Intact Vesicle Adsorption and Supported Biomembrane Formation

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Intact Vesicle Adsorption and Supported Biomembrane Formation from Vesicles in Solution: Influence of Surface Chemistry, Vesicle Size, Temperature, and Osmotic Pressure† Erik Reimhult,* Fredrik Hoö¨k, and Bengt Kasemo Department of Applied Physics, Chalmers University of Technology, S-412 96 Go¨ teborg, Sweden Received August 12, 2002. In Final Form: November 11, 2002 The adsorption kinetics of small unilamellar egg-yolk phosphatidylcholine vesicles was investigated by the quartz crystal microbalance-dissipation (QCM-D) technique, as a function of surface chemistry (on SiO2, Si3N4, Au, TiO2, and Pt), temperature (273-303 K), vesicle size (25-200 nm), and osmotic pressure. On SiO2 and Si3N4, the vesicles adsorb intact at low coverage, followed by transformation to a bilayer at a critical coverage. On TiO2, oxidized Pt, and oxidized Au, the vesicles adsorb intact at all coverages and all studied temperatures. Variation of vesicle size does not change the qualitative behavior on any of the surfaces, but the quantitative differences provide important information about surface-induced vesicle deformation. In the low-coverage regime (where vesicles adsorb intact on all surfaces), the deformation is much larger on SiO2 than on the surfaces where bilayer formation does not occur. This is attributed to stronger vesicle-surface interaction on SiO2. The bilayer formation is thermally activated with an apparent activation energy of 63-78 kJ/mol. Osmotic pressure promotes bilayer formation, especially when the external salt concentration is higher than the internal one. Depending on preparation conditions, a varying amount of nonruptured vesicles are trapped in the saturated bilayer on SiO2, but the fraction can be efficiently reduced to below the detection level using elevated temperature and/or high osmotic stress.

Introduction The cell membrane (biomembrane) is one of the most important constituents in living organisms. Via specialized biomolecules, incorporated in the phospholipid bilayer that forms the membrane, it mediates all communication between the intracellular and extracellular spaces, and thus also cell-cell communication (Figure 1a). The interest in understanding the properties of the cell membrane has spurred intense research to build simplified yet representative experimental model systems1-3 with the aim to promote both fundamental understanding of membranerelated processes and utilization of them in synthetic systems. Nonruptured (intact) surface-bound unilamellar phospholipid vesicles and supported phospholipid bilayers (SPBs) are two simple model systems for biological membranes.1 Planar lipid bilayers were first reported by Mueller et al.4 and have since then received increasing scientific and practical attention. The early work focused primarily on preparation and fundamental properties of these model membranes. Today both surface-bound vesicles and SPBs are subject to research ranging from mechanistic studies of how SPBs are formed,1,5-10 to curiosity-driven attempts to understand fundamental * Corresponding author. E-mail: [email protected]. Fax: +46317723134. † Part of the Langmuir special issue entitled The Biomolecular Interface. (1) Sackmann, E. Science 1996, 271, 43-48. (2) Tampe´, R.; Dietrich, C.; Gritsch, S.; Elender, G.; Schmitt, L. Biofunctionalized membranes on solid surfaces. In Nanofabrication and Biosystems: Integrating Materials Science, Engineering, and Biology; Hoch, H. C., Jelinski, L. W., Craighead, H. G., Eds.; Cambridge University Press: Cambridge, U.K. 1996; pp 201-221. (3) Plant, A. L. Langmuir 1999, 15, 5128-5135. (4) Mueller, P.; Rudin, D. O.; Tien, H. T.; et al. Nature 1962, 194, 979-980. (5) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397-1402.

Figure 1. (a) Cell membrane. (b) Phospholipid vesicle (liposome). (c) Supported phospholipid membrane.

processes occurring in living organisms,8,11-17 and further to emerging applications in life sciences, such as biosensing,2,6,16,18-23 diagnostics, drug screening, biomimetic (6) Steinem, C.; Janshoff, A.; Ulrich, W.-P.; et al. Biochim. Biophys. Acta 1996, 1279, 169-180. (7) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806-1815. (8) Keller, C. A.; Glasma¨star, K.; Zhdanov, V. P.; et al. Phys. Rev. Lett. 2000, 84, 5443-5446. (9) Puu, G.; Gustafsson, I. Biochim. Biophys. Acta 1997, 1327, 149161. (10) Pignataro, B.; Steinem, C.; Galla, H.-J., et al. Biophys. J. 2000, 78, 487-498. (11) Burgess, J. D.; Rhoten, M. C.; Hawkridge, F. M. J. Am. Chem. Soc. 1998, 120, 4488-4491. (12) McConnell, H. M.; Watts, T. H.; Weis, R. M.; et al. Biochim. Biophys. Acta 1986, 864, 95-106. (13) Terrettaz, S.; Stora, T.; Duschl, C.; et al. Langmuir 1993, 9, 1361-1369. (14) Nollert, P.; Kiefer, H.; Ja¨hnig, F. Biophys. J. 1995, 69, 14471455. (15) Heyse, S.; Vogel, H.; Sa¨nger, M.; et al. Protein Sci. 1995, 4, 2532-2544. (16) Henrickson, S. E.; Misakian, M.; Robertson, B., et al. Phys. Rev. Lett. 2000, 85, 3057-3060. (17) Krysin˜ki, P.; Tien, H. T.; Ottova, A. Biotechnol. Prog. 1999, 15, 974-990. (18) Ziegler, C.; Go¨pel, W. Curr. Opin. Chem. Biol. 1998, 2, 585-591. (19) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773-14781.

