Positively Charged Supported Lipid Bilayers as a Biomimetic Platform

Aug 24, 2012 - ABSTRACT: The supported lipid bilayer (SLB) is a well- known system for studying the cell membrane and membrane proteins. It is also ...
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Positively Charged Supported Lipid Bilayers as a Biomimetic Platform for Neuronal Cell Culture Dzmitry Afanasenkau and Andreas Offenhaü sser* Peter Grünberg Institute/Institute of Complex Systems Bioelectronics (PGI-8/ICS-8), Research Center Juelich, D-52425 Juelich, Germany S Supporting Information *

ABSTRACT: The supported lipid bilayer (SLB) is a wellknown system for studying the cell membrane and membrane proteins. It is also promising as a platform for studying cell processes: the cell adhesion, the cell membrane receptors, and the intercellular signaling processes. SLBs made of natural lipids appeared to be protein and cell repellent. Thus, to use the SLB as a substrate for cells, one should functionalize them to provide adhesion. In the present paper, we describe a simple approach to promote adhesion of neuronal cells to the SLB without using proteins or peptides, by introducing positively charged lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) into the SLB made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). We show that neurons adhere to these bilayers and grow for at least 10 days. The SLBs themselves were found to degrade with time in cell culture conditions, but maintained fluidity (as revealed by fluorescence recovery after photobleaching), demonstrating the possibility of using SLBs for studying neuronal cells in culture.

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INTRODUCTION The supported lipid bilayer (SLB) is an interesting system for studying cell adhesion on an artificial substrate due to the structural similarity with the cell membrane. This similarity provides the possibility to create a cell-mimicking surface, or “phantom cell”, on the solid support and to study cell adhesion and other processes taking place at the cell surface using advanced methods designed for surface analysis such as ellipsometry, SPR, QCM-D, TIRF, and AFM. SLB is a lipid membrane system which consists of a single lipid bilayer built on a hydrophilic substrate, surrounded by aqueous solution and separated from the substrate by a very thin hydration layer (see reviews, refs 1−3). It has been widely used as a model for fundamental studies of the cellular membrane and membrane proteins,4−7 for developing biosensors,8−10 as well as a model system for drug delivery.11,12 However, the number of experiments using SLBs for studying cells is low, perhaps, due to the fact that SLBs made of natural lipids, such as phosphatidylcholines (PC), are protein and cell resistant; cells cannot adhere to it. This effect was shown for the SLB prepared by a vesicle fusion method13 and earlier for lipid films prepared by spin-coating.14,15 It was concluded from these studies that cell adhesion to SLBs is hindered by surface electrostatics, surface hydration, and lateral mobility of the lipid molecules, which do not provide an anchor for the cell’s focal adhesion sites. Interestingly, the fluidity can play an important role. As was shown by Oliver et al.,16 human retinal pigment epithelial cells did adhere and grow nicely on solidlike lipid monolayers, while no adhesion was found on lipid bilayers in the fluid state. © 2012 American Chemical Society

To use the SLB as a platform for cell culture one has to functionalize it with special molecules that can provide adhesion. These functional molecules can be membrane proteins responsible for adhesion between cells in vivo. However, the protein incorporation step makes the whole procedure to prepare the functionalized SLB more complicated. Nevertheless, a few attempts have been made to use SLB as a platform for cell culture. Among papers describing the use of SLB for studying cells, several recent ones can be mentioned. Thid et al.17 used IKVAV peptide to promote adhesion of neural progenitor cells on SLB. Ananthanrayanan et al.18 functionalized SLB with RGD peptides to study neural stem cells. Huang et al.19 used collagen functionalized SLB as a platform for smooth muscle cells. Berat et al.20 used an approach in which Annexin-A5 peptides were stereoselectively linked to proteins self-assembling in a rigid two-dimensional matrix on SLBs. This platform was found to promote specific adhesion of human saphenous vein endothelial cells and mouse embryonic stem cells. Pautot et al.21 used glycosylphosphatidylinositol (GPI) anchored neuroligin to promote adhesion between the SLB and HEK cells expressing neuroxin. Furthermore, Baksh et al.22 produced silicon microspheres covered with neuroligin doped bilayers and showed that adding them to neuronal cell culture could promote synapse formation. Another example is formation of immunological synapses between T cells and a SLB.23 Received: June 20, 2012 Revised: August 7, 2012 Published: August 24, 2012 13387

