Adhesion of Phospholipid Bilayers to Hydroxylated Silica: Existence

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Adhesion of phospholipid bilayer to hydroxylated silica: existence of nanometer thick water interlayer Aleksey Vishnyakov, Ting Li, and Alexander V. Neimark Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03582 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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Adhesion of phospholipid bilayer to hydroxylated silica: existence of nanometer thick water interlayer Aleksey Vishnyakov, Ting Li, Alexander V. Neimark* Department of Chemical & Biochemical Engineering, Rutgers University

Abstract: Lipid bilayers attached to solid surfaces play important role in bioinspired materials and devices and serve as model systems for studies of interactions of cell membranes with particles and biomolecules. Despite active experimental and theoretical studies, the interactions of lipid membranes with solid substrates are still poorly understood. In this work, we explore, using atomistic molecular dynamics simulations, equilibrium and stability of a phospholipid DMPC membrane supported on hydroxylated amorphous silica. We reveal two distinct types of thermodynamically stable states, characterized by the different width of the water layer between the membrane and the substrate. In α-states, the membrane is closely attached with the lipid head groups interacting directly with surface hydroxyls, however due to the molecular level roughness of amorphous silica surface, there exists an inhomogeneous water layer trapped between the substrate and the membrane. In β -states, the membrane is separated from the silica surface by a water film of ~2.5 nm in thickness. The thermodynamic equilibrium is quantified in terms of the disjoining pressure isotherm as a function of membrane-substrate separation, which has a double sigmoidal shape with two minima and one maximum, which correspond to the limits of stability of α and β states. The thermodynamic properties and bilayer structure are compared with experimental findings and simulation results for relevant systems. Keywords: Lipid bilayer, silica, adhesion, molecular simulation, disjoining pressure

* Corresponding author; email

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Introduction Lipid membranes attached to solid surfaces are ubiquitous in nature and are of immense importance for fabrication of new biomimetic materials 1 and nanoparticle-based biotechnologies. 2,3 However, despite active research, the physico-chemical mechanisms of lipid bilayer (LB) adhesion are still poorly understood. Here, we explore using atomistic molecular dynamics (MD) simulations the equilibrium and stability of a DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine) LB supported on amorphous silica focusing on the multiplicity of states, which differ by the proximity of the membrane to the surface. We show that in addition to the states of close contact, the adhesion equilibrium may involve the existence of suprananometer thick water interlayer between the membranes and surface. Formation of an equilibrium nanometer thick water film, frequently called a hydration layer, trapped between LB and solid substrate was observed in various experiments with spontaneous adhesion of LBs to hydrophilic substrates. 4-8 Bayerl et al 4 estimated the thickness of the hydration layer between DMPC bilayer and silica as 1.7±0.5 nm in proton 1H NMR studies of LB covered glass beads. Similar values were found by specular reflection of neutrons on DMPC LB adsorbed to silica plate. 6 The hydration interlayer mediates lipid-substrate interactions, provides fluidity of the supported lipid bilayer (SLB), facilitates trans-membrane transport, and enables LB sliding over the substrates. 5,7 It is known that phospholipid vesicles tend to adsorb and spread on hydrophilic surfaces, such as hydroxylated mica or silica, that leads to their coalescence, rupture, and, ultimately, to the formation of a SLB with a nanometer thick hydration film. 7 This process, that occurs in the absence of external forces pushing the membranes towards the surface, points toward the presence of effective long-range attraction forces which, as the distance between the membrane and surface decreases, are compensated by repulsive hydration


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forces; the equilibrium thickness of the hydration layer corresponds to the balance of these forces. The authors, 4,8 who first observed the nanometers thick hydration layers in SLB systems, explained their stability by an interplay of the long-range repulsive hydration forces due to restructuring of confined water, electrostatic and attractive van der Waals lipid-substrate interactions. However, a quantitative theoretical or modeling explanation of this phenomena is still lacking. The thickness of water layers squeezed between surfaces is quantified in terms of the disjoining pressure Π that has to be applied to maintain given separation h between the surfaces. 9