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photosynthesis,19 and various medical applications such as nonfouling surfaces (see Andersson et al.24 and Glasma¨star et al.25 and references therein). The latter is primarily motivated by the biological inertness of pure SPBs,24,25 while the former originates from the fact that they at the same time can be functionalized with both water-soluble2 and transmembrane proteins.12,19,26,27 Obviously, these properties are also a useful platform for various bioanalytical sensor applications.1,18,20,28,29 Unilamellar phospholipid vesicles (liposomes)30,31 (Figure 1b) and supported phospholipid membranes (SPBs)6,16,32 (Figure 1c) were mentioned above as attractive and useful model systems. The former consist of a bilayer of amphiphilic phospholipid molecules in the shape of a spherical (in the bulk phase) shell, separating an “intracellular” liquid volume from the “extracellular” space, while the SPBs are planar, extended bilayers of the same composition as vesicles, but adsorbed on a suitable solid surface. In both systems, a shell of tightly bound water molecules (the “hydration shell”) exists around the lipid headgroups and on the surface (indicated schematically in Figure 1b,c). In early studies, SPBs were mostly formed by sequential Langmuir-Blodgett (LB) deposition of two lipid monolayers on a hydrophilic substrate.2,6,20,33 However, on suitably prepared surfaces, vesicles adsorbed from a solution undergo rupture and fusion to an extended, adsorbed, planar bilayer (Figure 1b f Figure 1c). Spontaneous self-assembly of supported continuous bilayers from vesicles in solution was pioneered by McConnell et al.12 and is today as common for creation of SPBs as LB deposition.2,5,12,34-36 The primary reason to choose the vesicle f SPB transformation route, rather than LB deposition, is its experimental simplicity and reproducibility, leading to an almost defect-free surface coverage of a fluid bilayer. In addition, this method is attractive for integration of functional components, for example, proteins19 or coupling sites,37 and the planar membranes are easily patterned (20) Stelzle, M.; Weissmu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (21) Tien, H. T.; Barish, R. H.; Gu, L. Q.; et al. Anal. Sci. 1998, 14, 3-18. (22) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; et al. Science 1997, 387, 580-583. (23) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. Rev. Phys. Chem. 2000, 51, 41-63. (24) Andersson, A.-S.; Glasma¨star, K.; Sutherland, D.; et al. J. Biomed. Mater. Res., submitted. (25) Glasma¨star, K.; Larsson, C.; Hoö¨k, F.; et al. J. Colloid Interface Sci. 2002, 246, 40-47. (26) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159-6163. (27) Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400-1414. (28) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; et al. Nature 1997, 387, 580-583. (29) Schmidt, C.; Mayer, M.; Vogel, H. Angew. Chem., Int. Ed. 2000, 39, 3137-3140. (30) Parmar, M. M.; Edwards, K.; Madden, T. D. Biochim. Biophys. Acta 1999, 1421, 77-90. (31) Curran, A. R.; Templer, R. H.; Booth, P. J. Biochemistry 1999, 38, 9328-9336. (32) Nielsen, L. K.; Vishnyakov, A.; Jørgensen, K.; et al. J. Phys.: Condens. Matter 2000, 12, A309-A314. (33) Lahiri, J.; Fate, G. D.; Ungashe, S. B.; et al. J. Am. Chem. Soc. 1996, 118, 2347-2358. (34) Groves, J. T.; Ulman, N.; Cremer, P. S.; et al. Langmuir 1998, 14, 3347-3350. (35) Mueller, H.; Butt, H.-J.; Bamberg, E. J. Phys. Chem. B 2000, 104, 4552-4559. (36) Hinterdorfer, P.; Baber, G.; Tamm, L. K. J. Biol. Chem. 1994, 269, 20360-20368. (37) Hoö¨k, F.; Ray, A.; Nordeń, B.; et al. Langmuir 2001, 17, 83058312.