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Figure 1. (A) Chemical structure of lipids used in this study. POPC is a zwitterionic lipid which has both positive and negative charge. In contrast, DOTAP has only positive charge. (B) PDMS wells on glass slides used for supported lipid bilayer (SLB) preparation. (C) Scheme of vesicles and SLB preparation including several steps. Lipids, dried on the walls of a glass flask, are reconstituted in PBS and form multilamelar vesicles. These vesicles are extruded through polycarbonate membrane to get small unilamelar vesicles of around 100 nm in diameter. They are deposited on the clean hydrophilized glass surface and rupture forming a SLB. Cells with negatively charged surfaces adhere to the positively charged SLB due to electrostatic interaction. nium-propane (DOTAP), 1-oleoyl-2-{6-[(7-nitro-2−1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoethanolamine (NBDPE), and Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE). The structures of POPC and DOTAP are shown in Figure 1A. Texas Red DHPE was purchased from Invitrogen GmbH as powder, and all the other lipids were purchased from Avanti Polar Lipids, Inc. dissolved in chloroform. Neurabasal medium, B27 supplement, L-glutamine, Vybrant DiI, and DPBS were purchased from Invitrogen as well as Calcein AM used for live-dead cell staining. Salts for phosphate buffer saline (PBS), gentomicin, trypan blue (0.4% solution), and poly-L-lysine (PLL) were purchased from Sigma-Aldrich GmbH. Hellmanex for cleaning glass coverslips was bought from Hellma Analytics. Surface Preparation. Glass coverslips (30 mm, from Thermo Fisher Scientific Inc.) were used as substrates for preparation of the SLB. The coverslips were washed extensively in 2% Helmanex in an ultrasonic bath until the surface was clean as indicated by a uniformly spread very thin film of liquid, on which lines of interference of light could be seen. Afterward, glass was rinsed extensively with Milli-Q water and dried at 60 °C. Glass slides were activated by oxygen plasma in Plasma system PICO (Diener Electronics) for 5 min, at 0.4 mbar, 40% power of the generator immediately before bilayer preparation. Preparation of Small Unilamellar Vesicles. Lipids were mixed to the desired ratios while dissolved in chloroform. The final concentration of fluorescently labeled lipids was 1%−2%. Lipids were dried on the walls of a glass flask under a stream of nitrogen and then left in vacuum for 1 h to remove residues of solvent. PBS was added to the dried lipids for a final concentration of 5 mg/mL. The solution was extruded 11 times through a 100 nm polycarbonate membrane using an Avanti mini extruder (Avanti Polar Lipids, Inc.).

The use of SLBs modified with appropriate proteins, as described above, would be the most natural way to mimic cell membranes. However, difficulties of protein purification and incorporation limit this concept. Alternatively, cell adhesion can be enhanced by nonspecific interactions, such surface electrostatics. Due to the net negative charge of cell membranes, they adhere well to positively charged surfaces, such as selfassembled monolayers or protein layers. In the present paper, we propose to use positively charged mixed lipid bilayers to increase cell adhesion. We studied growth and development of primary neuronal cell culture on SLBs containing different concentrations of DOTAP (a positively charged lipid) and showed that lipid bilayers made of mixtures of POPC (a zwitterionic lipid) and DOTAP support growth of neurons in culture for up to 10 days, providing a basic platform for investigation of cell adhesion and membrane interactions. We characterized our SLBs by fluorescence recovery after photobleaching (FRAP) to prove their quality and calculated the diffusion coefficient for SLBs of different types using an analytical solution of the Dirichlet problem corresponding to the second Fick’s law. Although we did not focus on the stability issues of SLBs in this study in detail, we show that they provide enough robustness to maintain neuronal cell culture for days or even weeks.

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EXPERIMENTAL SECTION

Materials. Lipids used in this study were 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), 1,2-dioleoyl-3-trimethylammo13388