In earlier works, Derjaguin and co-workers (see ref 9 for review) have shown using

ellipsometry that the disjoining pressure isotherm Π(h) of water films adsorbed on quartz and other surfaces exhibit a sigmoidal shape with two regions of equilibrium states at positive disjoining pressures: molecularly thin α-films and supra-nanometer thick β-films, Figure 1a. The region of stability of β-films is limited from below by a certain minimum thickness ℎ∗ , which corresponds to a disjoining pressure maximum, Π(ℎ∗ ), above which β-film spontaneously transforms into a much thinner α-film. The thickness of α-films is limited from above by a certain maximum thickness ℎ ∗ . Π(ℎ ∗ ) is negative and corresponds to a minimum on the disjoining pressure isotherm; at Π < Π(ℎ ∗ ), α-film spontaneously grows into macroscopically thick layer. At Π = 0, α-film of finite thickness ℎ is in equilibrium with the bulk water. α-films of thickness ℎ < h 0) at very close distances changes to attraction ( Π(ℎ) < 0) as the lipidsurface distance increases. The equilibrium thickness of α-film achieved at Π = 0 was ℎ ≈ 0.4 nm. The upper limit of stability of α-films (ℎ ∗ ≈ 0.45 nm) was reached at the negative disjoining pressure Π ≈ − 0.7kbar at the minimum and was followed the region of thermodynamically unstable films with increasing disjoining pressure extending to the lower limit of β-films stability found at ℎ∗ = 0.9 nm. The range of β-films explored by the authors was however relatively narrow between 0.9 nm (lower limit of β-films stability) and 1.5 nm, so that the possible equilibrium state ℎ corresponding to supra-nanometer thick hydration layer at Π = 0 may not have been reached. Concluding the above brief review, we may state that while the presence of nanometer size hydration films in SLBs is well established experimentally as well as in molecular simulations, a quantitative analysis of LB-substrate interactions in the whole diapason of interlayer thicknesses from atomistic to nanometer scales is still lacking. Below, we explore these interactions using atomistic MD simulations drawing on the example of DMPC (1,2-dimyristoyl-sn-glycero-3phosphocholine, Figure 2), a very common saturated zwitterionic lipid, one of the major components of cell membranes, and hydrophilic amorphous silica. Silica is presented by a realistic atomistic model that accounts for the inherent surface roughness of amorphous solids, which substantially affects the surface adsorption properties and the structure of the hydration layer. The molecular models of the amorphous silica and lipids, the simulation setup, and the computational methodology employed for calculating the disjoining pressure isotherm are


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presented in Section 2. The results of simulations are analyzed in Section 3. We show that there exist two distinct adhesion states, corresponding to equilibrium molecularly thin (ℎ ~ 0.4 nm) α-films and nanometer thick (ℎ ≈ 2.5 nm) β-films, which are stable in the absence of applied pressure. We also determine the limits of α- and β-films stability and reveal that the β-films can be observed within a limited diapason of thicknesses, ℎ∗ < ℎ < ℎ∗∗ , that is bound not only from below but also from above. The LB-silica interactions are quantified by the disjoining pressure isotherm, which has a double sigmoidal shape, schematically presented in Figure 1b, with two regions of stable and metastable α- and β-films separated by the region of thermodynamically unstable states, ℎ ∗ < ℎ < ℎ∗ , that cannot be observed experimentally. The molecular structure of the hydration film and the inner and outer leaflets of the LB is analyzed in detail. In α-states, the membrane is found to be closely attached with the lipid head groups interacting directly with surface hydroxyls, however due to the molecular level roughness of amorphous silica, the hydration film is highly inhomogeneous and patchy with pockets of water trapped between the substrate and the membrane. Conclusions are summarized in Section 4. Models, methods and simulation details Simulation set-up for disjoining pressure calculations. The setup for MD simulations of the thermodynamic equilibrium of a LB adhered to a solid substrate is shown in Figure 2a. The LB and the substrate are modeled on the atomistic level. Simulations are performed in an orthorhombic box with periodic boundary conditions applied in all three dimensions. The substrate representing a slab of amorphous silica is pinned by anchors at position Zs to prevent its translational movement as a whole, while allowing for local movements on the atomistic scale. In the initial state, LB is placed above a slab of silica at the distance exceeding expected range of