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using different supports38 and/or stamping techniques.39 They are also compatible with a wide range of surfaceanalytical tools which have been extensively used to study both the kinetics of SPB formation and their physical properties. These tools include surface plasmon resonance,40 quartz crystal microbalance (QCM),5,41 impedance spectroscopy,6 Fourier transform infrared spectroscopy,42 scanning probe microscopy,7 and fluorescence recovery after photobleaching.14,19,43,44 Theoretical and experimental efforts are now accelerating to understand and control the formation kinetics and properties of the SPBs.1,2,5-10 Despite the accumulated efforts to understand the spontaneous SPB-formation process, several questions are still open. Among these are how the bilayer formation is influenced (i) by the surface properties, (ii) by the composition, size, and so forth and solution properties of the vesicles, and (iii) by temperature. In addition, neither the detailed kinetics of vesicle f SPB transformation nor the state of the SPB on different surfaces (e.g., fluidity/mobility) is understood in detail. For example, it is not clear why the vesicle to bilayer transformation for egg-yolk phosphatidylcholine vesicles is so far limited to a small set of hydrophilic surfaces (mainly SiO2 and mica)5,7,10,14,19,34,36,38,39,45 and why seemingly similar surfaces (e.g., TiO2) only adsorb intact vesicles.5,14,34 In the present work, we have extended our previous studies with the QCM-D technique by increasing the temperature range, by adding new surfaces, and by including osmotic pressure as a new variable. Membrane spreading on glass surfaces has previously been reported to possess a temperature dependence,46 and a very weak temperature effect on the electrical properties of hybrid bilayers has also been reported.47 We report a strong temperature dependence of the vesicle f SPB process. To the best of our knowledge, the present work is the first systematic study of the influence of osmotic stress on SPB formation, even if osmotic effects may very well have been observed previously. In the Discussion, the new results are combined with recent results from our group (regarding e.g. the size dependence of vesicle adsorption and SPB transformation46) and other groups in an attempt to give a coherent picture of our present understanding of vesicle adsorption and vesicle f SPB transformation. Experimental Section Quartz Crystal Microbalance-Dissipation (QCM-D) Experiments. The QCM-D Technique. Adsorption experiments were performed using the QCM-D technique.48-51 The experi(38) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651653. (39) Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 894-897. (40) Salamon, Z.; Huang, D.; Cramer, W. A.; et al. Biophys. J. 1998, 75, 1874-1885. (41) Tja¨rnhage, T.; Puu, G. Colloids Surf., B 1996, 8, 39-50. (42) Cheng, Y.; Boden, N.; Bushby, R. J.; et al. Langmuir 1998, 14, 839-844. (43) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 25542559. (44) Starr, T. E.; Thompson, N. L. Langmuir 2000, 16, 10301-10308. (45) Egawa, H.; Furusawa, K. Langmuir 1999, 15, 1660-1666. (46) Nissen, J.; Gritsch, S.; Wiegand, G.; et al. Eur. Phys. J. B 1999, 10, 335-344. (47) Lingler, S.; Rubinstein, I.; Knoll, W.; et al. Langmuir 1997, 13, 7085-7091. (48) Rodahl, M.; Hoö¨k, F.; Krozer, A.; et al. Rev. Sci. Instrum. 1995, 66, 3924-3930. (49) Rodahl, M.; Kasemo, B. Rev. Sci. Instrum. 1996, 67, 3238-3241. (50) Rodahl, M.; Hoö¨k, F.; Kasemo, B. Anal. Chem. 1996, 68, 22192227. (51) Hoö¨k, F.; Rodahl, M.; Brzezinski, P.; et al. Langmuir 1998, 14, 729-734.

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Figure 2. Schematics of one of the QCM-D measurement cells. The second one is similar in design. mental setup used for the measurements on vesicle size dependence is a home-built exchange cell (Figure 2), where the sensor crystal surface is parallel with the inlet and outlet of the cell so that the bulk liquid flows parallel with the surface. For all the other experiments, a commercially available prototype QCM-D exchange cell from Q-Sense AB (Go¨teborg, Sweden) was used, where the crystal is mounted horizontally and the inlet tube axis is orthogonal to the crystal surface. Accurate temperature control of the Q-Sense cell is achieved by using a Peltier element in the cell and by placing the cell in a home-built temperature-controlled chamber. Since the resonant frequency, f, of the QCM depends on the total oscillating mass, a mass adsorbed on the surface can be detected as a decrease in f. At ideal (see below) conditions, there is a linear relation between the change in frequency and the adsorbed mass:

C ∆m ) ∆f n

(1)

where C ()17.7 ng/(cm2 Hz) at f ) 5 MHz) is the mass-sensitivity constant and n ()1,3,...) is the overtone number. Combined frequency and energy dissipation measurements obtained at a repetition rate of about 0.5 Hz, as used in the present work, give information about both the amount of adsorbed mass (through f) and the viscoelastic properties (through D) of the adsorbed film. Data were collected using Q-Soft (Q-Sense AB). All data shown for the frequency shifts are normalized to the response of a 5 MHz crystal. If the adsorption-induced change in dissipation is large, it may indicate that the mass of the adlayer is not just a “dead” or rigid mass but a mass that deforms during the shear oscillatory motion, which then signals that the Sauerbrey relation might no longer be valid.52 In such situations, a Voight-Kelvin-based model is used to analyze the data.52,53 In the model, the adsorbed film is approximated by an adlayer of homogeneous thickness, viscosity, and complex shear modulus. In the present work, the Sauerbrey relation holds for the complete SPBs (close to zero D) of typical thickness 4-5 nm, while the mass estimated using eq 1 is underestimated for adsorbed, nonruptured vesicles (this will be shown in the Discussion). The effective thickness referred to in this article is the thickness obtained from this type of modeling using multiple overtones of the QCM-D sensor (n ) 3, 5, and 7). Measurement Procedure. Before each experiment, all crystals are UV/ozone treated two times for 10 min in a home-built UV/ ozone chamber. Between the two UV/ozone treatments and after the second treatment, the crystals are rinsed with plenty of (52) Hoö¨k, F.; Kasemo, B.; Nylander, T.; et al. Anal. Chem. 2001, 73, 5796-5804. (53) Voinova, M. V.; Rodahl, M.; Jonson, M.; et al. Phys. Scr. 1999, 59, 391-396.

