Langmuir 2004, 20, 4621-4628
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In Situ Sensing of Salinity in Oriented Lipid Multilayers by Surface X-ray Scattering Heinz Amenitsch, Michael Rappolt, Cilaˆine V. Teixeira,† Monika Majerowicz,‡ and Peter Laggner* Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Schmiedlstraβe 6, 8042 Graz, Austria Received December 9, 2003. In Final Form: March 4, 2004 The influence of LiCl solutions on liposomal and surface-supported phosphatidylcholine/water systems (dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), respectively) has been studied by small-angle X-ray techniques. In liposomal dispersions of DPPC, an osmotically stressed liquid-crystalline phase, denoted as LR osm, forms readily after rapid mixing with salt solutions. The transition from LR f LR osm proceeds in two steps. The first step takes place within seconds and is due to water diffusion from the liposome into the bulk solution. The second, slower process (minutes) can be attributed to the relaxation of initially deformed intermediate liposomes into spherical ones. In experiments with aligned lipid bilayers supported on silicon wafers, it was possible to reproducibly exchange different concentrations of LiCl solutions on a single sample and to determine the lattice changes by time-resolved X-ray scattering at grazing incidence. Independently of the deposition technique (spray- or spin-coating, respectively), none of the investigated POPC samples displayed an osmotically stressed liquid-crystalline phase. While liposomes can be considered nearly defect-free, supported bilayer stacks show a high abundance of defects, such as oily streaks typical of the LR phase. Thus, the alkali ions are free to diffuse into the interbilayer water regions and to cause a slight increase of the bilayer separation (about 1 Å). It is concluded that low to medium concentrations of Li+ ions partially screen the attractive van der Waals force between adjacent membrane layers. However, upon annealing the defect regions or regions of high curvature in the oriented lipid matrix, e.g. by low amounts of oleyl alcohol (OA), the system is able to sense osmotic stress upon addition of a salt solution.
1. Introduction Substrate-supported lipid membranes provide a fascinating approach to biofunctionalize solid surfaces, to study the structure and dynamics of biomimetic systems, and to carry out in situ surface chemistry. As an important model system of biological membranes, phospholipid bilayers are the natural host and binding matrix for an abundance of biomaterials.1-4 Moreover, apart from biological and biochemical concerns, oriented lipid membranes deposited on solid substrates offer unique experimental possibilities to study self-assembly phenomena, fluctuations, and interactions,5 both with and without additional membrane-active molecules such as amphiphilic peptides or membrane proteins.6-8 For diffraction analysis, samples of stacked and oriented multilamellar membranes are particularly useful, since the scattering signal is greatly enhanced as compared to single-bilayer or monolayer preparations. Due to their orientation they also allow for a distinction between the normal and the * To whom correspondence should be addressed. Telephone: ++433164120302.Fax: ++433164120390.E-mail: Peter.Laggner@ oeaw.ac.at. † Current address: Department of Physical Chemistry, Åbo Akademi University, 20500 Turku, Finland. ‡ Current address: Institute for Medical Physics and Biophysics, Universtiy of Leipzig, 04103 Leipzig, Germany. (1) Sackmann, E. Science 1996, 271, 43. (2) McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95. (3) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773. (4) Safinya, C. R. Colloids Surf., A 1997, 128, 183. (5) Parsegian, V. A.; Rand, R. P. In Structure and Dynamics of Membranes; Lipowsky, R., Sackmann, E., Eds.; Elsevier/North-Holland: Amsterdam, 1995; p 643. (6) White, S. H.; Ladokhin, A. S.; Jayasinghe, S.; Hristova, K. J. Biol. Chem. 2001, 276, 32395. (7) Yang, L.; Weiss, T. M.; Lehrer, R. I.; Huang, H. W. Biophys. J. 2000, 79, 2002. (8) Bechinger, B. J. Membr. Biol. 1997, 156, 197.
