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/m², 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.     

Adhesion of giant vesicles mediated by weak binding of sialyl-LewisX to E-selectin in the presence of repelling poly(ethylene glycol) molecules

Prior to establishing tight contact with the endothelium, cells such as leukocytes or cancer cells use the recognition between sialyl-LewisX ligands and E-selectin receptors to establish weak, reversible adhesion and to roll along the vessel wall. We study the physical aspects of this process by constructing a mimetic system that consists of a giant fluid vesicle with incorporated lipid-anchored sialyl-LewisX molecules that bind to E-selectin that is immobilized on the flat substrate. The vesicles also carry a certain fraction of repelling PEG2000 molecules. We analyze the equilibrium state of adhesion in detail by means of reflection interference contrast microscopy and find that the adhesion process relies purely on the formation of one or more adhesion domains within the vesicle-substrate contact zone. We find that the content of ligands in the vesicle must be above 5 mol % to establish specific contacts. All concentrations of sialyl-LewisX above 8 mol % provide a very similar final state of adhesion. However, the size and shape of the adhesion domains strongly depend on both the concentrations of E-selectin (0-3500 molecules/microm2) and PEG2000 (0-5 mol %). At 3500 E-selectin molecules/microm2 and small concentrations of PEG2000, the vesicle-substrate contact is maximized and fully occupied by a single adhesion domain. At concentrations of 5 mol %, PEG2000 completely impedes the specific binding to any substrate. Lastly, an increase in the adhesion strength is observed in systems with identical compositions if the reduced volume of the vesicles is larger.     

Adhesive interactions between vesicles in the strong adhesion limit

We consider the adhesive interaction energy between a pair of vesicles in the strong adhesion limit, in which bending forces play a negligible role in determining vesicle shape compared to forces due to membrane stretching. Although force?distance or energy?distance relationships characterizing adhesive interactions between fluid bilayers are routinely measured using the surface forces apparatus, the atomic force microscope, and the biomembrane force probe, the interacting bilayers in these methods are supported on surfaces (e.g., mica sheet) and cannot be deformed. However, it is known that, in a suspension, vesicles composed of the same bilayer can deform by stretching or bending, and can also undergo changes in volume. Adhesively interacting vesicles can thus form flat regions in the contact zone, which will result in an enhanced interaction energy as compared to rigid vesicles. The focus of this paper is to examine the magnitude of the interaction energy between adhesively interacting, deformed vesicles relative to free, undeformed vesicles as a function of the intervesicle separation. The modification of the intervesicle interaction energy due to vesicle deformability can be calculated knowing the undeformed radius of the vesicles, R0, the bending modulus, k(b), the area expansion modulus, k(a), and the adhesive minimum, W(P)(0), and separation, D(P)(0), in the energy of interaction between two flat bilayers, which can be obtained from the force?distance measurements made using the above supported-bilayer methods. For vesicles with constant volumes, we show that adhesive potentials between nondeforming bilayers such as |W(P)(0)| 5 × 10(?4) mJ/m2, which are ordinarily considered weak in the colloidal physics literature, can result in significantly deep (>10×) energy minima due to increase in vesicle area and flattening in the contact region. If the osmotic expulsion of water across the vesicles driven by the tense, stretched membrane in the presence of an osmotically active solute is also taken into account, the vesicles can undergo additional deformation (flattening), which further enhances the adhesive interaction between them. Finally, equilibration of ions and solutes due to the concentration differences created by the osmotic exchange of water can lead to further enhancement of the adhesion energy. Our result of the progressively increasing adhesive interaction energy between vesicles in the above regimes could explain why suspensions of very weakly attractive vesicles may undergo flocculation and eventual instability due to separation of vesicles from the suspending fluid by gravity. The possibility of such an instability is an extremely important issue for concentrated vesicle-based products and applications such as fabric softeners, hair therapeutics and drug delivery.     