Milli-Q water. The treatment yields an oxidized surface free of organic contaminants (the ozone effectively removes hydrocarbon and organic contaminants by oxidizing them to H2O and CO2) as verified by X-ray photoelectron spectroscopy (XPS).54 After each experiment, a used crystal is rinsed thoroughly with Milli-Q water and usually cleaned in a solution of detergents (sodium dodecyl sulfate (SDS) or ordinary machine dish detergent). In all measurements, the cell is initially filled with buffer and rinsed a couple of times until a stable baseline is established at the desired temperature. The temperature is set to 295 K for all measurements, except for the series of experiments on the temperature dependence, where the temperature is varied. When the baseline is stable, the buffer is exchanged for a solution containing vesicles and the adsorption is monitored as a function of time by recording the changes in f and D. Preparation of Surfaces. The QCM crystals were cleaned before the deposition of a new surface-coating film. The crystals were immersed in a 6:1:1 (v/v/v) solution of H2O/NH3 (25%)/H2O2 (30%) and heated to 70 °C for 10 min followed by thorough rinsing with water and drying in a stream of nitrogen gas. This process creates a surface clean from hydrocarbon contaminants. As quickly after cleaning as possible, the crystals were placed in a thin film deposition chamber (AVAC HVC-600 thin film deposition system) and the chamber was pumped down to high vacuum pressure. At ∼2 × 10-6 Torr, a thin film was deposited on the substrate by e-beam evaporation. The surfaces for adsorption of vesicles/SPBs used in this study, produced by evaporation, were Ti (spontaneously oxidized to TiOx upon air exposure), Pt, and SiO2, all of high purity (Unaxis, Switzerland). All deposited films were 100 nm in thickness with a 3 nm layer of titanium underneath for increased adhesion to the gold electrode (except for the titanium film, which is a continuously deposited 100 nm film of titanium). The Si3N4 surface was grown using molecular beam epitaxy in a deposition chamber after plasma cleaning on top of the QCM crystal. As gold surfaces, the original electrodes of the QCM crystals were used. The UV/ozone treatment used to clean all surfaces before a measurement also completes (saturates) the oxide films. The XPS signature of the deposited silicon oxide film becomes close to that of SiO2, and that of in-air spontaneously oxidized titanium becomes close to that of TiO2. The Pt and Si3N4 surfaces are also oxidized, and the Si3N4 actually seems to form a thin film of SiO2 on top of the nitride, according to XPS measurements. For Pt, which does not easily oxidize, the surface should probably be viewed as a Pt surface with a saturated chemisorption layer, or possibly as a two-dimensional oxide film of monolayer thickness. We call this surface “oxidized” Pt. All surfaces show radically increased wettability after ozone treatment. Materials. QCM-D Sensors. All water used in the experiments was ultrapure Milli-Q water (Millipore, Sweden) with a resistivity of 18.2 MΩ/m. It was used for all cleaning and for making buffers. Several kinds of AT-cut QCM-D crystals were used. (AT-cut crystals oscillate in the thickness shear mode; i.e., the oscillatory motion is parallel to the sensor surface with an amplitude at the solid-liquid interface well below 10 nm.) (The evanescent shear acoustic wave emitted into the liquid is damped with an extinction length of ca. 200 nm.) For the measurements of the size dependence of vesicle adsorption, crystals with a fundamental resonance frequency of 3 MHz were specially ordered from ValpeyFisher (MA) for the measurements on SiO2 surfaces, while 5 MHz crystals from Maxtek (CA) were used for the studies on all other surfaces. For the studies of temperature dependence and osmotic stress, crystals from Q-Sense AB, with a fundamental resonance frequency of 5 MHz, were used. Lipids, Buffers, and Other Chemicals. Egg-yolk phosphatidylcholine (eggPC) from Sigma-Aldrich (Sweden) was used as the lipid mixture in all experiments (melting temperature, 258 K), except for the cycled temperature measurements where POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Avanti Polar Lipids, USA) was used (the melting temperature for this mixture is lower than for eggPC). Tris[hydroxymethyl]aminomethane (Tris) and sodium chloride for making buffers were purchased (54) Krozer, A.; Rodahl, M. J. Vac. Sci. Technol., A 1997, 15, 17041709.