lateral component of the scattering vector, thus allowing one to retrieve even mechanical properties such as the bending rigidity of the single bilayer and the bulk compression modulus.9 The investigation of membranemimetic surface coatings is attracting strong interest, because of their potential as carriers of various functional groups, like antibodies for the development of biosensors10 or other protein-based biotechnological application, for instance for the study and design of new antibiotics in pharmacological research.11,12 Although surface X-ray scattering on supported lipidwater systems is a relatively young field, a great variety of techniques has been employed in aligning synthetic and biological membrane stacks.13-16 Roughly speaking, these may be divided into three categories: first, aligned multilayers hydrated from vapor with relative humidities (RH) below 100% (e.g., refs 17 and 18); second, lipid samples under 100% RH condition;19 third, fully hydrated specimens at an excess of water.20,21 Under low-humidity (9) Lyatskaya, Y.; Liu, Y.; Tristram-Nagle, S.; Katsaras, J.; Nagle, J. F. Phys. Rev. E 2001, 63, 011907. (10) Clark, L. C., Jr. Biosens. Bioelectron. 1993, 8 (1), 3. (11) Lohner, K. In Development of Novel Antimicrobial Agents: Emerging Strategies; Lohner, K., Ed.; Horizon Scientific Press: Wymondham, U.K., 2001; p 149. (12) Epand, R. M.; Vogel, H. J. Biochim. Biophys. Acta 1999, 1492, 11. (13) Smith, G. S.; Sirota, E. B.; Safinya, C. R.; Clark, N. A. Phys. Rev. Lett. 1988, 60, 813. (14) Clark, N. A.; Rothschild, K. J.; Luippold, D. A.; Simon, B. A. Biophys. J. 1980, 31, 65. (15) Prosser, R. S.; Hunt, S. A.; DiNatale, J. A.; Vold, R. R. J. Am. Chem. Soc. 1996, 118, 269. (16) Hentschel, M. P.; Rustichelli, F. Phys. Rev. Lett. 1991, 66, 903. (17) Hristova, K.; White, S. H. Biophys. J. 1998, 74, 2419. (18) Sengupta, K.; Raghunathan, V. A.; Katsaras, J. Phys. Rev. E 2003, 68, 031710. (19) Katsaras, J.; Watson, M. J. Rev. Sci. Instrum. 2000, 71, 1737. (20) Katsaras, J. Biophys. J. 1997, 73, 2924. (21) Vogel, M.; Mu¨nster, C.; Fenzl, W.; Thiaudie`re, D.; Salditt, T. Physica B 2000, 283, 32.
10.1021/la036319p CCC: $27.50 © 2004 American Chemical Society Published on Web 04/29/2004
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conditions the samples are very stable and thus easy to handle. Therefore most of the structural and dynamical studies have been carried out at RH below 100%, e.g., investigations on lipid/protein interaction (for a review see ref 6) or time-resolved diffraction studies on the effect of additives.22 To be of “biological relevance”, it is desirable that the lipid bilayer stacks are fully hydrated and in the liquid crystalline LR phase and that the aqueous environment is adjustable to the physiological conditions of interest, e.g., variation of pH or salinity. The immersion of aligned multilayers in water may lead to the spontaneous detachment of sample patches from the substrate.23 In the present study, therefore, particular attention has been paid to the preparation of the solid support as well as on the coating technique itself (cf. to refs 24 and 25). Biological membranes in contact with buffers can exhibit quite different ionic concentrations inside and outside the compartment. Their role in the electrostatic interaction between the saline environment and the lipid membrane surface26 is very important for the events taking place in living systems, e.g., formation of nonlamellar phases, membrane fusion, or transport phenomena across the bilayers. Therefore, the biologically most abundant alkali ions have been extensively investigated with respect to their influence on cellular and subcellular systems. In former studies on liposomal suspensions, we had focused on the alkali-ion-induced LR-phase separation,27,28 and particular attention had been given to the lightest alkali metal, lithium, also because of its neuropharmalogical potential.29 Similar questions are addressed in the present study, but now under the perspective of solid-supported phosphatidylcholine systems. To our knowledge, we have been able for the first time to exchange reproducibly different salt concentrations on a single sample and determine the structural and dynamical changes by time-resolved surface X-ray scattering measurements. The experiments show that aligned lipid systems display completely different hydration kinetics as compared to liposomal dispersions. The paper is organized as follows: sample preparation, experimental details, and a short description of the data analysis are given in section 2, in section 3 results of the effects of LiCl solutions on liposomal dispersions as well as aligned membrane stacks are presented, the response kinetics of these systems are discussed on the basis of different inherent defects in section 4, and finally, the conclusions are drawn in section 5. 2. Materials and Experimental Methods 2.1. Sample Preparation. 1,2-Dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) were purchased from Avanti Polar Lipids, Inc., Alabaster, AL (purity > 99%), and used without further purification. The additives oleyl alcohol (OA; purity > 98%) were (22) Rapp, G.; Funari, S. F.; Richter, F.; Woo, D. In Lipid Bilayers: Structure and Interactions; Katsaras, J., Gutberlet, T., Eds.; Springer: Berlin, 2001; p 165. (23) Pabst, G.; Katsaras, J.; Raghunathan, V. A. Phys. Rev. Lett. 2002, 88, 128101. (24) Brzoska, J. B.; Azouz, I. B.; Rondelez, F. Langmuir 1994, 10, 4367. (25) Mennicke, U.; Salditt, T. Langmuir 2002, 18, 8172. (26) Bo¨ckmann, R. A.; Hac, A.; Heimburg, T.; Grubmu¨ller, H. Biophys. J. 2003, 85, 1647. (27) Rappolt, M.; Pressl, K.; Pabst, G.; Laggner, P. Biochim. Biophys. Acta 1998, 1372, 389. (28) Rappolt, M.; Pabst, G.; Amenitsch, H.; Laggner, P. Colloids Surf., A 2001, 183, 171. (29) Birch, N. J. Lithium and the Cell. Pharmacology and Biochemistry; Academic Press: London, 1991.
