Solid-Supported Lipid Multilayers under High Hydrostatic Pressure

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Solid supported lipid multilayers under high hydrostatic pressure Benedikt Nowak, Michael Paulus, Julia Nase, Paul Salmen, Patrick Degen, Florian Josef Wirkert, Veijo Honkimaki, and Metin Tolan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04554 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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Solid-supported lipid multilayers under high hydrostatic pressure Benedikt Nowaka ,∗,† Michael Paulus,† Julia Nase,† Paul Salmen,† Patrick Degen,‡ Florian J. Wirkert,† Veijo Honkimäki,¶ and Metin Tolan† Fakultät Physik/DELTA, TU Dortmund, 44221 Dortmund, Germany, Physikalische Chemie II, TU Dortmund, 44221 Dortmund, Germany, and European Synchrotron Radiation Facility, F-38043 Grenoble, France E-mail: [email protected]

Abstract In this work, the structure of solid-supported lipid multilayers exposed to increased hydrostatic pressure was studied in situ by x-ray reflectometry at the solid-liquid interface between silicon and an aqueous buffer solution. The layers’ vertical structure was analyzed up to a maximum pressure of 4500 bar. The multilayers showed phase transitions from the fluid into different gel phases. With increasing pressure, a gradual filling of the sublayers between the hydrophilic head groups with water was observed. This process was inverted when the pressure was decreased, yielding finally smaller water layers than in the initial state. As it is commonly known, water has an abrasive effect on lipid multilayers by the formation of vesicles. We show that increasing pressure can reverse this process, so that a controlled switching between multiand bilayers is possible. ∗ To

whom correspondence should be addressed Dortmund, Fakultät Physik ‡ TU Dortmund, Fakultät Chemie ¶ ESRF † TU

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Introduction In all living organisms, cell membranes regulate the mass transfer between the intracellular and extracellular region. 1,2 The basic membrane structure consists of a lipid bilayer 3 that is interstratified by cholesterol and proteins. These systems undergo pressure- and temperature-induced phase transitions. While extreme conditions in general are unfavorable for animate beings, some organisms are able to withstand surprisingly harsh conditions. Although the structural properties of lipid membranes are altered by high pressure, some species are adapted to live at low temperatures and high pressures, e.g. in the Mariana trench. 4 The high flexibility in lipid membranes, based on the high lateral mobility that is provided by the liquid phase (“liquid mosaic model” 5 ), and the existence of so-called lipid rafts, is essential to fulfill specific functionalities. In previous studies on lipid bulk systems in water, it has been proven that there are phase transitions from the fluid phase into different gel phases at elevated pressures e.g. for DPPC (dipalmitoylphosphatidylcholine) 6 or DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). 7 For DMPC, several gel phases were observed besides the fluid phase (Lα ), which are the so called ripple phase (Pβ ′ ) 10 and the lamellar gel phases Lβ ′ , Lβ i, and GEL III. The Lβ i-phase is also called interdigitated phase, see e.g. 8 and references therein, or this reference 9 for DMPC. In the GEL-III-phase, the hydrocarbon tails are completely stretched and not kinked as in the Lβ ′ -phase. The tails are not tilted and, in contrast to the Lβ i phase, not interdigitated. Note that the Lβ i-phase and the GEL-III-phase only exist at high pressures and do not occur in lipid membrane systems at ambient pressure and temperature. High resolution structural investigations of lipid membranes under realistic conditions, which means in direct contact with the aqueous phases, are a non-trivial task. Small angle scattering techniques provide structural information with sub-Ångstrom resolution on bulk systems like (multilamelar) vesicles in aqueous solutions. 6,10,11 If the structure of highly ordered flat systems, like lipid membranes bound to solid substrates, is to be determined with sub-Ångstrom resolution, they can be analyzed by X-ray reflectometry (XRR). In the past, these samples were stored under a high humidity (98%) gas phase and 2 ACS Paragon Plus Environment