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Table 1. Mean Size of Distributions for SUVs and EUVs Measured by PCS pore diameter (nm)

mean (PCS) diameter (Ønom) (nm)

25 30 50 100 200

25 35 70 110 200

from Sigma-Aldrich. All buffers, except the ones used for the osmotic stress experiments, were made with Milli-Q water, 10 mM Tris, and 100 mM NaCl. The pH was set with hydrogen chloride (Merck, Sweden). The pH of all buffers was 8.0. For the experiments conducted on the influence of osmotic stress, the vesicles were made using a buffer with 150 mM NaCl. To change the osmotic stress, buffers ranging in NaCl concentration from 115 to 300 mM NaCl were used. Other chemicals used in the experiments (mostly for cleaning) were chloroform, hydrogen peroxide (30% in water, Merck), ammonia (25% in water, Merck), Hellmanex (Hellma Worldwide, U.K.), ethanol (95.5%, Kemetyl, Sweden), SDS (Aldrich, Sweden), and nitrogen gas (N48, Air Liquide, Sweden). Preparation of Vesicles. Unilamellar vesicles were prepared according to two different protocols. Small sonicated unilamellar vesicles (SUVs) with a narrow size distribution were prepared according to the protocol by Barenholz et al.55 Extruded unilamellar vesicles (EUVs) were prepared with a range of sizes. The procedure is the same regardless of size. For each vesicle size, a solution of ∼13 mg/mL lipids was extruded 31 times through two polycarbonate membranes of the desired nominal size, placed in series. The nominal pore sizes were 30, 50, 100, and 200 nm (Avestin, Canada; Structure Probe, USA). The extrusion was performed using a LiposoFast extruder from Avestin and a home-built setup to achieve constant pressure during the extrusion. The extrusion yields vesicles of approximately the nominal size in a narrow size distribution.56,57 Determination of Size Distributions. Actual size distributions were determined for the measurements on vesicle size dependence using photon correlation spectroscopy (PCS) on a home-built system. All sizes in the text, data, figures, and calculations for the investigations of vesicle size dependence refer to the mean size from the PCS measurements. SUVs have a very narrow size distribution with a mean diameter of ∼25 nm. Wider but still very sharp size distributions were found for the EUVs. The results are found in Table 1.

Figure 3. Adsorption kinetics of lipid vesicles are displayed as ∆f(t) and ∆D(t) for vesicles extruded through 30 nm pores. Adsorption curves are presented for (a) Si3N4, (b) oxidized Pt, (c) TiO2, and (d) SiO2.

Dependence of Vesicle Adsorption and SPB Formation on Surface Chemistry. The understanding is incomplete regarding how vesicle adsorption and SPB formation depend on surface chemistry. On hydrophobic surfaces, a single monolayer of lipids tends to form,1,5,47 which is understandable on thermodynamic grounds. On hydrophilic surfaces, both bilayer formation (e.g., on SiO2 and mica)5,7,38 and intact vesicle adsorption5,38 have been observed, and it is obvious from the measured data that kinetic factors are important and superimposed on the thermodynamic driving forces. To collect more data with the aim of obtaining a better understanding of the role of surface chemistry and the nature of vesicle-surface interactions, Pt and Si3N4 surfaces were added to the previously investigated58 surfaces SiO2 and TiO2. Figure 3 shows typical temporal variations of ∆f and ∆D upon exposure of (a) oxidized Si3N4 and (b) oxidized Pt surfaces to solutions of vesicles extruded through 30

nm filters (25 nm SUVs for Si3N4). For comparison, the same type of experiments on (c) TiO258 and (d) SiO258 are also shown. The observed kinetics (diffusion limited almost up to saturation)8,59 is qualitatively different on SiO2 and Si3N4 compared to TiO2 and Pt. On the former, f initially decreases (increasing mass load) and D initially increases (increasing dissipative losses) and then f eventually starts to increase and D starts to decrease. Thus, a minimum, ∆fmin, develops in f (maximum in coupled mass) along with a corresponding maximum in D, ∆Dmax (we use ∆ to indicate changes from the initial value at t ) 0). This behavior is different from that of the TiO2 and Pt surfaces where the signals grow monotonically. The Si3N4 result is similar to that reported previously for SiO2,5,8,58 while the Pt result is similar to that previously reported for oxidized gold5 and TiO2.58 These two behaviors correspond to the following kinetics: (i) On TiO2 and oxidized Pt, nonruptured vesicles adsorb over the whole coverage range up to saturated coverage (t ≈ t3 for 30 nm EUVs in Figure 3c). (ii) On SiO2 and Si3N4, vesicles initially adsorb without rupturing (for t < t1 for 30 nm EUVs in Figure 3d), but at a critical surface coverage, Θc, rupture is initiated and a supported bilayer is formed (t1 < t < t2).5,8 The bilayer is completed, stable, and saturated at t > t2; that is, there is no further mass uptake or release. Few or no (see below) intact vesicles are incorporated in the adlayer. (No significant desorption was observed upon rinsing.) The change in sign of df/dt at t1 on SiO2 and Si3N4 originates from the fact that the mass uptake at this point is dominated by rupture of vesicles causing release of trapped water. The trapped water inside and between vesicles is detected as a mass gain by the QCM-D during adsorption of intact vesicles and as a mass loss when the

(55) Barenholz, Y.; Gibbes, D.; Litman, B. J.; et al. Biochemistry 1977, 16, 2806-2810. (56) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168. (57) MacDonald, R. C.; MacDonald, R. I.; Menco, B. P. M.; et al. Biochim. Biophys. Acta 1991, 1061, 297-303. (58) Reimhult, E.; Hoö¨k, F.; Kasemo, B. J. Chem. Phys. 2002, 117, 7401-7404.