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palpable LB-substrate interactions, and the system is filled with water. The silica slab is perforated with several channels of roughly cylindrical shape which allow water exchange between the bulk water atop LB and the gap between LB and substrate. The system is equilibrated in the NPT ensemble, and we monitor the density of water inside the reservoir to be close enough to that of bulk water at ambient conditions. Then, a gravity-type driving field g is applied to all lipid atoms. Under this force, LB moves toward the substrate until the driving force is counterbalanced by the disjoining pressure due to long-range LB-substrate interactions: f (h) = g ∑ m i = Π (h) × L x L y

(1)

i

where Lx and Ly are box sizes in lateral dimensions, and mi are masses of the atoms to which the gravity field is applied. When the LB moves in z direction under external force, water transfers through the substrate holes from the gap between LB and substrate to the reservoir atop LB until the equilibrium is reached.


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Figure 2. (a) Simulation setup. Silica slab substrate, modeled atomistically, is perforated through by transport channels and immobilized by “anchors” restricting its vertical motion. LB is placed parallel to the substrate and a constant gravity-type field is applied to all atoms in LB in z direction. Water can migrate between the gap and reservoir through the channels to provide the thermodynamic equilibrium in the system due to the periodic boundary conditions in z-direction. (b) Typical time dependence of the hydration film thickness h between LB and substrate during equilibration (c) snapshot of amorphous silica slab (d) chemical structure of DMPC lipid (e) snapshot of freestanding DMPC bilayer (water not shown). Atom colors on snapshots: Si in green-yellow, O in red, Cs in cyan, H in white, N in blue, phosphorus in bright yellow.

The position of LB is monitored by the coordinate ZL of the LB center of mass. After the driving field is applied, the LB starts moving from its initial position until its location stabilizes and fluctuates around a constant value. The statistics is collected over the stable part. The effective water film thickness h is calculated from integration of the water density profile ZL

−1 h = ρ WB ∫ ρ W ( z )dz ZS

(2)

where ρW(z) is the local number density of water molecules at normal coordinate z, ρWB is the density of bulk water, and ZS and ZL are the average locations of the middle planes of the silica block and the LB correspondingly. The water molecules located in the holes drilled through the


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substrate are excluded from this calculation. The effective thickness h defined by Eq.2 represents the thickness of the plane layer of bulk water that has the same number of molecules as the inhomogeneous hydration film under consideration. Figure 2b shows a typical time dependence of the water film thickness with the equilibrium achieved in about 30 ns. The size of the simulation box is 5.21 × 5.276 × 19.08 nm3 in average; periodic boundary conditions are applied in all directions. The length of the box in z-direction is large in order to ensure that the interaction of the LB with the other face of the silica slab (due to periodic boundary conditions) is negligible. MD simulations are performed with LAMMPS 29 software. The simulation length depends on the system and ranges from ~ 20 ns (10 ns for equilibration and 10 ns for averaging) for α-films at high disjoining pressure Π to ~100ns for βfilms at negative Π with equilibration time of 30 ns and averaging time of 70 ns. We should note that in our simulation (unlike GCMC modeling 28) we do not fix the film thickness h and calculate the disjoining pressure Π, but rather determine h at a given Π. The system has mechanically unstable intervals of h where (∂Π/∂h)P,T > 0, which are expected between the regions of stable α- and β-films. These values of h cannot be probed in our simulations. If a simulation starts from a LB position that corresponds to the unstable film, LB moves fast and irreversibly in the direction of the field, and a plateau on h(t) curve like the one shown in Figure 2(b) cannot be reached. (Typical shapes of h(t) curves are shown in detail in Supporting Information, section S1). The same behavior may happen in a system corresponding to a metastable configuration, if the limit of stability is very close and the energy barrier separating metastable and unstable states is low enough to be overcome due to thermal


