Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

A silicate gel for promoting deposition of lipid bilayers

A simple method for modifying a polymer surface to induce lipid bilayer formation by vesicle fusion is described. A silicate gel was prepared by condensation of tetraethyl orthosilicate (TEOS) in the presence of acid. When applied to a poly(methylmethacrylate) substrate, either a rough or a smooth layer could be produced, depending on the method used for the application. The smooth surface induced formation of a supported lipid bilayer by fusion of lipid vesicles; the rough silicate surface induced adsorption of a vesicle layer. A high-frequency acoustic waveguide device was used to follow the initial adsorption of vesicles, the transition from a vesicle layer to a bilayer, and the formation of a complete bilayer; the time required to form a bilayer was determined as a function of lipid concentration in suspension. The presence of a bilayer on the smooth silicate surface was confirmed by fluorescence recovery after photobleaching. An additional procedure is described to modify a gold surface to induce bilayer formation.     

Simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption

Recent experimental investigations of the kinetics of vesicle adsorption in solution on SiO2 demonstrate a thermally activated transition from adsorbed intact vesicles to a supported lipid bilayer. Our Monte Carlo simulations clarify the mechanism of this process. The model employed is an extension of the model used earlier to describe vesicle adsorption at room temperature. Specifically, it includes limitations of the adsorption rate by vesicle diffusion in the solution, and adsorption- and lipid-membrane-induced rupture of arriving and already adsorbed vesicles. Vesicles and lipid molecules, formed after rupture of vesicles, are considered immobile. With these ingredients, the model is able to quantitatively reproduce the temperature-dependent adsorption kinetics, including a higher critical surface concentration of intact vesicles for lower temperatures, and the apparent activation energy for the vesicle-to-bilayer transition E(a) approximately 5 kcal/mol.     

Stabilization of Liposomes by Polyelectrolytes: Mechanism of Interaction and Role of Experimental Conditions

ζ-potential measurements on LUVs allow to evidence the influence of pH, ionic salt concentration, and polyelectrolyte charge on the interaction between polyelectrolyte (chitosan and hyaluronan) and zwitterionic lipid membrane. First, chitosan adsorption is studied: adsorption is independent on the chitosan molecular weight and corresponds to a maximum degree of decoration of 40% in surface coverage. From the dependence with pH and independence with MW, it is concluded that electrostatic interactions are responsible of chitosan adsorption which occurs flat on the external surface of the liposomes. The vesicles become positively charged in the presence of around two repeat units of chitosan added per lipid accessible polar head in acid medium down to pH = 7.2. Direct optical microscopy observations of GUVs shows a stabilization of the composite liposomes under different external stresses (pH and salt shocks) which confirms the strong electrostatic interaction between the chitosan and the lipid membrane. It is also demonstrated that the liposomes are stabilized by chitosan adsorption in a very wide range of pH (2.0 < pH < 12.0). Then, hyaluronan (HA), a negatively charged polyelectrolyte, is added to vesicles; the vesicles turn rapidly negatively charged in presence of adsorbed HA Finally, we demonstrated that hyaluronan adsorbs on positively charged chitosan-decorated liposomes at pH < 7.0 leading to charge inversion in the liposome decorated by the chitosan-hyaluronan bilayer. Our results demonstrate the adsorption of positive and/or negative polyelectrolyte at the surface of lipidic vesicles as well as their role on vesicle stabilization and charge control.     

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle–vesicle and vesicle–substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.     

Simulations of lipid vesicle adsorption for different lipid mixtures

Numerous experimental studies of lipid vesicle adsorption on solid surfaces show that electrostatic interactions play an important role for the kinetics and end result. The latter can, e.g., be intact vesicles or supported lipid bilayers (SLB). Despite an accumulated quite large experimental data base, the understanding of the underlying processes is still poor, and mathematical models are scarce. We have developed a phenomenological model of a vesicle adsorbing on a substrate, where the charge of the surface and the charge and polar state of the lipid headgroup can be varied. With physically reasonable assumptions and input parameters, we reproduce many key experimental observations, clarify the details of some experiments, and give predictions and suggestions for future experiments. Specifically, we have investigated the influence of different lipid mixtures (different charges of the headgroups) in the vesicle on the outcome of a vesicle adsorption event. For different mixtures of zwitterionic lipids with positive and negative lipids, we investigated whether the vesicle adsorbs or not, and--if it adsorbs--to what extent it gets deformed and when it ruptures spontaneously. Diffusion of neutral vesicles on different types of negatively charged substrates was also simulated. The mean surface charge density of the substrate was varied, including or excluding local fluctuations in the surface charge density. The simulations are compared to available experiments. A consistent picture of the influence of different lipid mixtures in the vesicle on adsorption, and the influence of different types of substrates on vesicle diffusion, appear as a result of the simulation data.     