(59) In the present case, the kinetics of adsorption, i.e., the supply to the surface of lipid mass in the form of vesicles, is diffusion limited for most of the time. Diffusion-limited adsorption often prevents the true adsorption kinetics from being observed, unless the transport to the surface is reliably modeled and corrected for. However, in the present case the kinetics on the surface, e.g., vesicle to SPB transformation, which is of prime interest here, can still be followed thanks to the information contained in f and D.

Results


Figure 4. Asymptotic frequency shifts for saturated adsorption of intact vesicles on TiO2 as a function of mean bulk vesicle diameter (filled circles). The “hard sphere” line shows the expected frequency shifts according to the Sauerbrey relation, assuming that the vesicles adsorb as rigid spheres with the density of water (this is obviously not the case (see the main text) but is valuable as a reference). The “corrected” line shows ∆f after the data have been corrected for viscoelastic losses, as described in the text.

vesicles rupture, release water, and form a SPB. After the minimum in f, the rate of mass loss due to release of trapped water exceeds the rate of mass addition due to continuing vesicle adsorption, causing an overall negative rate of mass change. The change in sign of dD/dt at approximately the same time as that in df/dt reflects the transition from an adlayer composed of soft dissipative vesicles (causing high ∆D) to the much more rigid and less dissipative flat bilayer.5,8 On TiO2 and Pt, there is obviously no spontaneous bilayer formation, since both |∆f| and ∆D increase monotonically. Note that the asymptotic ∆f and ∆D values at saturation on TiO2 and Pt are much larger than the largest ∆f and ∆D values at their minimum/maximum during adsorption on SiO2 and Si3N4. Even if a surface-induced deformation of adsorbed vesicles is occurring on SiO2 (and Si3N4), which would in turn reduce the magnitude of |∆f| and ∆D, this observation suggests that the vesicle f SPB transformation on the latter surfaces starts well before the surface is saturated with vesicles. Dependence on Vesicle Size. From the results in the previous paragraph, it is clear that the vesicle to SPB transformation kinetics is influenced by the interaction strength with the surface (since e.g. SiO2 and TiO2 give different results) and also by vesicle-vesicle interaction on the surface (since a critical coverage is required for the SPB formation to begin). For both these interactions, the actual outcome (intact vesicles or SPB) will depend on the elastic properties and cohesive strength (stability) of the vesicles themselves. One available control parameter of obvious interest in this context is the vesicle radius (size) which has been investigated previously by us for SiO2 and TiO2.58 These experiments have been repeated here on the oxidized Pt surface, demonstrating a behavior very similar to that of TiO2 (not shown). In brief, these investigations demonstrate that the qualitative adsorption kinetics, including the vesicle rupture and bilayer formation kinetics on SiO2, is independent of vesicle size in this size regime. However, there are interesting quantitative size-dependent effects. One of the most important is that the deformation of adsorbed vesicles appears to increase with increasing vesicle size.58 A summary of these data is shown in Figure 4 and Figure 5, displaying a linear increase of |∆f∞| for TiO2 (Figure 4) and |∆fmin| for SiO2 (Figure 5) with increasing mean bulk vesicle diameter, Ønom. The observed linear increase in mass uptake (∆f) can be understood from the fact that the mass (proportional to the volume) per (nondeformed) vesicle increases as r3. Furthermore,

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Figure 5. Maximum frequency and dissipation shifts (at t ) t1) as a function of the vesicle size on SiO2. The maximum values occur in the regime where intact vesicles start to transform to a supported bilayer.