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fluctuations during the MD run. As the simulation results, we present only the configurations that have been reliably stabilized with the water film thickness h unequivocally estimated. Model of the silica substrate. To account for molecular level roughness of silica substrates, we constructed a slab-shaped block of amorphous silica in the following fashion. A cristobalite cubic periodic crystal of 5.17 × 5.17 × 5.17nm3 was build using Materials Studio crystal builder. The crystal was melted in NVT MD simulations at 4000K with the forcefield from ref 30 for 40ps, and then annealed to 300K temperature for 300ps. After that, a 5.17nm × 5.17nm × 3nm slab was cut out of the glassy block, and transport channels of about 0.6-0.8nm in radius were created by removing the atoms that occupied that space. That left the neighboring atoms with unoccupied valences, and a proper number of Si-O bonds was created manually to "heal" the structure. Hydroxyl groups were attached to make a hydroxylated surface with 4 OH groups per nm2, according to Katoh. 31 After 30ps equilibration at ambient temperature and pressure, we proceeded the simulation with the CHARMM force field 32 and the standard Nose thermostat, for 100ps to obtain an equilibrated slab of amorphous silica to be used in further simulations. The transport pores are somewhat deformed in the process of simulation yet remain passable to water. The details of the amorphous silica slab construction are given in Supporting Information Section S2. The slab is periodic on xy plane, and non-periodic in z direction. The final lateral size of the slab is 5.12nm × 52.3nm and the thickness is about 3nm; the slab is shown in Figure 2c. The substrate is to be kept static during the simulations despite the force applied to it. Therefore, we stabilized the slab by “anchors” (Figure 2a): immobile particles of 0.6 nm in diameter that interact only with Si atoms via r−12 repulsive potentials and did not interact anyhow with other components. The substrate therefore cannot move past the anchor plane.


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It is important that the prepared substrate has a molecularly rough surface typical for amorphous silica with characteristic fluctuations of the positions of surface molecules on the level of intermolecular distance of ~ 0.2 nm. Detailed characterization of the silica density at the substrate edge is presented in Supporting Information Section S2. The roughness parameter, which characterized the half-width of the density fluctuation region, agrees reasonably with previous MD simulations of amorphous silica surfaces (e.g ref 33, see Supporting Information Section S3). Model of the lipid bilayer. The DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) LB was modelled using the CHARMM forcefield. 34 The forcefield provides good agreement with experimental density elasticity and structural characteristics of LB on the molecular level. Water was described by three-center SPC 35 model as recommended in ref 34. A fragment of a freestanding LB was composed of 92 lipid molecules (46 in each leaflet) and equilibrated in a separate simulation in pure water at zero tension in NPT ensemble (Figure 2d). The area per lipid head is 0.599nm2, in good agreement with experimental data and simulations. 34 The LB is fluidlike in all simulations. The lipids could flip-flop (change the leaflet) during the simulations, but none such event was ever recorded. This might be one of the reasons why no gel structure was observed in the inner leaflet even in the direct contact with the substrate. On the other hand, it is possible that LB does not freeze at a disordered silica surface. Results and discussion. The simulations reveal two regions of hydration films equilibrated with the required precision: molecularly thin α-films and nanoscale thick β-films. The constructed disjoining


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pressure isotherm that has a double sigmoidal shape and the characteristic snapshots are given in Figures 3 and 4.