Phase Coexistence in a Dynamic Phase Diagram

Metastability and phase coexistence are important concepts in colloidal science. Typically, the phase diagram of colloidal systems is considered at the equilibrium without the presence of an external field. However, several studies have reported phase transition under mechanical deformation. The reason behind phase coexistence under shear flow is not fully understood. Here, multilamellar vesicle (MLV)‐to‐sponge (L₃) and MLV‐to‐L_α transitions upon increasing temperature are detected using flow small‐angle neutron scattering techniques. Coexistence of L_α and MLV phases at 40 °C under shear flow is detected by using flow NMR spectroscopy. The unusual rheological behavior observed by studying the lamellar phase of a non‐ionic surfactant is explained using ²H NMR and diffusion flow NMR spectroscopy with the coexistence of planar lamellar–multilamellar vesicles. Moreover, a dynamic phase diagram over a wide range of temperatures is proposed.     

Photophysical characterization of 1,8 naphthalimide in micelle-diblock copolymer nano-composite: a case of morphological transformation and vesicle formation

This paper reports a morphological transition of the spherical colloidal structures of the sodium dodecyl sulfate-polyethylene-b-polyethylene glycol (SDS-PE-b-PEG) complex and anionic micelle (SDS) to "rod-shaped" colloidal structures induced by a charge transfer dye, 1,8-naphthalimide (NAPMD) (forms anions in aqueous solution by intermolecular charge transfer). The distinct steady-state results of NAPMD in the above two media point toward the formation of a new microenvironment. SDS and SDS-PE-b-PEG form unilamellar (ULV) and multilamellar vesicles (MLV), respectively, along with the rod-shaped colloidal structures as observed from transmission electron microscopy (TEM) images. This dye causes a variation in the hydrophilic/hydrophobic ratio and forms a hydrogen bond with the copolymer in the SDS-PE-b-PEG complex and subjected to electrostatic interaction with the SDS micelle in aqueous solution, which causes this morphological transformation. These vesicles show complete encapsulation of a hydrophobic dye in its interior as evident from the TEM images. ULV get ruptured at low pH, pointing toward their lower stability over MLV at low pH value. The formation of these vesicles with complete idea of its mechanism, encapsulation of bioactive molecules and its rupture at lower pH raise hope as a potential nanoscale vehicle for biologically relevant compounds and their release at low pH medium.     

Theoretical analysis of adhering lipid vesicles with free edges

A theoretical model for describing the adhesion of lipid vesicle with free edges is developed. For adhesion in contact potential or in finite-range potential, the total energy functional is defined as the sum of elastic free energy, the surface energy, the line tension energy and the contact potential or the long-ranged potential. The equilibrium differential equation and boundary conditions for opening-up lipid vesicles are derived through minimizing the total energy functional. Numerical solutions to these equations are obtained under the axial symmetric condition. These numerical solutions can be used to qualitatively explain the influence of the substrate on the open-up lipid vesicles.     

Elucidating driving forces for liposome rupture: external perturbations and chemical affinity

Atomic force microscopy (AFM) studies under aqueous buffer probed the role of chemical affinity between liposomes, consisting of large unilamellar vesicles, and substrate surfaces in driving vesicle rupture and tethered lipid bilayer membrane (tLBM) formation on Au surfaces. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio) propionate] (DSPE-PEG-PDP) was added to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles to promote interactions via Au-thiolate bond formation. Forces induced by an AFM tip leading to vesicle rupture on Au were quantified as a function of DSPE-PEG-PDP composition with and without osmotic pressure. The critical forces needed to initiate rupture of vesicles with 2.5, 5, and 10 mol % DSPE-PEG-PDP are approximately 1.1, 0.8, and 0.5 nN, respectively. The critical force needed for tLBM formation decreases from 1.1 nN (without osmotic pressure) to 0.6 nN (with an osmotic pressure due to 5 mM of CaCl(2)) for vesicles having 2.5 mol % DSPE-PEG-PDP. Forces as high as 5 nN did not lead to LBM formation from pure POPC vesicles on Au. DSPE-PEG-PDP appears to be important to anchor and deform vesicles on Au surfaces. This study demonstrates how functional lipids can be used to tune vesicle-surface interactions and elucidates the role of vesicle-substrate interactions in vesicle rupture.     