since the frequency shift of the QCM-D is proportional to the mass uptake per unit surface area, which scales as r2, ∆f is expected to be proportional to r (or Ønom). This qualitatively explains the linear relation between |∆f∞| for TiO2 and |∆fmin| for SiO2 with increasing vesicle diameter. It is noticeable that the almost straight lines describing |∆f| and ∆D versus Ønom do not pass through the origin when they are extrapolated to zero vesicle size in Figure 4 and Figure 5. The deviation from the origin is much larger for ∆f than for ∆D, possibly indicating that there is a component of the adsorbed mass that does not contribute to ∆D in the low-coverage limit. One possible, but very speculative, explanation might be a constant contribution of mass per area from the water shell surrounding the vesicles. The lipid headgroups coordinate a relatively stiff shell of water molecules.60 This shell has a constant thickness essentially independent of vesicle size, and its mass contribution will scale approximately with the vesicle area, that is, as r2, yielding an expected constant contribution of the water shell with respect to Ønom. There are, however, also several other possible factors that could cause the nonorigin intercept of the |∆f| and ∆D versus Ønom plots. For example, the QCM-D might sense the water trapped between the parts of the vesicle membranes in contact with the surface as a rigid mass contribution even at large vesicle separations. This contribution would be the same regardless of vesicle size. Also, if the decreased height of the vesicles due to their deformation on the surface is taken into account the deviation from the origin will be substantially decreased. We believe that the latter correction and the contribution of coupled water explain most of the deviation. Dependence on Temperature. The data for SiO2 above show that a critical coverage is needed to induce vesicle rupture and SPB formation on this surface, which in turn suggests an activated process where vesiclevesicle interaction reduces the activation energy. Thus, the influence of temperature is of interest. Figure 6 shows one representative set of QCM-D measurements of vesicle adsorption and bilayer formation on SiO2 as a function of temperature. The adsorption kinetics (previously published by Reimhult et al.)61 is displayed as ∆f and ∆D versus time62 at five different temperatures, for EUVs approximately 40 nm in diam(60) Gennis, R. B. Biomembranes: Molecular structure and function; Springer-Verlag: New York, 1989. (61) Reimhult, E.; Hoö¨k, F.; Kasemo, B. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 2002, 66, 051905. (62) The time scale is renormalized to take into account that the supply of vesicles to the surface is diffusion limited (ref 8). Therefore the real (clock) time is multiplied by T1/2, to account for the T-dependent diffusion rate. This correction has only a minor influence on the results discussed below. At t ) 40 (n.u.) the clock time is 144 and 138 s at 278 and 303 K, respectively.

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Figure 6. ∆f(t) (left) and ∆D(t) (right) for vesicle adsorption on SiO2 at five different temperatures. The time axis has been normalized to remove the effect of temperature on the bulk diffusion rate (ref 62). Note that decreasing temperature delays the transition from intact vesicles to bilayer to larger times/ higher coverages. These data are further analyzed in Figure 13.

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Figure 8. ∆f(t) (left axis) and ∆D(t) (right axis) for vesicle adsorption on Au at six different bulk concentrations of NaCl. The initial salt concentration inside the vesicles was 150 mM in all experiments.

eter.63 With increasing temperature, the minimum in ∆f and maximum in ∆D are shifted to shorter exposure times with smaller amplitude. Since the initial vesicle adsorption is mass transport limited, this means that a lower surface coverage of intact vesicles is required to induce rupture and bilayer formation at higher temperatures. Since a higher coverage of vesicles is required to induce rupture and fusion at lower temperatures, these data clearly demonstrate the importance of lateral vesicle-vesicle interactions. Note in Figure 6 that a slightly different kinetics is observed at 303 K, with a reproducible “spike”, not caused by the fluid exchange, and with a higher maximum/minimum in ∆D/∆f than at 295 K. Unfortunately, limitations of the experimental setup prevented reproducible experiments at even higher temperatures than 303 K. At the lowest temperature, the data show that the system is near the point where vesicles do not spontaneously form a SPB at all. Therefore, the behavior of the system was investigated at the lowest possible temperature, which for the present system is 273 K (the freezing point of the buffer is lower than 273 K). The result is shown in Figure 7, with the adsorption starting at t1 ) 0 min. Interestingly, at this temperature the adsorption kinetics displays a behavior similar to that on TiO2 and Pt (cf. Figure 3); that is, the spontaneous bilayer formation process is prevented. After saturated adsorption of intact vesicles at 273 K, the bulk solution was rinsed with pure buffer (t ) 27 min) which was followed by an increase in solution temperature to 283 K (t2 ) 30 min). The temperature-induced change in density and viscosity of

the bulk solution64 results in large changes in ∆f and ∆D, preventing continuous recording of the process during heating/cooling. Therefore, to make a comparison possible between the state of the adlayer before and after heating, the temperature was subsequently decreased to 273 K again (reached at t3 ) 80 min, Figure 7). The shifts in frequency and dissipation with regard to the baseline before heating are ∆f ) -41 Hz and ∆D ) 2.5 × 10-6, that is, a substantial increase in ∆f (mass loss) and decrease in ∆D (increased stiffness) compared to data before cycling the temperature. This indicates that a substantial part of the vesicle layer ruptured and formed a SPB during the heating cycle. However, a comparison with the expected change for a pure bilayer (∆f ∼ -26 Hz and ∆D ∼ 0.2 × 10-6) shows that the conversion during the heating cycle is only partial, thus pointing toward the importance of having vesicles present in the bulk for the rupture process to be efficient. Interestingly, subsequent addition of vesicles after the heating-cooling cycle (t ) 93 min, Figure 7) caused no detectable adsorption or other change of the adsorbed film. Thus, the lipid film existing at 273 K after the heating-cooling cycle is saturated. We take this as evidence that the mass loss during the heating cycle is not caused by vesicle desorption but rather is induced by release of coupled water during vesicle rupture. These results also demonstrate that addition of vesicles to the bulk (at 273 K) does not induce rupture of the remaining intact vesicles on the surface, since a net mass loss due to release of coupled water would then have been observed. The state of the surface after the heating-cooling cycle is thus most likely a mixture of SPB and intact vesicles. Dependence on Osmotic Stress. The above results clearly show that the vesicle to bilayer transformation is kinetically hindered and thermally activated. Further information can be obtained by using other available variables that can influence the stability of the system of intact vesicles on the surface and induce bilayer formation. One such available variable is the osmotic stress. Figure 8 shows results from exposing an oxidized gold surface to vesicles prepared in 150 mM NaCl at different (115-300 mM) concentrations of NaCl in the bulk. (Oxidized gold behaves similarly to TiO2 and adsorbs only, or at least primarily, intact vesicles.) The gradient in salt concentration across the vesicle membrane produces an osmotic stress and probably a small change in the volume of the vesicle, known to be stable for significantly longer than the time scale of our experiment.65 As shown in Figure 8, there is no major qualitative difference in the results at the different osmotic stresses, and the adsorption kinetics is consistent with the previously demonstrated intact vesicle adsorption on gold surfaces.5 The osmotic