Characterization of phospholipid bilayer formation on a thin film of porous SiO2 by reflective interferometric Fourier transform spectroscopy (RIFTS)

Classical methods for characterizing supported artificial phospholipid bilayers include imaging techniques such as atomic force microscopy and fluorescence microscopy. The use in the past decade of surface-sensitive methods such as surface plasmon resonance and ellipsometry, and acoustic sensors such as the quartz crystal microbalance, coupled to the imaging methods, have expanded our understanding of the formation mechanisms of phospholipid bilayers. In the present work, reflective interferometric Fourier transform spectrocopy (RIFTS) is employed to monitor the formation of a planar phospholipid bilayer on an oxidized mesoporous Si (pSiO(2)) thin film. The pSiO(2) substrates are prepared as thin films (3 μm thick) with pore dimensions of a few nanometers in diameter by the electrochemical etching of crystalline silicon, and they are passivated with a thin thermal oxide layer. A thin film of mica is used as a control. Interferometric optical measurements are used to quantify the behavior of the phospholipids at the internal (pores) and external surfaces of the substrates. The optical measurements indicate that vesicles initially adsorb to the pSiO(2) surface as a monolayer, followed by vesicle fusion and conversion to a surface-adsorbed lipid bilayer. The timescale of the process is consistent with prior measurements of vesicle fusion onto mica surfaces. Reflectance spectra calculated using a simple double-layer Fabry-Perot interference model verify the experimental results. The method provides a simple, real-time, nondestructive approach to characterizing the growth and evolution of lipid vesicle layers on the surface of an optical thin film.     

Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels

We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively.     

Adsorption behavior of DODAB/DPPC vesicles on silica

The interaction between composite dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) bilayer vesicles in the gel state and silica is investigated over the 0-20% DODAB range from determination of adsorption curves, silica sedimentation, particle sizing and zeta-potentials. At 1 mg/mL silica, 0% DODAB, pH 6.3, over the 0-150 mM NaCl range of ionic strengths, high affinity adsorption curves were barely affected by ionic strength and all of them exhibited limiting adsorption values above the level expected for single bilayer deposition. At 1 mg/mL silica, 2% DODAB, pH 6.3 and 1 mM NaCl, high affinity adsorption curves fortuitously presented limiting adsorption indicative of one bilayer deposition on each silica particle. At %DODAB<2% or %DODAB>2%, limiting adsorption was above and below the level expected for bilayer deposition, respectively. Increasing %DODAB in the vesicle composition negatively modulated the limiting adsorption on silica despite the increasing surface charge on vesicles and electrostatic attraction between vesicles and particles. The results point out the difficulty of closed vesicle disruption (required for bilayer deposition from vesicles) when the bilayer is tightly packed in the rigid gel state and might be of interest for analytical applications of immobilized vesicles on silica.     

Influence of nanotopography on phospholipid bilayer formation on silicon dioxide

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.     

A thermodynamic study of protein-induced lipid lateral phase separation. Effect of lysozyme on mixed lipid vesicles

In this work we study by differential scanning calorimetry (DSC) the lateral phase separation induced by a globular protein (lysozyme) on vesicles built-up by charged (phosphatidic acid) and neutral (phosphatidylcholine) lipids.The adsorption of the positively charged protein onto the negative vesicle surface induces the formation of micro-domains richer in the charged lipid component. This phenomenon is revealed as a splitting of the excess heat capacity peak associated to the melting of the lipid hydrocarbon chains.Also, the peak associated to the protein denaturation is shifted, suggesting the presence of adsorbed proteins onto the vesicle surface. The surface electrostatic potentials, both of proteins and vesicles, have been modulated by pH and ionic strength variations, showing a deep influence of the electric charges in modifying protein adsorption, rate of denaturation (related to unfolding enthalpy variation), and lipid micro-domain formation.Some of the present results have been rationalized on the basis of a theoretical model recently developed by the authors.     

Condensation properties of vesicles formed from an amphiphilic N-phosphorylamino acid

Stable unilamellar vesicles were formed in water under appropriate pH from dispersions of N-(O,O-di-n-hexadecyl)phosphorylalanine, an amphiphilic N-phosphorylamino acid. We found that condensation occurred in the vesicle solution after incubated at 40 degrees C, which may contribute to the stability of the vesicular system. Dipeptide derivative in the vesicle solution was identified by electrospray ionization mass spectrometry (ESI-MS), which suggests the peptide formation without any coupling reagents. Hydrogen bond and electrostatic interactions play important roles in the process of vesicle formation, while the suitable orientation and packing of the amphiphilic molecules at the vesicle/water interface together with certain conformational freedom in the vesicular bilayer are considered to be most favorable for the condensation in ordered systems as vesicles.     