Real-time membrane fusion of giant polymer vesicles

Membrane fusion is very important for the formation of many complex organs in metazoans throughout evolution, such as muscles, bones, and placentae. Lipid vesicles (liposomes) are frequently used as model membranes to study the fusion process. This work demonstrates for the first time the real-time membrane fusion of giant polymer vesicles by directly displaying a series of high-resolution and real-time transformation images of individual vesicles. The fusion process includes the sequential steps of membrane contact, forming the center wall, symmetric expansion of fusion pore and complete fusion, undergoing the intermediates of "8" shape with a protruding rim at the contact site, peanut (pear) shape, and oblate sphere. The vesicle swells during fusion, and the fusing vesicle only deforms in the neck domain around the fusion pore in the lateral direction, which verifies the importance of the lateral tension on the fusion pore at the vesicle deformation level. The successful fusion of the synthetic and protein-free polymer vesicles reported here also supports that vesicle proximity combined with membrane perturbation suffices to induce membrane fusion, and that the protein is not necessary for the fusion process.     

Geometric theory for adhering lipid vesicles

This paper aims at constructing a general mathematical model for the equilibrium theory of adhering lipid vesicles from a geometrical point of view. Based on the generalized potential functional, a few differential operators and their integral theorems on curved surfaces, the general normal and tangential equilibrium differential equations and boundary conditions are given at the first time for inhomogeneous lipid vesicles. A general boundary condition is first put forward including line tension. No assumptions are made either on the symmetry of the vesicle or on that of the substrate. The physical and biological meaning of the equilibrium differential equations and the boundary conditions are discussed. Numerical simulation results based on the Helfrich energy for adhering lipid vesicles under the axial symmetric condition show the effectiveness and convenience of the present theory.     

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, micro 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.     

Effective interactions in colloid-semipermeable membrane systems

We investigate effective interactions between a colloidal particle, immersed in a binary mixture of smaller spheres, and a semipermeable membrane. The colloid is modeled as a big hard sphere, and the membrane is represented as an infinitely thin surface, which is fully permeable to one of the smaller spheres and impermeable to the other one. Within the framework of the density functional theory, we evaluate depletion potentials and we consider two different approximate theories: the simple Asakura-Oosawa approximation and the accurate White-Bear version of the fundamental measure theory. The effective potentials are compared with the corresponding potentials for the hard, nonpermeable wall. Using statistical-mechanical sum rules, we argue that the contact value of the depletion potential between a colloid and a semipermeable membrane is smaller in magnitude than the potential between a colloid and a hard wall. A heuristic argument is provided that the colloid-semipermeable membrane effective interactions are generally weaker than these near a hard nonpermeable wall. These predictions are confirmed by explicit calculations, and the effect is more pronounced for smaller osmotic pressures. The depletion potential for a colloidal particle inside a semipermeable vesicle is stronger than the potential for the colloidal particle located outside of a vesicle. We find that the asymptotic decay of the depletion potential for the semipermeable membrane is similar to that for the nonpermeable wall and reflects the asymptotics of the total correlation function of the corresponding binary mixture of smaller spheres. Our results demonstrate that the ability of the membrane to change its shape as well as specific interactions constitute an important factor in determining the effective interactions between the semipermeable membrane and the colloidal macroparticle.     

Novel method for measuring the adhesion energy of vesicles

Adhering vesicles with osmotically stabilized volume are studied with Monte Carlo simulations and optical microscopy. The simulations are used to determine the dependence of the adhesion area on the vesicle volume, the surface area, the bending rigidity, the adhesion energy per membrane area, and the adhesion potential range. The simulation results lead to a simple functional expression that is supplemented by a correction term for gravity effects. The obtained equation provides a new tool to analyze optical microscopy data and, thus, to measure the adhesion energy per area by analyzing the geometry of the adhering vesicle. The method can be applied in the weak and ultra-weak adhesion regime, where the adhesion energy per area is below 10(-6) J/m(2). By comparing the shapes of adhering vesicles with different reduced volumes, the bending rigidity can be estimated as well. The new approach is applied to experimental data for lipid vesicles on (i) an untreated and (ii) a monolayer-coated glass surface, providing ultra-weak and weak adhesion strength, respectively.     