Figure 3. Dependence of the disjoining pressure on the effective thickness of the water layer. αstates (direct contact between substrate and lipid heads) are shown in blue, β-states (suprananometer hydration films) are shown in red. Lines are drawn to guide the eye. The disjoining pressure isotherm has a double sigmoidal shape. Arrows show limits of stability: at these pressures LB experiences irreversible transitions between α- and β-states. The equilibrium thicknesses of α- and β-films at Π = 0 are ℎ =0.37 nm and ℎ =2.6 nm, respectively. The estimated upper limit of stability at α-films, ℎ∗ 0.48 ± 0.2 nm. The lower and upper limits of stability of β-films are ℎ∗ 2.4 nm and ℎ∗∗ >3.2 nm, respectively. Regions of states that could not be stabilized in simulations, are shown by dash lines. The two most right points with negative disjoining pressure correspond to β-states, which cannot be measured in SFA and AFM experiments with monotonically increasing applied force. The existence of these states indicates the supra-nanometer region of attraction between LB and the substrate that causes spontaneous adhesion of LB in a β-state with the equilibrium hydration film of thickness ℎ =2.6 nm. The insert shows the simulation points in the region of β-films with the error bars.


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Figure 4. Snapshots of LB over hydrophilic silica surface (a) stable α-state configuration with direct contact between bilayer heads and silica at Π = 3kbar, h = 0.3nm. (b) stable configuration with a film of approximately 2.5 nm between the lipid bilayer and substrate; Π = 0. Substrate is drawn in lines: Si in orange, O in red, H in white. Water atoms are drawn as balls (transparent O in red, H in white) on panels (a) and (c), solid dark blue in panel (b)). DMPC is drawn in solid balls (C in light blue, P in yellow, N in violet, O in rose); (c) top view on the α-state contact configuration presented in (a) with water and silica only; water forms patches on silica surface; (d) Density profiles of water, substrate and LB for α-state at Π = − 0.5 kbar. The profile shows deformation of the inner leaflet due to adsorption at the substrate. Water forms an inhomogeneous patchy film between the substrate and LB with the local density not exceeding 0.5 g/cm3.

α-films. α-films correspond to contact configurations. A snapshot of the stable configuration of α-film obtained at Π = 3kbar (the leftmost point in Figure 3) is shown in Figure 4a. At high applied pressures, LB is pushed into a direct contact with the substrate and pressed against the rough wall. Almost all water molecules in the hydration layer between LB and substrate are


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squeezed out. LB interacts with the substrate directly via van der Waals and electrostatic forces; hydrogen bonds form between Si-OH hydroxyls and phosphate groups of DMPC molecules. Water film is highly heterogeneous and patchy, composed of pockets of water that fills surface indentations and solvates the lipid head groups. A typical snapshot of water on the top of the substrate is shown in Figure 4c (LB is not shown). Geometrical analysis performed similarly to ref 36 show that water molecules in the pockets forms hydrogen bonds to both the substrate and LB and are practically immobile The close contact with the substrate distorts the LB structure that is discussed below. A positive disjoining pressure means a strong repulsion between LB and substrate. As the pressure is released, LB relaxes and more water molecules penetrate into the hydration film. The disjoining pressure monotonically decreases with h (Figure 3), and achieves zero at ℎ 0.37 nm that corresponds to the equilibrium α-film in the absence of applied pressure. Further increase of the hydration film thickness is achieved with the disjoining pressure becoming negative that means the attraction interaction between LB and substrate. It is worth noting that, due to the surface roughness and the direct contact of some lipid head groups with the substrate, all α-films are highly disordered and consist of interconnected pockets of waters. For example, the density profiles for Π = − 0.5kbar is shown in Figure 4d. We can see that the water local density never exceeds 0.5 g/ml (that is, half on the bulk value) and the effective thickness obtained with eq. 2 is h = 0.42nm (that is, about 1.5 molecular layers of water on average). Generally, the surface roughness and hydroxylation level significantly affect the geometry and stability of α-films. Π(h) monotonically decreases till Π reaches about −1kbar at h = 0.455 nm. If the pressure further decreases, the system becomes unstable and LB irreversibly separates from the substrate