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temperature-induced phase transitions where studied. 12–14 However, working in a high humidity gas phase, the investigation of phase transitions that appear at high hydrostatic pressures, as e.g. in the deep sea, 15 is not possible. To do so, the samples need to be fully immersed in water. In this work, the pressure dependence of solid-supported multilayers has been investigated under full hydration. Multilayers were prepared on silicon wafers by spincoating and were transferred into aqueous solution. In contrast to vesicles in solution, there is an additional interaction between substrate and lipid layers, 16–18 which might alter the pressure response that is observed in bulk systems. The influence of the substrate however becomes weaker with more bilayers deposited. Above some 10 bilayers, the interaction between the substrate and the lipid layer becomes negligible. With a system consisting of 6 to 9 sublayers, we place ourselves at the limit of this boundary condition. Using x-ray reflectivity measurements, we were able to analyze pressure-induced structural changes with high resolution in situ. The application of pressure stabilizes multilayers in an aqueous environment. We observed the successive penetration of water molecules into the sublayers of the multilayers, more exactly between the hydrophilic headgroups. In a second experiment, we found that when a lipid bilayer is in contact with a DMPC-enriched aqueous solution, it is possible to trigger multilayer formation. Since this process is reversible, it is possible to switch between biand multilayers via pressure de- and increase.

Sample preparation and methods Silicon wafers from Wacker (Burghausen, Germany) were cut to a size of 7.6 x 7.6 mm2 . The wafers were cleaned in an ultrasonic bath consecutively in chloroform, acetone and 2-propanol for 15 minutes. Afterwards, the surface of the wafers was etched by piranha solution (75% sulfuric acid and 25% hydrogen peroxide) to prepare a clean hydrophilic SiO2 -surface. The wafers were stored under ultrapure water (MilliQ, resistivity 18.2 MΩ cm) until use. DMPC (Sigma-Aldrich, Munich, Germany) was dissolved in 2-propanol at a concentration of


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10 mg/ml. 60 µ l of the lipid solution was pipetted onto the wafer and spincoated for 30 seconds at 4000 rpm 13 on a SCC-200 spincoater by Novocontrol Technologies. Solvents were purchased from Sigma Aldrich. After evaporation of the solvent, a DMPC multilayer remained on the wafer, with the number of sublayers proportional to the lipid concentration. 13 The layer’s quality was checked by atomic force microscopy and x-ray reflectivity measurements. In figure S1 of the supporting information, an AFM measurement of a DMPC multilayer system and a corresponding height profile are shown. Holes in the upper layer lead to an increased surface roughness that is accounted for in the data analysis. For in situ measurements, pressure-resistant Bis-Tris buffer solution with a concentration of 25 mmol/l was used as liquid phase so that the pH value was constant at 7 also at high pressures. 19

X-ray reflectometry With x-ray reflectometry, it is possible to determine the laterally averaged electron density profile

ρ (z) perpendicular to the sample surface or interface. 20 To perform a measurement, the scattered intensity I(qz ) is recorded as a function of the incident angle αi under specular condition αi = αf , where αf is the exit angle. In this scattering geometry, the wave vector transfer q has only one component perpendicular to the samples surface qz = 4π sin(αi )/λ with the wavelength λ of the X-ray radiation. The reflected intensity is 21 Z 2 1 dρe (z) exp(iqz z) dz . I(qz ) ∝ 4 qz dz

(1)

For the in situ study of multilayer systems at the solid-liquid interface, a photon energy of several 10 keV is required to ensure sufficient transmission through the bulk liquid phase. XRR measurements were carried out at beamline ID15a of ESRF/Grenoble, France, 22 with a photon energy of 70 keV, at beamline BL9 of DELTA/Dortmund, Germany, 23 with an energy of 27 keV and at beamline I07 of Diamond Light Source/Oxfordshire, England, with an energy of 30 keV. 24 With our custom-made high hydrostatic pressure cell, it is possible to carry out in situ XRR measure-


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ments up to a maximum pressure of 5000 bar. A detailed description of this cell can be found in. 26 The temperature is controlled through a built-in cooling circuit and a chiller. All measurements have been performed at a temperature of 37 ◦ C. At this temperature, the system is at 1 bar in the fluid Lα -phase and a pressure increase should lead to consecutive transitions into the P′β -phase (at 700 bar), Lβ′ -phase (at 2750 bar) and GEL-III-phase (at 3380 bar). 7 As lipid multilayers on solid supports are unstable under water due to the formation of vesicles, 18 the pressure was increased to 50 bar directly after sample preparation. This results in a stabilization of the multilayers. Likely, the same mechanisms are at play that were discovered in the context of the formation of solidsupported biomembrane formation: increased (osmotic) pressure leads to deposition of vesicles to a solid substrate and promotes the formation of bilayers. 25 A typical x-ray reflectivity scan including the measurement of the diffusely scattered radiation took approximately 6 minutes recording time. The x-ray beam was blocked during motor movements to avoid radiation damage. As the lipid samples are very sensitive to x-ray radiation, the system was laterally shifted after each scan. X-ray scans at selected conditions were repeated to probe the stability of the respective phase over time.