Force-induced de-adhesion of specifically bound vesicles: strong adhesion in competition with tether extraction

A theoretical study of the thermodynamic equilibrium between force-induced tether formation and the adhesion of vesicles mediated by specific ligand-receptor interactions has been performed. The formation of bonds between mobile ligands in the vesicle and immobile receptors on the substrate is examined within a thermodynamic approximation. The shape of a vesicle pulled with a point force is calculated within a continuous approach. The two approaches are merged self-consistently by the use of the effective adhesion potential produced by the collective action of the bonds. As a result, the shapes of the vesicle and the tether, as well as the number of formed bonds in the contact zone, are determined as a function of the force, and approximate analytic expressions for them are provided. The de-adhesion process is characterized by the construction of a phase diagram that is a function of the density of the ligands in the vesicle, the surface coverage by receptors, the ligand-receptor binding affinity, and the reduced volume of the vesicle. In all cases, the phase diagram contains three regions separated by two nonintersecting lines of critical forces. The first is the line of onset forces associated with a second-order shape transition from a spherical cap to a tethered vesicle. The second line is attributed to the detachment forces at which a first-order unbinding transition from a tethered shape to a free vesicle occurs.     

The adhesion kinetics of sticky vesicles in tension: the distinction between spreading and receptor binding

We investigate the kinetics of spreading and adhesion between polymer vesicles decorated with avidin and biotin, held in micropipettes to maintain fixed tension and suppress membrane bending fluctuations. In this study, the density of avidin (actually Neutravidin) and biotin was varied, but was always sufficiently high so that lateral diffusion in the membrane was unimportant to the adhesive mechanism or rate. For a stunning result, we report a concentration-dependent distinction between adhesion and spreading: At low surface densities of avidin and biotin, irreversible vesicle adhesion is strong enough to break the membrane when vesicle separation is attempted, yet there is no spreading or "wetting". By this we mean that there is no development of an adhesion plaque beyond the initial radius of contact and there is no development of a meaningful contact angle. Conversely, at 30% functionalization and greater, membrane adhesion is manifest through a spreading process in which the vesicle held at lower tension partially engulfs the second vesicle, and the adhesion plaque grows, as does the contact angle. Generally, when spreading occurs, it starts abruptly, following a latent contact period whose duration decreases with increasing membrane functionality. A nucleation-type rate law describes the latency period, determined by competition between bending and sticking energy. The significance of this result is that, not only are membrane mechanics important to the development of adhesion in membranes of nanometer-scale thickness, mechanics can dominate and even mask adhesive features such as contact angle. This renders contact angle analyses inappropriate for some systems. The results also suggest that there exist large regions of parameter space where adhesive polymeric vesicles will behave qualitatively differently from their phospholipid counterparts. This motivates different strategies to design polymeric vesicles for applications such as targeted drug delivery and biomimetic scavengers.     

Interaction of propyl paraben with dipalmitoyl phosphatidylcholine bilayer: a differential scanning calorimetry and nuclear magnetic resonance study

The influence of the preservative, propyl paraben (PPB) on the biophysical properties of dipalmitoyl phosphatidyl choline (DPPC) vesicles, both in multilamellar vesicle (MLV) and unilamellar vesicle (ULV) forms, has been studied using DSC and (¹H and ³¹P) NMR. The mechanism by which PPB interacts with DPPC bilayers was found to be independent of the morphological organization of the lipid bilayer. Incorporation of PPB in DPPC vesicles causes a significant depression in the transition temperature and enthalpy of both the pre-transition (PT) and the gel to liquid crystalline transition. The presence of the PPB also reduces the co-operativity of these transitions. However, at high PPB concentration the PT disappears. DSC and NMR findings indicate that: (i) PPB is bound strongly to the lipid bilayer leading to increased headgroup fluidity due to reduced headgroup–headgroup interaction and (ii) the PPB molecules are intercalated between the DPPC polar headgroups with its alkyl chain penetrate into the co-operative region. MLV incorporated with high PPB concentration shows additional transitions whose intensity increases with increasing PPB concentration. This phase segregation observed could probably be due to co-existence of PPB-rich and PPB-poor phospholipid domains within the bilayers. The effect of inclusion of cholesterol in the PPB-free and PPB-doped DPPC dispersion was also studied. Equilibration studies suggest that PPB molecules are very strongly bound and remain intercalated between the polar headgroup for prolonged time.     