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Before using, vesicles were kept in glass vials closed with Teflon lids sealed with parafilm at 4 °C for up to 1 month. Preparation of Supported Lipid Bilayers. SLBs were prepared in wells made of PDMS that were attached to clean glass slides (Figure 1B). The size of one well is 9 mm × 9 mm, with four wells on each slide. These wells were used for two purposes: (1) to create a pattern for producing SLB of different types and a control surface in the same Petri dish and (2) to allow solution exchange without the danger of exposing the surface to air. The procedure of SLB preparation (including preparation of vesicles) is schematically depicted in Figure 1C. Vesicle solution was diluted to the concentration of 0.3 mg/mL with PBS. In the case of vesicles made of POPC with a DOTAP content less than 50%, DPBS containing Ca2+ and Mg2+ was used (concentration of Ca2+ was 0.901 mM, and that of Mg2+ was 0.493 mM). Formation of CaPO4 was not observed. A total of 100 μL of this solution was applied to each well and left for at least 30 min to allow vesicle fusion and formation of SLBs. The bilayers were rinsed with a stream of PBS using a syringe (20 mL of PBS per well). For the experiments with cells, after preparation the bilayers were left overnight at 4 °C in Petri dishes filled with PBS. Before cell plating, PBS was replaced with cell culture medium in such a way that the bilayers were never exposed to air, and afterward the PDMS wells were carefully removed by using tweezers. Preparation of Poly-L-lysine Coated Glass. Control substrates were coated with the known cell adhesive protein poly-L-lysine (PLL). Poly-L-lysine dissolved in Gey’s balanced salt solution (GBSS) at a concentration of 10 μg/mL was applied to one of the four wells on the coverslip. After 20 min of incubation at room temperature, the PLL coating was carefully washed 2 times with GBSS. Fluorescence Microscopy and FRAP. SLBs with incorporated NBD-PE or Texas Red DHPE and stained cells were imaged by fluorescence microscopy. NBD-PE was used for bleaching experiments because the wavelength of the available laser comes into its spectrum of excitation. Texas Red DHPE was used for experiments with cells. The microscopes Axio Scope and Apotome from Carl Zeiss GmbH were used. Bleaching was done by using a 473 nm laser from Rapp OptoElectronics coupled into a Zeiss Axio Scope. A series of images were made to track the recovery of fluorescence. Analysis of recovery was done by the method similar to Jönsson et al.,24 which provides the possibility to calculate the diffusion coefficient from images of the recovery without any assumption about initial shape of the fluorescence profile. Using a fitting procedure (similar to the one described in ref 25), one can find a value of the diffusion coefficient such that the analytically calculated curves fit the experimental ones. Data processing was performed by custom developed Matlab based software (the code is available on request). Cell Culture. Rat embryonic cortical neurons were prepared as described elsewhere.26 Briefly, embryos were recovered from pregnant Wistar rats at 18 days gestation (E18). Cortices were dissected from the embryonic brains; cells were mechanically dissociated by trituration in 1 mL Hank’s balanced salt solution (HBSS) (without Ca2+ and Mg2+), 1 mM sodium bicarbonate, 1 mM sodium pyruvate, 10 mM HEPES, 20 mM glucose, pH 7.4 with a fire-polished silanized Pasteur pipet. One milliliter of HBSS (with Ca2+ and Mg2+, supplemented as above) was added to the dispersed cells. Nondispersed tissue was allowed to settle for 3 min, and then the supernatant was centrifuged at 200g for 2 min. The pellet was resuspended in 1 mL of neurobasal medium (NB) containing 1% B27, 0.5 mM L-glutamine, and 50 ug/mL gentomicin. An aliquot was diluted such that the final mixture was 1:2:1; cells/NB/trypan blue and the dye-excluding cells were counted in a Neubauer counting chamber. The remaining cells were diluted in neurobasal medium with the above supplements and plated onto the substrates at a density of 8 × 104 cells per 32 mm Petri dish. Half of the medium was changed 4 h after preparation and subsequently every 3−4 days. Cell Staining and Counting. For staining live cells, Calcein AM was used. The dye was diluted 1:1000 in PBS. The cell culture medium was changed to this solution, and the cells were incubated for 5 min at 37 °C. Cells were then washed with PBS without Calcein AM, and

images were made. Counting of cells was done using ImageJ software with the Cell Counter plug in. In some experiments, cells together with the SLB were stained with Vybrant DiI cell labeling solution.

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RESULTS SLB Characterization. Prepared SLBs consisted of zwitterionic POPC and positively charged DOTAP mixed in various ratios. A small amount of fluorescently labeled lipids was added to visualize the SLB. To check 2D fluidity of the prepared supported bilayers, and to compare bilayers of different types, FRAP measurements were performed and the diffusion coefficients were calculated. A laser was used to bleach a small area of the bilayer and the recovery of the fluorescence was observed. The process of recovery is shown in Figure 2 for

Figure 2. Time series of micrographs showing fluorescence recovery after photobleaching (FRAP) of a spot bleached in the SLB of different types. The scale bar is identical for all images.