Diffusive dynamics of vesicles tethered to a fluid supported bilayer by single-particle tracking

We recently introduced a method to tether intact phospholipid vesicles onto a fluid supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). Once tethered, the vesicles can diffuse in two dimensions parallel to the supported membrane surface. The average diffusion coefficient, D, is typically 0.2 microm(2)/s; this is 3-5 times smaller than for individual lipid or DNA-lipid conjugate diffusion in supported bilayers. In this article, we investigate the origin of this difference in the diffusive dynamics of tethered vesicles by single-particle tracking under collision-free conditions. D is insensitive to tethered vesicle size from 30 to 200 nm, as well as a 3-fold change in the viscosity of the bulk medium. The addition of macromolecules such as poly(ethylene glycol) reversibly stops the motion of tethered vesicles without causing the exchange of lipids between the tethered vesicle and supported bilayer. This is explained as a depletion effect at the interface between tethered vesicles and the supported bilayer. Ca ions lead to transient vesicle-vesicle interactions when tethered vesicles contain negatively charged lipids, and vesicle diffusion is greatly reduced upon Ca ion addition when negatively charged lipids are present both in the supported bilayer and tethered vesicles. Both effects are interesting in their own right, and they also suggest that tethered vesicle-supported bilayer interactions are possible; this may be the origin of the reduction in D for tethered vesicles. In addition, the effects of surface defects that reversibly trap diffusing vesicles are modeled by Monte Carlo simulations. This shows that a significant reduction in D can be observed while maintaining normal diffusion behavior on the time scale of our experiments.     

Dissipation-enhanced quartz crystal microbalance studies on the experimental parameters controlling the formation of supported lipid bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.     

Composition and asymmetry in supported membranes formed by vesicle fusion

The structure and formation of supported membranes at silica surfaces by vesicle fusion was investigated by neutron reflectivity and quartz crystal microbalance (QCM-D) measurements. The structure of equimolar phospholipid mixtures of DLPC-DPPC, DMPC-DPPC, and DOPC-DPPC depends intricately on the vesicle deposition conditions. The supported bilayer membranes exhibit varying degrees of compositional asymmetry between the monolayer leaflets, which can be modified by the deposition temperature as well as the salt concentration of the vesicle solution. The total lipid composition of the supported bilayers differs from the composition of the vesicles in solution, and the monolayer proximal to the silica surface is always enriched in DPPC compared to the distal monolayer. The results, which show unambiguougsly that some exchange and rearrangement of lipids occur during vesicle deposition, can be rationalized by considering the effects of salt screening and temperature on the rates of lipid exchange, rearrangement, and vesicle adsorption, but there is also an intricate dependence on the lipid-lipid interactions. Thus, although both symmetric and asymmetric supported bilayers can be prepared from vesicles, the optimal conditions are sensitive to the lipid composition of the system.     

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.     

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.     

Quantitative analysis of tethered vesicle assemblies by quartz crystal microbalance with dissipation monitoring: Binding dynamics and bound water content

To implement the molecular recognition properties of membrane proteins for applications including biosensors and diagnostic arrays, the construction of a biomimetic platform capable of maintaining protein structure and function is required. In this paper, we describe a tethered phospholipid vesicle assembly that overcomes the major limitations of planar supported lipid bilayers and alternative biomimetic membrane platforms and characterize it using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy. We provide evidence of a one-step mechanism for bilayer formation and monitor the subsequent adsorption and binding of streptavidin, vesicles, and streptavidin-coated microspheres. For all three species, we identify a critical surface density above which a significant amount of coupled interstitial water contributes to the response of the quartz resonator in a phenomenon similar to dynamic coupling due to surface roughness. A Sauerbrey-type analysis is sufficient to accurately interpret the QCM-D results for streptavidin binding if water is treated as an additional inertial mass, but viscoelastic models must be invoked for vesicle and microsphere binding. Additionally, we present evidence of vesicle flattening, possibly enhanced by a biotin-mediated membrane-membrane interaction.     

Vesicle adsorption on mesoporous silica and titania

Lipid bilayer formation via vesicle fusion on mesoporous silica and mesoporous titania was investigated using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescent recovery after photobleaching (FRAP). Results showed that lipid bilayers were formed on mesoporous silica and that intact vesicle adsorption was obtained on mesoporous titania. From the FRAP results, it could be concluded that the lipid bilayer was fluid; however, it had a smaller diffusivity constant compared to bilayers supported on a nonporous silica.     

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.