Correction of random surface roughness on colloidal probes in measuring adhesion

Atomic force microscopes (AFM) are commonly used to measure adhesion at nanoscale between two surfaces. To avoid uncertainties in the contact areas between the tip and the surface, colloidal probes have been used for adhesion measurements. We measured adhesion between glass spheres and silicon (100) surface using colloidal probes of different radii under controlled conditions (relative humidity of < 3%, temperature of 25 +/- 1 degrees C). Results showed that the adhesion forces did not correlate with the radii of the spheres as suggested by elastic contact mechanics theories. Surface roughness and random surface features were found on the surfaces of the colloidal probes. We evaluated various roughness parameters, Rumpf and Rabinovich models, and a load-bearing area correction model in an attempt to correct for the roughness effects on adhesion, but the results were unsatisfactory. We developed a new multiscale contact model taking into account elastic as well as plastic deformation in a successive contacting mode. The new model was able to correct for most of the surface roughness features except for surface ridges with sharp angular features, limited by the spherical asperity assumption made in the model.     

Thermodynamic Study on Vesicle Formation and Adsorption of Decyltrimethylammonium Decyl Sulfate

The surface tension of an aqueous solution of decyltrimethylammonium decyl sulfate (DeTADeS) was measured as a function of temperature T at various molalities &mcirc; under atmospheric pressure. DeTADeS has been found to form equilibrium multilamellar vesicles (MLV) spontaneously. The surface density, the entropies of adsorption, and the entropy of vesicle formation are evaluated. The mechanism of formation of equilibrium vesicles is investigated from the standpoint of thermodynamics and from the comparison of the results with those of the micelle-forming systems. From the relatively small change of the surface density Gamma;(H) on T at a given &mcirc;, the adsorbed film is implied to be tightly packed due to the strong electrostatic attraction between the polar headgroups. The energy change associated with adsorption from the vesicular state per mole of surfactant Delta(V)(H)u is positive in the entire temperature range; thus, the curved bilayer in MLV is energetically more favorable than the planar adsorbed film. From the negative values of the entropy of vesicle formation Delta(W)(V)s, it is concluded that vesicle formation is driven by enthalpy whereas micelle formation is mostly entropy driven. Copyright 2001 Academic Press.     

Vesicle growth and deformation in a surfactant solution below the Krafft temperature

We have studied vesicle growth and deformation in aqueous solutions of nonionic surfactant C(16)E(7) below the Krafft temperature by means of an optical microscope. It has been found that vesicles become larger by fusing together, and that the growth rate is slower than that of the unilamellar vesicle or emulsion systems due to the multilamellar structures of shells in a vesicle. The deformation of the vesicles depends on the temperature quench depth, and we found the transformation from spherical vesicles to string-like domains at a certain quench-temperature. From the small angle X-ray scattering and confocal microscope experiments, it can be deduced that the deformation of vesicles would be induced by osmotic pressure due to the micellar concentration difference between inside and outside of vesicles.     

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 Ca(2+) 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. Copyright 2000 Academic Press.     

Shear-induced structural transformation of pentaethylene glycol <Emphasis Type="Italic">n</Emphasis>-dodecyl ether and lithium perfluorooctane sulfonate mixed-surfactant lamellar solution

The viscoelastic behavior of the shear-induced structural transformation from the lamellar phase to multilamellar vesicles (MLVs) of a mixed-surfactant system was investigated. The transformation was divided into two processes on the basis of the strain dependence of the apparent viscosity. The first stage is a lamellar-to-intermediate structure transformation. It was found that a strain, not an applied shear rate, governed this process. The second stage is an intermediate-to-MLV phase transformation, which was not controlled by the strain. These structure developments were found in the shear-thickening viscosity regime. The MLV phase formed by applying shear flow exhibited shear-thinning viscosity behavior and reversible response to shear flow. The viscoelastic properties of the MLV phase were investigated by dynamic viscoelastic measurements. Under oscillating shear deformation, the amplitude dependence of the dynamic modulus indicated that the viscoelasticity of the MLV depended on the initial structure, such as the number of vesicle shells and the size of the MLV, which is governed by the preshear rate.