilized solid supported integrin receptors αIIbβ3 of blood platelets and lipid-coupled cyclic peptides containing arginine−glycine−aspartate (RGD) tripetide ligands, embedded in the vesicle, that are recognized selectively by the integrins. We find that unbinding takes place at force levels far below those found in single-molecule studies. We interpret our findings in terms of the fracturing of the receptor−ligand pairs by the torque generated by the force which is much more effective than the traction.

Directly observed membrane fusion between oppositely charged phospholipid bilayers.

A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4-20 micron in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion; the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs in cells, in the absence of proteins.

Morphology and dynamics of vesicles

The shapes and dynamics of vesicles are governed by the competition between curvature energy, geometrical constraints and viscous dissipation in the surrounding liquid. Recent progress in this field has occurred in three major areas. Firstly, detailed comparisons between theory and experiment have been performed for various shape transformations such as the budding transition, conformai diffusion of vesicles of higher genus, and starfish vesicles. Secondly, the novel experimental technique of using laser tweezers to excite membranes locally has revealed non-equilibrium properties, such as pearling of cylindrical vesicles, which can be explained theoretically using the concept of marginal stability. Thirdly, the quantitative study of shape fluctuation of adhering vesicles motivated a theoretical investigation which includes self-consistent shape-dependent steric repulsion.

Molecular and collective dynamics of lipid bilayers

The unique advantages of the incoherent quasielastic neutron scattering technique (in particular in combination with NMR relaxation methods and molecular dynamics simulations) have been used to unravel different modes of the hierarchical superposition of motional processes in the fluid and solid states of lipid bilayers. New theoretical and experimental developments have taken place that analyze bending excitations of adhering vesicles and measure undulation forces.

P-F3-07 Shape changes of adhering vesicles: effect of nonspecific forces: Nardi J., Sackmann E. Biophysics Group E22, Tech. Univ. Munich (D)

P-F3-07 Shape changes of adhering vesicles: effect of nonspecific forces: Nardi J., Sackmann E. Biophysics Group E22, Tech. Univ. Munich (D)

Interactions between Dioctadecyldimethylammonium Chloride or Bromide Bilayers in Water

Interactions between dioctadecyldimethylammonium bromide (DODAB) or chloride (DODAC) closed bilayers in water are investigated. First, bilayers from vesicles are deposited onto oppositely charged polystyrene microspheres. Thereafter, a further increase in amphiphile concentration leads to vesicle adhesion to the bilayer-covered latex. For DODAC vesicles, the extent of vesicle adhesion to the covered latex is much higher than for DODAB vesicles. There is no rupture of adhered DODAC vesicles. In contrast, adhesion of DODAB vesicles is followed by vesicle rupture. Rupture for DODAB is associated with the absence of hydration repulsion whereas the absence of rupture for DODAC is related to its occurrence. A high extent of interdigitation in the DODAC bilayer causes the long-ranged hydrophobic attraction responsible for adhesion and can be associated with a long-ranged hydrophobic attraction between bilayers. The possibility of adhesion at a secondary minimum is ruled out for interactions in water.

Imaging optical thicknesses and separation distances of phospholipid vesicles at solid surfaces

We present the application of reflection interference contrast microscopy (RICM) (1) to map the optical density of supported bilayers and vesicles and (2) to image the contact profile of phospholipid vesicles at surfaces. The resolution in the surface profile is 0.2 $\mu$m laterally and 1 nm out of plane. The optical thickness of the membrane can be determined with 0.2 nm accuracy. We outline the theoretical basis of RICM and derive the interference intensities of adhering vesicles from first principles. An analytical expression for the decaying contrast of the intrference fringes is given. The contact contour of vesicles is determined for various substrates. We further demonstrate that deposition of a magnesium fluoride layer on the glass substrate enhances the contrast and allows the optical density of adsorbed membranes to be imaged. By contrast variation of the buffer solution, the layer thicknesses and the indices of refraction can be measured. The novel method was applied to image lipid domains of different chain lengths in a substrate supported monolayer.

Entropy-driven tension in vesicle membranes and unbinding of adherent vesicles

Entropy-driven tension in vesicle membranes and unbinding of adherent vesicles