SLBs of different types. One can see that the bleached spots diffused with time and dissipated almost completely after several minutes for all SLB types. The recovery, which is due to diffusion, was analyzed using Fick’s second law. The profiles of intensity at different time points were extracted from the images (Figure 3A, black curves), and the corresponding Dirichlet problem (see Supporting Information) was solved. The parameters of this solution were fit in such a way that the calculated curves (Figure 3A, gray curves) match the experimental ones. The determination coefficient for most of the fits was higher than 0.90. Good fits prove that recovery was mostly due to diffusion. The diffusion coefficient given by the fitting procedure for a pure POPC bilayer was 1.8 um2/s, which is in good agreement with data from literature (for example, 1.88 um2/s17). As shown in Figure 3B, the diffusion coefficient decreased with increasing amounts of DOTAP in the mixed bilayer and reached a value of 0.6 um2/s for pure DOTAP bilayers, which is 3 times smaller than the value we determined for POPC alone. The observed reduced fluidity of the bilayers containing positively charged lipids is discussed below. Adhesion of Neurons to Charged SLB. SLBs were prepared within the PDMS wells as described before. This method allows the preparation of different bilayers on one glass 13389

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Figure 4. Fluorescence micrographs of neurons on the supported lipid bilayers made of POPC (A) and 70% POPC/30% DOTAP mixture (B) on DIV1. The bilayers are stained with Texas Red-PE (red). Live cells are stained with Calcein AM (green).

Figure 3. (A) Experimentally determined intensity profiles (black curves) and a fit of analytical solutions of the diffusion equation (gray) for the SLB made of 70%POPC/30%DOTAP. (B) Dependence of the diffusion coefficient on the concentration of DOTAP in the bilayer. The bars represent the average for each concentration. The circles represent single measurements.

cells on PLL coated glass. The number of live cells on positively charged SLBs after 1 day was high and comparable with cells grown on PLL, while the number of neurons growing on pure DOTAP bilayers was even higher than on the PLL-control. Increasing the amount of DOTAP leads to an improved adhesion and outgrowth on the bilayer that can be attributed to the increased amount of positive charge. The spread of the experimental data is quite broad, due to the various factors affecting cell adhesion as discussed below. A few cells were found on the pure POPC bilayer. However, we cannot rule out that they might adhere due to rare defects in the SLB. Investigation of some phenomena of cell physiology (such as electric activity or synapse formation) may require long-term cell culture. For this reason, we studied cells on SLB for up to 10 days. Micrographs of cells on DIV4 and DIV10 are shown in Figure 6. After 4 days, the neurons on all surfaces except the POPC bilayer showed good growth and development of neurites. After 10 days, the cells on positively charged SLBs form a dense network although some neurospheres are observed. In comparison, on PLL the cells form denser networks with a large number of highly branched processes. Those cells growing on glass without any coating show a very different morphology from those grown on PLL or positively charged lipids. The neurons on glass form many clumps connected to each other by a few bundles of neurites. Stability of SLB in Neuronal Cell Culture. Holes appear in the SLB within the first few days that become bigger with time (see Figures 7 and 8). This effect seems to be independent of the presence of neurons or cell culture medium, as we observed the same degradation even in PBS and Milli-Q water. Degradation appeared to be enhanced in the presence of the

slide (usually complemented with one region covered by PLL as a control, which also ensures the right amount of growth factors excreted from cells in case the neuronal growth on SLBs was very limited). In addition, one can compare the effect of different surface coatings within one dish. The wells were removed just before cell plating. The cells were observed starting from the first day in vitro for up to 10 days. The bilayers made of pure POPC appeared to be cell resistant, as was previously reported in literature.13 We found just a small amount of cells adhering to and growing on the POPC. In Figure 4A, one can see the edge of POPC bilayer covered area that is almost cell free, while neurons are growing on areas not covered by the bilayer. In contrast, charged bilayers support adhesion and growth of neurons. In Figure 4B, one can see a positively charged SLB made of 70% POPC and 30% DOTAP. The cells appear to grow both on glass and on the bilayer. Both figures show neurons after 1 day in vitro (DIV1). The PDMS well leaves a residue on the glass that may be cell repulsive.27 Therefore, to create substrates with areas of glass and areas of SLB within a single well, one side of the bilayer was exposed to air to destroy it just before plating cells. Influence of Charge Density on Cell Attachment and Growth. Next we tested the influence of DOTAP concentration in the SLB on the adhesion and outgrowth of neurons. Figure 5 shows bar plots representing the number of live cells on bilayers containing different concentrations of DOTAP and on uncoated glass. All data are normalized to the number of live 13390

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Figure 6. Microscopy images of neurons of DIV4 and DIV 10 growing on the supported lipid bilayers made of different mixtures of POPC and DOTAP (the percentage of DOTAP in the mixture is indicated at the left side of the figure), as well as PLL. Live cells were stained by calcein AM. The scale bar is the same for all images.