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in a snap-off transition, as observed in AFM and SFA experiments. The system is not stabilized until the film thickness becomes essentially macroscopic, comparable with the half-size of the simulation box, ½ Lz. We estimate that the upper limit of stability of α-films corresponds to the film thickness of ℎ∗ 0.48 ± 0.2 nm in thickness and correspondingly, to −1.3 < Π < −1.0 kbar. Although stable α-states with the direct contact between LB and solid surface were observed in AFM 19 and SFA experiments 20 a direct comparison with our simulation results is hardly possible because experimental hydration film thickness is not exactly known. Similar contact configurations were obtained in MC simulations by Pertsin and Grunze 28 for DPLE lipids on mica. However, due to the smooth surface of mica, the snapshots of water film appeared more homogeneous than in our work, although some lipid heads contacted mica surface. Nevertheless, we attempted to compare our Π(h) isotherm for α-states to that from ref 28 and, despite the different substrate and LB chemistry, found reasonable agreement. β-films. If LB is placed in the initial configuration at about 3nm from the silica surface in the absence of applied field (Π = 0), the LB equilibrates at ℎ = 2.6nm; that is, a hydration film equivalent to about 8.5 molecular layers of water forms between LB and substrate (Figures 3, 4b). This distance is within the limits estimated experimentally in the systems with spontaneous adhesion of LBs. 5,19 Thermodynamically stable β-films are observed in our simulations around ℎ with increase and decrease of the disjoining pressure between 0.25 and −0.1 kbar. These simulations were quite long: the β-film at Π = − 0.1 kbar was equilibrated over about 30 ns and averaged over 80 ns. The simulation was started from the same starting configuration as in simulation of β-configuration obtained at Π = 0 and resulted in higher than initial h, while a simulation at Π = 0 resulted in lower than initial h. The system spent overall about 110ns in β-


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form with negative Π. As a control experiment, we performed a simulation at the same Π = − 0.1 kbar and the same starting configuration with h = 3nm, but replaced water with a LJ fluid (LB parameters were modified to preserve the LB integrity). After equilibration at NPT condition, the LB position seemingly stabilized, and the force corresponding to Π = − 0.1 kBar was applied to the LB. The LB in that reference system almost immediately snapped off from the substrate in a fast irreversible motion (Supporting Information, Section S1). This make us suppose that the stability of spontaneously formed β-films is related to the specific properties of supra-nanometer thick hydration film between the silica and LB that cause the observed region of LB-substrate attraction The range of Π that stabilize β-films corresponds to the variation of the β-film thickness between 2.4 and 3.2 nm. These values approximate the lower and upper limits of nanoscale thick β-film: ℎ∗ < 2.4 nm and ℎ∗ > 3.2 nm. At 2.4 nm < h < 3.2 nm, and respectively between 0.25 < Π < −0.1 kbar, β-films are thermodynamically stable: the disjoining pressure Π decreases with h. If Π is set below −0.125 kbar, LB starts irreversible motion away from the substrate. If Π increases up to 0.5kbar, the LB starts irreversible motion towards to substrate (red arrows in Figure 3). The films in the region of thicknesses ℎ ∗ = 0.5 nm < h < ℎ∗ =2.4 nm, as well as the films thicker than ℎ∗∗ =3.2 nm could not be stabilized in simulations: they are inherently unstable (correspond to the increasing disjoining pressure, dΠ/dh>0, or very close to the limits of stability). In the region of stable β-films, the Π(h) dependence appears to qualitatively resemble those observed between supported lipid bilayers, 15 although the precision of our data does not allow for a detailed comparison.


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Figure 5. Dependence of the predominant orientation of P-N vectors of DMPC molecules with ####, %̅'() ) where %̅'() is the unit vector parallel to respect to z axis. The order parameter cos(PN z axis and directed from the center of the bilayer towards the top of the leaflet (that is, towards the substrate for the inner and from the substrate for the outer leaflet). Π = −0.5kbar for α state and Π = 0 for β state.