The pressure-dependent structure of DMPC multilayers Results Figure 1 shows x-ray reflectivity curves that were obtained on a DMPC multilayer at different pressures. They are normalized by the Fresnel reflectivity of an ideally flat silicon - water interface and shifted vertically for better visualization. Bragg reflections that originate from multiple reflections at the multilayer are clearly visible on the reflectivity curve. Error bars represent the statistical error of the measurement. The individual bilayer thickness d of the DMPC multilayer system can be determined from the position of the Bragg reflexions qz, Bragg as

d=

2π qz, Bragg

.


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50 bar 1500 bar 3500 bar 4500 bar 50 bar

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Figure 1: XRR data of a DMPC multilayer at 50 bar, 1500 bar, 3500 bar, 4500 bar and 50 bar after pressure decrease with simulated reflectivities. The reflectivities are shifted vertically for better visualization. Fits to the data are shown as solid lines. The position qz, Bragg has been determined by fitting a Gaussian profile to the Bragg reflexions after a linear background was subtracted from the relevant region of the curve. The corresponding bilayer thickness d of the repetition unit is shown in figure 2 as a function of pressure. When considering the pressure dependence of d, three different regions can be identified. The first part covers a pressure region from 50 bar to 750 bar with a bilayer thickness of 56.5 Å. At this temperature and these pressures, the system is in the fluid Lα -phase. At 750 bar, a phase transition into the Pβ ′ -phase occurs. The bilayer thickness in this second region shows a non-monotonic slope. Starting at d = 63.7 Å at 1000 bar, the bilayer thickness increases slightly up to 64.5 Å at 2000 bar and decreases continuously by 2.5 Å to 62.0 Å during pressure increase. Above 3500 bar, the system becomes thicker again and at 4500 bar, it has a thickness of 64.0 Å. The phase transitions are known for bulk solutions 7 and are indicated in the figure as dashed lines. These systems show a phase transition at 2750 bar and enter the Lβ ′ phase, while at 3400 bar the GEL-III-phase is reached. 7 These two phase transitions are not clearly separated in our solid-supported multilayers; only a minimum of the bilayer thickness at 3500 bar


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Figure 2: Measured bilayer thickness at 37 ◦ C. Red markers correspond to measurements at increasing pressure and blue markers at decreasing pressure. Error bars (calculated from the Gaussian fit of the qz position) are smaller than the marker size. Dashed lines represent phase boundaries in bulk solutions. 7 is observed. If this minimum layer thickness is attributed to the L′β -phase, a shift of the phase boundaries to higher pressure might be deduced. Reducing the pressure to 2750 bar again results to roughly the same layer thickness as during pressure increase. After a pressure reduction to 50 bar, the layer system has a thickness of 52.0 Å, thus 4.5 Å less than before pressure exposure. We will discuss later the reason for this decrease from the exact electron density profiles. Discussion In the literature, phase transitions in DMPC systems were vastly studied, so that a comparison to earlier results is appropriate. Mennicke et al. investigated a DMPC multilayer system consisting of 10 layers at a relative humidity of 98%. 13 Their samples were prepared by spin-coating as in the present work. The authors observed a layer thickness of 53.9 Å in the Lα phase, i.e. 2.6 Å thinner than in the present work. The smaller layer thickness can be explained by the lower hydration of the layers, so that less water was included between the hydrophilic head groups in each bilayer. In that reference, phase transitions were triggered by varying tempera7 ACS Paragon Plus Environment