Figure 5. Number of live neurons growing on mixtures of POPC and DOTAP, as well as on uncoated glass for DIV1 (A) and DIV10 (B). The number of cells is normalized to the number of cells on PLL. The bars represent the average for each concentration. The circles are single measurements.

cells as they can damage the SLB mechanically or by chemical factors. An example of a bilayer made of 70%/30% POPC/ DOTAP mixture on DIV 16 is shown in the Figure 7. However, the observed degradation after several days, and even weeks, seems not to harm the structure of the double layer, as the rest of the SLB maintained fluidic properties as revealed by FRAP (Figure 8). In Figure 8, one can see a POPC SLB after 19 days in cell culture conditions. Recovery of fluorescence in the bleached area was clearly seen, indicating that the bilayer was still fluid.

Figure 7. Lipid bilayer made of a 70%/30% POPC/DOTAP mixture after 16 days in neuronal cell culture conditions. Bright lines are neurites of cells, gray areas show the mixed SLB stained. The bilayer and the cells were stained with Vybrant DiI.

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DISCUSSION Fluidity of Mixed SLB. We demonstrate that the SLBs made of POPC and DOTAP prepared in our experiments show typical fluidity as evaluated by FRAP. The diffusion coefficient of the SLBs appeared to depend on the DOTAP/POPC ratio and decreased with increasing DOTAP concentrations. This decrease is not very high for SLBs with concentrations of DOTAP 30% and 70% in POPC. For the SLB made of DOTAP only, the diffusion coefficient is around 3 times lower than for POPC. We propose several hypotheses to explain this

effect: (1) This effect may arise from electrostatic interaction of the labeled lipids with positively charged lipid molecules, which suppresses their mobility. (2) Another explanation of this dependence may be the increase in density of the lipid bilayer due to interaction of positively charged heads of DOTAP molecules with zwitterionic POPC molecules as described in literature.28 (3) It is also possible that positively charged lipids have stronger interactions with the negatively charged surface 13391

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Figure 8. Fluorescence recovery after photobleaching (FRAP) in POPC bilayer after 19 days in neuronal cell culture conditions. The scale bar is the same for all images.

adhesion of the cells to the surface compared to cell−cell interactions. The number of clumps on SLBs was lower than on glass. The number of clumps increased slightly on SLB with a low concentration of DOTAP, which is probably due to degradation of the SLB and appearance of areas of uncovered glass to which cells adhere poorly. The neurons on the SLB made of DOTAP only do not form clumps. Although the number of cells is around 2 times lower than that on PLL, the neurons form dense networks. Stability of SLB. Defects were observed in SLBs, the number and size of which increased with time (data not shown). This effect does not depend on cell culture medium or on the presence of cells as it was also observed in PBS under sterile conditions. The formation of holes in the SLB was previously described by Tamm and McConnell30 for SLBs made by the Langmuir−Blodgett technique. This effect was explained by stretching of the SLB. The effect was also dependent on the temperature since the surface pressure of the SLB is temperature dependent. The same effect can also take place in our SLB, as we do not have any border to prevent its stretching. We think that defects on the surface of the glass slides or small impurities may facilitate this effect, as in the case of stretching the lipid molecules tend to move to the places where their interaction with the surface is stronger. Additionally, cells growing on the SLB affect the bilayer by destroying it mechanically or by excreted biochemical factors (an example is shown in Figure 7). At the same time, the rest of the SLB stays fluidic as confirmed by fluorescence recovery in bleached spots even in SLBs of several weeks old (Figure 8).