Specifics of molecular structure of the LB leaflets and hydration film. It is expected that the molecular structure and orientation of lipids in inner and outer leaflet of LB may differ due to the interactions with the substrate. Figure 5 demonstrates the predominant orientation of P-N vector of the lipid heads in the inner and outer leaflets in α-state at Π = −0.5 kbar. In the free LB, the predominant angle between PN vector and z axis is 70-80 deg. 28,37 For the inner layer in the αstate, the predominant orientations of P-N vector is flat, i.e. parallel to xy plane. The deformation of the contact leaflet is due to the contact with the substrate. The P-N orientations for the outer leaflet shows a very broad maximum. That is, orientations from flat (parallel to xy plane) to about 50 deg with z axis are equally probable. The average angle is about 75 deg, which corresponds well to that obtained by of Hogberg and Lyubartsev 37 for the free-standing bilayers.


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The outer leaflet for the β-state obtained at Π = 0 has practically the same orientation distribution of P-N angles. This indicates that the contact with the surface only affects the headgroups of the contact leaflet, and the 2.6 nm water film between the LB and support is thick enough to make the influence of the latter on the LB negligible. Nevertheless, the disjoining pressure for β -films increases steeply as h decreases below 2.5nm. This effect may be related to water structuring in the vicinity of the LB and silica surfaces. The existence of the structural component of the disjoining pressure that is responsible for the LB-substrate repulsion implies a distortion of water structure in the confined film and can be evaluated by the analysis of the distribution of orientation of water molecules. Figure 6 shows density profile of water, LB and silica for B-state at Π = 0 and the predominant orientation of water characterized with the average cosine of the angle between water dipoles and z axis (note, that here z vector is always pointed from the substrate, unlike zLB vector in Figure 5. Water molecules located close to the LB core (essentially in the space between the heads) are predominantly oriented with oxygens looking towards the LB, due to hydrogen bonds between those water and the carboxyls of the lipids. Water molecules in between the surfaces but close to the LB are predominantly oriented with oxygen atoms towards the substrate. Near the substrate, water orientation to with oxygens towards the bilayer is predominant on the contact layer (mainly due to hydrogen bonds between silica and water. Thus, water molecules form two poorly defined layers near the LB and substrate with opposite predominant orientation of water dipoles. These layers are divided by at least 1 nm thick water film with no apparently preferred orientation as the average cosine between water dipole and z axis equals zero. Yet, the interaction between the oriented layers is apparently sufficient to produce strong repulsive


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interactions between LB and the substrate, despite a seemingly isotropic uniform film in between.

Figure 6. Water density profile (red line, right y-axis) for β-state at Π = 0 and the average cosine of the angle between water molecule dipole and z axis (blue dotted line, open circles, left axis). The normal coordinate is shifted so that the interface between water and silica corresponds to z = 0 (green dashed vertical line)

4. Conclusion With atomistic MD simulations of DMPC lipid membrane supported on amorphous silica, we demonstrated the presence of two distinct regions of thermodynamically stable adhesion states, which differ by the thickness of the hydration water layer between the membrane and substrate: α-states of close contact with the hydration layer of molecular dimensions (0.3-0.45 nm) and βstates, in which the membrane is separated from the substrate by a supra-nanometer thick water