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ture. The first transition from the fluid Lα -phase to the Pβ ′ -phase resulted in a layer thickness of 66.2 Å at ambient pressure, 13 somewhat larger than the value of approximately 64 Å that we observed at increased pressures and at full hydration. This result is in accordance with literature, where it was shown that the bilayer spacing of DMPC oligo-layers is decreasing with increasing osmotic pressure. 16 Likewise, Petrache et al. found that the thickness of a fully hydrated DMPC bilayer at ambient pressure is 62.7 Å and decreases to 51.5 Å at an osmotic pressure of p = 27 atm at 30◦ C. 27 At ambient pressure, Kucerka et al. have measured a thickness of 62.6 Å , also for DMPC at 30◦ C. 28 Another possible reason for the smaller bilayer thickness that we observe lays in the fact that the number N of bilayers in a multilayer system influences the bilayer thickness. It was shown that low N suppresses vertical fluctuations within the multilayers 16 due to a strong coupling between the layer and the substrate. Consequently, the reduced layer thickness in our system can be attributed to reduced vertical fluctuations. A strong coupling can also cause the complete suppression of the Pβ ′ -phase in the case of a single bilayer. 17 Concerning the gel phase, Tristram-Nagele et al. 29 determined a bilayer spacing of 59.9 Å for a DMPC multilayer system under full hydration in the gel phase. This is somewhat smaller than the values between approximately 62 Å and 64.5 Å that we detected at increased pressure. When comparing these results, one has to keep in mind the different preparation methods. While TristramNagele et al. prepared the samples on cleaved mica or glass microscope slides by letting the system evaporate for one day, our samples were prepared by spin-coating. In addition, our system is immersed in Bis-Tris buffer and not in pure water as in the literature. Small variations in the absolute d values might be based on these different conditions. Comparing to bulk systems, Trapp et al. investigated fully hydrated multi-lamellar vesicles (MLVs) at high hydrostatic pressure. 30 The measured repeat distance in the Lα phase was approximately 62 Å and thus 5 Å larger than in the presented work. The repeat distance in the Pβ phase is around 69 Å in Trapp’s investigation and thus approximately 4 Å larger than in our work. Again, this can be interpreted as a signature of the coupling between substrate and lipid layers. As mentioned before, the fact that Trapp et al. work in pure water, in contrast to our Bis-Tris buffer environment, might also play a role.


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Figure 3: Electron density profiles of a DMPC multilayer with a sublayer system consisting of six layers obtained by stochastic variation of the fits (red lines). The solid colored lines represent the start profiles. The profiles are shifted vertically for better visualization. Inset: Schematic layer model with six sublayers. Layer I is the water layer, layers II and VI represent the headgroups of the lipids, layers III and V their alkyl tails. Layer IV is the gap between the lipids. In order to obtain a more detailed insight into the layer structure, the reflectivities were simulated using the Parratt algorithm in combination with the effective density model. 31,32 First, the multilayer was divided into a set of identical bilayers, where the bilayer structure was modeled by six sublayers (see inset of figure 3). In addition, the resulting (idealized) electron density profile was varied stochastically to account for the imperfections of real multilayer systems in the fitting procedure. In figure 1, the fits to the data are shown as solid red lines. Figure 3 displays the corresponding electron density profiles. z = 0 marks the solid substrate, more precisely the interface between Si and SiO2 . z > 0 denotes the multilayer and the water phase. The electron density is normalized to Si and the curves at different pressures are shifted vertically for a better visibility. The start profiles are shown as colored lines and the profiles after stochastic variation as red lines. Now, we discuss the influence of pressure on this system as seen in figure 3. At the beginning of the measurement (p = 50 bar, brown line), the multilayer system consists of 9 bilayers. Since the upper bilayers do not cover the entire surface of the multilayer system, but only parts of it