on which the bilayer is formed, making mobility lower. (4) The structure of the fatty acid chains of DOTAP is different from that of POPC, which can affect mobility of the labeled molecules. All these effects may act simultaneously. Scattering of the magnitudes of the diffusion coefficient may be due to defects in the SLBs that introduce inhomogeneities, which could confound our computational approach based on the assumption of the uniform bilayer. Another reason for such scattering of results can be incomplete washing of liposomes that stay on the top of the bilayer and may slow down movement of the lipid molecules. The scattering is similar for all bilayer types which implies that if there are defects, they are similar for all types of SLB. Richter et al. have shown by QCM-D that pure DOTAP SLBs present a small amount of defects (around 5% of the surface).29 However, in the present study, we observed approximately equal number and size of holes for both POPC and DOTAP bilayers. Thus, we did not consider defects as a factor that would explain the observed difference in the diffusion coefficient of POPC and DOTAP bilayers. Adhesion of Neurons to Supported Lipid Bilayers. Lipid surfaces made of pure phosphatidylcholine are cell resistant, as was shown previously in several papers. There are several hypothesis proposed in literature to explain this phenomenon:13 (1) diminished electrostatic interaction between cells and the bilayer, (2) formation of a comparatively strongly bound water layer associated with phosphocholine, and (3) fluidity of the SLB. Our results show that two parameters of the SLB are changed with different concentrations of DOTAP: surface charge and fluidity of the SLB. We consider the effect of positive surface charge of the SLB to be the main reason for adhesion of cells. The outer surface of the cellular membrane is usually negatively charged so it is very likely that cells adhere to positively charged SLBs due to electrostatic interaction. The average number of cells on positively charged bilayers after the first day in culture was 6−10 times higher than on the POPC bilayer. At the same time, fluidity of the SLBs containing 10% and 30% DOTAP was not considerably changed. This implies that the charge of the SLB is the main factor influencing cell adhesion. For the bilayer made of pure DOTAP, the effect of fluidity on cell adhesion can be taken to consideration. The same two factors, charge and fluidity of the SLB, can influence morphology of neurons. Morphology of neurons on charged SLBs and PLL is quite similar after 4 days in vitro as can be seen from Figure 6. After 10 days in culture, the cell morphology differs between the various surfaces. Neurons on glass tend to form clumps (not shown), which indicate poor

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CONCLUSIONS In this work, we have studied the adhesion and outgrowth of neuronal cells on fluidic supported lipid bilayers. While zwitterionic (neutral) lipids do not promote neuronal cell adhesion or growth, as was also shown previously by other groups, successful cultures can be achieved by the addition of positively charged lipids. The data suggest that positive charges of the mixed lipid SLBs are most likely responsible for the adhesion and development of neurons. While the addition of positively charged DOTAP lipids reduces the fluidic properties of the SLB, this effect is small compared with fixed surface modification. The viability of the cells on the SLB was smaller than on control PLL coated surfaces, though the morphology of the neuronal network on the SLB was similar to controls. The reduced viability could be due to the increase in SLB defects over time, which decreases adhesion. The formation of defects was observed both for SLBs containing DOTAP and for pure POPC SLBs. Although degradation may influence the survival 13392

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rate of the neurons, we could demonstrate the unchanged fluidity of this model cell membrane, which makes it possible to use it as a biomimetic platform for neuronal cell culture.

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ASSOCIATED CONTENT

S Supporting Information *

Mathematical analysis of the FRAP data, images of DOTAP SLBs on DIV 11 in cell culture conditions, and images of FRAP in DOTAP SLBs on DIV 11 in cell culture conditions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +49-2461-61-2330. Fax: +49-2461-61-8733. Email: a.off[email protected]. Mailing address: Peter Grünberg Institute/Institute of Complex Systems Bioelectronics (PGI-8/ICS-8), Research Center Juelich, Leo-BrandStr., D-52425 Juelich, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank to Dr. Vanessa Maybeck and Alexey Yakushenko for reading and discussing the manuscript, and Ida Delac for the assistance with the experiments. The work was supported by DFG Research Training Group (GRK) 1572 Bionik.

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ABBREVIATIONS SLB, supported lipid bilayer; POPC, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; NBD-PE, 1-oleoyl-2-{6-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoethanolamine; Texas Red DHPE, Texas Red 1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine, triethylammonium salt; PC, phosphocholine; SPR, surface plasmon resonance; QCMD, quartz crystal microbalance with dissipation; TIRF, total internal reflection fluorescence microscopy; AFM, atomic force microscopy; GPI, glycosylphosphatidylinositol; FRAP, fluorescence recovery after photobleaching; PBS, phosphate buffered saline; PLL, poly-L-lysine; PDMS, polydimethylsiloxane; GBSS, Gey’s balanced salt solution; HBSS, hank’s balanced salt solution; DIV, days in vitro; NB, neurobasal medium

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