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film (2.4-3.2 nm). The membrane-substrate interactions are quantified in terms of the disjoining pressure Π that is equal to the external pressure needed to be applied to the membrane to maintain equilibrium hydration film of given thickness h. The isotherm of disjoining pressure Π(h) depends on an interplay of different forces acting between the membrane and substrate, including van der Waals, electrostatic, and hydration forces. The long-range hydration forces, or the structural component of disjoining pressure that originates from restructuring of water hydrogen network in the vicinity of interfaces, play the critical role in the formation of β-films. Disjoining pressure Π(h) may be either positive or negative depending on whether the membrane is repelled or attracted by the substrate at given h. We found that the disjoining pressure isotherm Π(h) exhibits a double sigmoidal shape with two minima and one maximum, which limit two regions of equilibrium states corresponding to α- and β-films, Figure 3. The region of α−films (0.3-0.45 nm) is separated from the region and β-films (2.4-3.2 nm) by the region of intermediate film thicknesses (0.45-2.4 nm) that cannot be stabilized by applying either positive or negative external pressure. In the absence of applied pressure at Π=0, there exist two equilibrium states: α−film of 0.37 nm, which is formed from a state of close contact upon release of applied pressure, and β−film of 2.6 nm, which is formed spontaneously due to long range membranesubstrate attraction if the membrane is placed sufficiently close to the substrate (~ 5nm). These states are separated by an energy barrier that can be overcome either by applying external force or due to natural thermal fluctuations. Stable films correspond to the regions of decreasing disjoining pressure (∂Π/∂h < 0), and respectively, the limits of stability of α- and β-films correspond to the minima and maximum of the disjoining pressure isotherm Π(h). The thickness of α-films is limited from above by


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maximum thickness of ~ 0.48nm achieved at the negative disjoining pressure of Π −1 kbar. Further decrease of disjoining pressure leads to the membrane snap-off. The region of stability of β-films is limited from below by minimum thickness ℎ∗ 2.4 nm achieved at positive disjoining pressure of Π(ℎ∗ ) 0.5 kbar, above which β-film spontaneously transforms into a much thinner α-film. The lower limit of β-films estimated as >3.2 nm corresponds to negative disjoining pressure Π −0.1 kbar; further reduction of disjoining pressure leads to the membrane snap-off. In is worth noting that the spontaneous transition from β-films to α−films upon the increase of the applied pressure that is associated with overcoming the repulsion barrier and the spontaneous snap-off of α−films in the reverse process of pressure release are observed in ATM and SFA experiments with various pairs of LBs and solid substrates. However, in these experiments with forced attachment of LB, the equilibrium supra-nanometer β-films are not stabilized. These states, at which LB adheres to the substrate without applied pressure, are observed in the experiments with spontaneous formation of supported LBs due to either selfassembly or deposition. Two physical factors play critical roles in the observed behavior of hydration films between the membrane and substrates. Firstly, due to the inherent molecular level roughness of silica surface, α-films are highly inhomogeneous. In these states, water congregates in pockets filling the surface indentations and the internal leaflet of the membrane is distorted with some lipid heads being in close contact with the solid. The role of surface roughness is very important and this factor has not been considered previously. Secondly, analyses of configurations of water molecules in β-films confirm the distortion of orientation of water molecules in the vicinities of


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both, solid (substrate) and soft (membrane), interfaces that gives rise to the long-range repulsion structural component of the disjoining pressure, which balances the van der Waals attraction. Transition from the long-range repulsion to attraction and existence of the attraction minimum, found in the simulations of β-films, are caused by complex cooperative interactions of water molecules in the hydration layer sandwiched between the LB and substrate, which include vdW, electrostatic and hydrogen bonding, the interplay of which is still to be better understood.

Acknowledgement. The authors thank Dr. Somisetti Sambasivarao for technical help. This work is supported by NSF grant No. 1264702 “Adhesion and Translocation of Nanoparticles through Lipid Membranes.” Calculations were performed using the Extreme Science and Engineering Discovery Environment (XSEDE) 38 (project DMR160162), which is supported by NSF grant No ACI-1053575. Supporting Information available: Supporting information includes: dependence of bilayer location on simulation time for several characteristic systems (Section S1), details of SiO2 simulations (Section S2), characterization of the roughness of amorphous silica surface (Section S3). Supporting Information is available free of charge at http://pubs.acs.org/.

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TOC

Two distinct types of thermodynamically stable states for DMPC lipid bilayer on hydroxylated silica: α-states, the membrane is closely attached to the substrate. In β -states, the membrane is separated from the substare by ~2.5 nm in thick water film.


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Adhesion of Phospholipid Bilayers to Hydroxylated Silica: Existence

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