Amenitsch et al. purchased from Fluka (Buchs, Switzerland) and LiCl (p.a.) from Merck (Darmstadt, Germany), respectively. For the rapid-mixing experiments, multilamellar liposomes were prepared by dispersing 20 wt % lipid in quartz-bidistilled and deionized water. To ensure complete hydration, the dispersions were incubated for about 2 h 25 °C above the main transition temperature. During the last 1/2 h the samples were vigorously vortexed. For the preparation of oriented multilamellar lipid stacks on silicon wafers two different protocols were applied. Solutions of 10 mg/mL POPC dissolved in 2-propanol (p.a.; Merck) were deposited on silicon wafers either by spray-coating 50 µL of solution by an air brush onto the wafer (IWATA HP-A, Dr Fr. Schoenfeld, Du¨sseldorf, Germany) or by spin-coating,25 i.e., pipetting 15 µL of solution on the rotating wafer (frequency ∼ 600 rpm).30 For preparing hydrophobic substrates, the Si surfaces were first cleaned and then silanized with dimethyldichlorosilane according to the following procedure.24 For cleaning, the silicon wafers were rinsed in ethanol (purity 99.8%; Merck), dipped for 15 s in 2% (v/v) hydrofluoric acid (40% (v/v); Carlo Erba Reagenti, Milano, Italy), washed carefully in distilled water, and then rinsed briefly in ethanol. The wafers were then immersed in a 1:1 solution of methanol (p.a.; Merck) and 7 N hydrochloric acid (37% (v/v); Sigma Aldrich, Steinheim, Germany) for approximately 30 min, rinsed 10 times in distilled water, and air-dried. Thereafter, they were put in sulfuric acid (volumetric standard 0.505 N; Aldrich, Milwaukee, WI) for 30 min, rinsed 10 times in distilled water, and dried under vacuum for at least 1 h. Finally after drying, they were dipped into a 1:1 mixture of tetrahydrofuran (THF) and dimethyldichlorosilane (both, Sigma Aldrich), and then briefly rinsed in THF. Until usage the silanized wafers were stored under vacuum. After the coating procedure, the wafers were kept under vacuum for at least 24 h in order to remove any residual contamination of the solvent. The lipid multilayers were optically blue and opaque: this corresponds to a thickness of about 1000 Å.31 Before and after experiments the purity of the phospholipid was checked by thin-layer chromatography using CHCl3/CH3OH/NH3,conc. (75:25:6 (v/v)) as solvent. Aluminum sheets, silica gel 60, purchased from Merck, were used as stationary phase. All phospholipid samples showed just one single spot before and after the measurements. 2.2. X-ray Measurements. All X-ray scattering patterns were recorded at the Austrian SAXS (small-angle X-ray scattering) beamline32,33 at the synchrotron light source “ELETTRA” (Trieste, Italy) using a one-dimensional position sensitive detector,34 which covered the s-range (s ) 2 sin(θ)/λ) of interest from about 1/300 to 1/15 Å-1 at an X-ray energy of 8 keV. Silver behenate (CH3(CH2)20-COOAg, d spacing ) 58.38 Å)35 was used as a standard to calibrate the angular scale of the measured intensity. Stop-flow mixing experiment measurements on lipid dispersions were performed with a commercial apparatus (SF400, Biologic, Grenoble, France) in combination with simultaneous time-resolved X-ray diffraction. The device consists of four reservoirs (each 10 mL; stepper motor driven), of which two have been operated. A shot volume of 50 µL each was chosen. When actuated by an electronic trigger signal, both the phosphatidylcholine dispersion and the salt solution, respectively, were injected into the mixing chamber and finally pressed into an X-ray quartz capillary with a diameter of 1 mm (specified dead time ∼ 1 ms). Fresh lipid dispersions (no salt) of 20% (w/w) DPPC were mixed 1:1 with 0, 0.1, 0.3, 0.5, 1.0, and 1.5 M LiCl (Table 1). For the first four concentrations the complete turnover from (30) In some cases a thin polymer foil was placed directly onto the lipid coating. While the lipid layers could be stabilized for longer time, the exchange rate of salt solutes was clearly reduced. Ideally, one should find a nanoporous and very weak X-ray scattering material to provide the lipid multilayer with a longer lifetime. (31) Seul, M.; Sammon, M. J. Thin Solid Films 1990, 185, 287. (32) Amenitsch, H.; Rappolt, M.; Kriechbaum, M.; Mio, H.; Laggner, P.; Bernstoff, S. J. Synchrotron Radiat. 1998, 5, 506. (33) Bernstorff, S.; Amenitsch, H.; Laggner, P. J. Synchrotron Radiat. 1998, 5, 1215. (34) Petrascu, A.-M.; Koch, M. H. J.; Gabriel, A. J. Macromol. Sci., Phys 1998, B37(4), 463. (35) Huang, T. C.; Toraya, H.; Blanton, T. N.; Wu, Y. J. J. Appl. Crystallogr. 1993, 26, 180.
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Table 1. Rapid Mixing Experiments of DPPC (20 wt %) with Different LiCl Solutions Carried Out at 50 °C (cp. Figure 3a) initial LiCl concn (M)
final LiCl concna (M)
osmotic pressure (atm)
final d spacing of the LR osm phaseb (Å)
0 0.1 0.3 0.5 1 1.5
0 0.06 0.18 0.29 0.58 0.87
2.9 8.7 13.4 28.7 42.9
66.5 58.9 56.8 56.1 54.7 53.9
a The final LiCl concentration estimation excluded the lipid volume as well as the lipid-bound water volume. b Measured 15 min after the rapid mixing.
Figure 1. (A) Schematic and (B) photograph of the cell constructed for surface X-ray scattering experiments under excess of water conditions. The two arrows indicate the incident and reflected X-ray beam, respectively (for details see text). fully hydrated to partially dehydrated bilayer stacks has been followed over a period of 15 min, whereas for the two highest concentrations only the initial and final states have been recorded. The temperature was controlled by a water bath to 50 ( 0.1 °C (Unistat CC; Huber, Offenburg, Germany), which was connected to the water circuit of the stop-flow apparatus. The sample cell for the surface X-ray scattering measurements is illustrated in Figure 1. The transmission cell (optical path, 3 mm) was especially designed for the purpose of carrying out in situ surface chemistry. It allows the remote-controlled exchange of bulk solutions by separated in- and outlets. The X-ray windows (w) of the sample holder were made of 10 µm thin poly(ethylene terephthalate) film (Kalle GmbH, Wiesbaden, Germany) and were tested to give negligible contribution to the measured intensities. The sample holder was held in copper jacket (Figure 1B), which provides good thermal contact with a water circuit. The temperature was controlled by a water bath within (0.1 °C (Unistat CC, Huber). All POPC samples were equilibrated at 25 °C. The complete sample unit was mounted on a high-precision cradle (angular resolution, 0.001°; BGM 80 PP Goniometric Cradle, Newport, de Saint Gue´nault, France), which allowed one to perform rocking scans of the sample (ω scans). Initial and final ω angles as well as the rocking speed were freely programmable. With the surface X-ray scattering cell, water and several LiCl solutions (0.1, 0.3, and 0.5 M) could be reproducibly exchanged on a single multilayer sample. To resolve the hydration kinetics, time-resolved X-ray patterns were recorded at fixed angles of incidence with samples that displayed a relatively large mosaic angular spread of the multilayers (1-5°). For specimens with low mosaic spread (