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(see figure S1 in the supporting information), the interface between the water phase and the whole multilayer system is rather extended in a lateral average, accounted for by a high roughness. After passing the first phase transition into the Pβ ′ -phase (p = 1500 bar, light green curve), three layers peel off. Furthermore, an increase of the electron density in the area of the water layer (region I in the inset of figure 3) between the lipid bilayers becomes visible when entering the Pβ ′ -phase. In figure 3, the effect is highlighted with black arrows. With increasing pressure, the electron density in region I of all sublayers gradually increases, meaning that the hydration between all the bilayers is increased. We analyzed in more detail the behavior of the water layer from the electron density profiles. The lower panel of figure 4 shows the maximum electron density of the water layer as a function of pressure. Values are the average over all bilayers in figure 3. The mean maximum electron density of the water layer increases strongly (approximately by 18.5%) when the system undergoes the transition into the gel phase. At higher pressures, the electron density increase seems to saturate. Thus, water molecules are included between the head groups when the system reaches the gel phase. When the pressure is decreased again (blue marker), the electron density recovers the original value. Further, we studied the thickness of the water layer dH2 O (see upper panel of figure 4). While it is obvious from figure 2 that the bilayer thickness increases strongly when the gel state is reached, the thickness of the water layer decreases at that point. Increasing further the pressure once the gel phase is reached, the water layer is only slightly compressed. The non-monotonous behavior of the bilayer thickness in the gel phase between 2000 bar and 4500 bar (figure 2) is not reflected in dH2 O . When the pressure is released back to 50 bar (blue marker), the thickness of the water layer is even smaller than the initial value (≈ 4 Å as compared to ≈ 10 Å in the beginning of the experiment). Since the thickness of the bilayer after pressure release is decreased by ≈ 5 Å, we can deduct that this reduction is mainly caused by the reduced thickness of dH2 O . Following the phase diagram by Prasad et al., 7 a bulk DMPC system at 37 ◦ C should show a transition from the Lβ′ to the GEL-III-phase at increased pressure, while the bulk DMPC system 10 ACS Paragon Plus Environment

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Figure 4: Upper panel: Thickness of the water layer in the multilayer system in figure 3. Lower panel: Maximum electron density of the water layer of the multilayer system in figure 3. The values are averaged over all bilayers. studied by Ichimori et al. 8 shows a transition into the interdigitated Lβ i-phase. Figure S2 in the supporting information shows an isolated repetition unit from the multilayer in figure 3 at different pressures. The non-symmetric shape of the electron density profiles is most likely an artifact of the calculation method. The curves were shifted horizontally so that the region of the hydrocarbon tails is centered at each pressure. From this figure, it is visible that the region of the hydrocarbon tails becomes slightly larger after the transition into the gel phase and then stays mainly constant. From this, we can deduct that the hydrocarbon tails are not interdigitated. The increase in the thickness of the hydrophobic region can be explained by a slight straightening of the acyl chains. However, the straightening is not complete as in the GEL-III-phase, since the increase in the bilayer thickness is too small. The transition from the Lβ ′ -phase to the GEL-III-phase would lead to an enlargement of more than 10%. 13 At the same time, the electron density raises slightly, indicating a more dense packing of the acyl chains at increased pressure. After reducing the pressure to 50 bar again at the end of the measurement series (blue curve in figure 3), the system returned to the Lα -phase. However, a reduced bilayer thickness of 52 Å was observed, 4 Å less than in the beginning of the experiment. As discussed before, this effect can 11 ACS Paragon Plus Environment

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be attributed to a decreased amount of water within the system, since the water layer between the lipid bilayers (region I) became thinner while the electron density remained constant.

Pressure-induced DMPC multilayer formation As mentioned above, solid-supported multilayers tend to desorb by the formation of vesicles in the bulk water. 18 This process can be used e.g for the production of vesicles in solution or to produce a lipid bilayer. The pressure-dependent response of such a lipid bilayer in contact with a vesicle solution will be discussed in the following section. The sample was prepared by spin-coating again a multilayer onto a silicon substrate. The substrate was introduced into the sample holder with buffer solution. Leaving the sample for some time at rest at ambient pressure, the multilayer was degraded. Finally, a single bilayer remained on the substrate, with the lipids in solution. Note that the DMPC concentration in the aqueous phase is not exactly known. Pressure-dependent x-ray reflectivity scans of the system are shown in figure 5. The first measurement of the bilayer system at 50 bar was fitted, the resulting electron density profile is shown in the supporting information in figure S3. The bilayer thickness of 44.5 Å corresponds to a bilayer system in the Lα -phase. 33 The subsequent reflectivity taken after a pressure increase to 750 bar (brown line) shows strong Bragg reflections, indicating that a multilayer was formed at the interface. Decreasing the pressure to 50 bar again (light green curve), the system switches back to a bilayer. This proves that indeed, we observe a pressure effect and not a time effect. Decreasing and re-increasing the pressure to 750 bar causes a disintegration and reformation of the layer system, demonstrating the reversibility of the observed effect. The electron density profiles of the system at 750 bar, 1500 bar and 3500 bar are shown in figure 6. No further phase transition was observed with increasing pressure. The layer thickness of 63.3 Å does not change. After decreasing the pressure from 3500 bar to 50 bar, the system is not completely recovering its initial shape. It was not possible to fit a consistent electron density 12 ACS Paragon Plus Environment

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profile to the final XRR curve, so that it is not in a bilayer state. At the same time, the absence of Bragg reflections proves that the multilayer is destroyed.

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The formation of mono- and bilayers via vesicle deposition is well known, see e.g., 34–37 while the effect of high hydrostatic pressure on the formation process is still unknown. One of the key processes during the formation of solid-supported lipid layers is the rupture of adsorbed vesicles. Studies on the effect of osmotic pressure on vesicle rupture showed that a positive osmotic pressure leads to a compression and deformation of the vesicles at an interface and thus promotes their rupture and adsorption kinetics. 25,38,39 The reversible multilayer formation that we report on in this article can be attributed to the same compression and deformation mechanisms. The switching between bi- and multilayer happens at the phase transition pressure between fluid and gel phase. Thus, the pressure-induced phase transition and possibly the corresponding change in the structure of the lipid layer might act as an additional destabilizing factor.

Summary and conclusion In summary, pressure-dependent structural changes of DMPC multilayers were investigated at the solid-liquid interface. For this purpose, high energy x-ray reflectometry was applied, which enabled us to study such systems in situ in a pressure range between 50 bar and 4500 bar. When exceeding the first phase boundary between the Lα - and Pβ ′ -phase, three of nine bilayers were lost. The phase behavior of the remaining solid-supported multilayer is similar to that of bulk systems, but the bilayer spacing is decreased because of the bounding to the substrate. The Lβ ′ -phase, which should cover a pressure range between 2750 bar and 3400 bar, is only suggested by a drop in the sublayer thickness, which reaches its lowest value at 3500 bar. This indicates that the phase boundaries might be shifted to higher pressures. The effect can be attributed to the interaction of the lipid layer with the substrate, which might stabilize the layer with regard to phase transitions. A detailed analysis of the electron density profiles shows a subsequent penetration of water molecules into the multilayer with rising pressure, starting with the top-most layers. This filling of the water layer between the lipid head groups was inverted by a pressure release, yielding a lower hydration compared to the initial system. It would be interesting to study in the future if the


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decreased hydration is recovering the initial state with time. Finally, it was shown that a pressure increase can control the formation of lipid multilayers in situ and we proved that formation and destruction are reversible when HHP is applied. While the system is completely reversible after a pressure in- and decrease to 750 bar, a pressure increase to 3500 bar introduces a non-reversibility. Going back to 50 bar, the multilayer is destroyed, but the initial bilayer shape is not recovered. The transition between bi- and multilayers is explained by the pressure-induced adsorption and destabilization of vesicles on the surface and was measured in this work for the first time. In that way, we obtain a way to switch the state of a lipid layer from bi- to multilayer and back in situ, without changing the sample. This can be helpful to study differences in the interaction between biomolecules and bi- and multilayers in the future, e.g. when proteins adsorb to lipid layers. We hope that our experiments will give input to colleagues with expertise in theory and numerical simulations, so that finally it will be possible to deduce information on interaction parameters and forces at play under increased pressure. Notably, it would be very interesting to understand in more depth how the phase transition influences the size of the water layer between the head groups.

Supporting information In the supporting information, we provide the following additional material: Figure S1 shows an AFM measurement of a DMPC multilayer system and a corresponding height profile. Figure S2 in the supporting information shows an isolated repetition unit from the multilayer in figure 3 at different pressures. The curves were shifted horizontally so that the region of the hydrocarbon tails is centered. The electron density profile corresponding to the bilayer at 50 bar from figure 5 is shown in figure S3. This material is available free of charge via the Internet at http://pubs.acs.org.


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Acknowledgement This work is supported by the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft. The authors thank the DFG (FOR 1979) for financial support. FJW thanks the NRW Forschungsschule Forschung mit Synchrotronstrahlung in den Nano- und Biowissenschaften for financial support. We acknowledge the European Synchrotron Radiation Facility ESRF, the DIAMOND light source and DELTA for providing synchrotron radiation. Further we acknowledge MPI/Stuttgart for the use of HEMD (high energy microdiffraction setup).

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Solid-Supported Lipid Multilayers under High Hydrostatic Pressure

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