(63) Qualitatively similar results were obtained for 25 nm SUVs (not shown), except that the curve shapes were somewhat different. The width and depth of the minimum in f/maximum in D generally depend on size, vesicle composition, and vesicle manufacturing method.

(64) Both f and D scale with the square root of density times viscosity when exposed to a Newtonian liquid. (65) Hauser, H.; Oldani, D.; Philips, M. C. Biochemistry 1973, 12, 4507-4517.

Figure 7. Directly recorded ∆f(t) and ∆D(t) curves for adsorption of POPC vesicles at 273 K. After completed adsorption, the temperature is increased to 283 K and then decreased to 273 K again, yielding huge changes in ∆f and ∆D. Comparison of the ∆f and ∆D values before and after the temperature change reveals a thermally induced incomplete bilayer formation.


Figure 9. ∆f(t) (left) and ∆D(t) (right) for vesicle adsorption on SiO2 at five different bulk concentrations of NaCl. The initial salt concentration inside the vesicles was 150 mM in all experiments.

Figure 10. Asymptotic frequency shifts, |∆f∞|, and dissipation shifts, ∆D, after completed bilayer formation on SiO2, indicating a small amount of trapped, intact vesicles. Note the magnified vertical scale. The total variation in ∆f is only about 10%.

stress thus did not lower the barrier for vesicle rupture enough to cause bilayer formation on oxidized Au. Figure 9 shows the same experiment as in Figure 8, but performed on a SiO2 surface. At all bulk salt concentrations, vesicle rupture and bilayer formation are observed. The bilayer formation kinetics is qualitatively similar at the different osmotic stresses. However, the exposure time and magnitudes of the ∆f and ∆D peaks decrease when the vesicles are osmotically stressed. The reduction in exposure time (coverage) before SPB formation occurs is largest for the highest salt concentration outside the vesicle, that is, when the vesicle volume is expected to shrink due to osmotic stress. There is also a small tendency of a simultaneous decrease in |∆f∞| and ∆D∞; that is, the asymptotic values of ∆f and ∆D after bilayer formation approach the theoretically predicted values when the salt concentration is increased. Discussion We now discuss the results from the four subsets of experiments presented above and combine them with previous data in order to describe our current mechanistic picture of vesicle adsorption and vesicle f SPB transformation. We first address the surface-induced conformation/deformation of intact vesicles that can be deduced from the adsorption and/or rupture behavior versus vesicle size on TiO2 and SiO2. We then complement the picture using the new results from the temperature and osmotic stress dependence. We also discuss the influence of surface chemistry and to what extent the formed bilayers are complete, that is, free from defects. Vesicle Deformation and Effective Adlayer Thickness: Vesicle to SPB Transformation Kinetics. Using a viscoelastic model for the measured QCM data, we were previously able to give an approximate quantitative measure of the magnitude of deformation of intact vesicles adsorbed on TiO2 (Figure 10).58 In brief, for Ønom ) 35 nm we obtained an effective vesicle height of Øeff ) 29 nm (i.e., ∼20% flattening) and for Ønom ) 200 nm we get Øeff ) 100 nm (i.e., ∼50% flattening), which means that there is, within this model, which excludes the possible effect of a hydration shell around the vesicles (see above), a

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Figure 11. It is useful for analysis to plot the ∆f(t) and ∆D(t) data of the type shown in Figure 4 (recorded and displayed versus time) in so-called D-f plots, where time is eliminated as an explicit variable. In this plot, ∆D is displayed versus ∆f for the adsorption process on (solid line) SiO2 and (dashed line) TiO2 for 110 nm EUVs.

successively increasing deformation with vesicle size.58 The degree of deformation/flattening on a particular surface results from the competition between vesicle attraction to the surface (promoting deformation) and the increase in elastic energy of the vesicle upon deformation, creating regions with a small radius of curvature (counteracting deformation). Figure 11 nicely illustrates the key results from comparing the bilayer-forming surface (SiO2) with the nonruptured vesicle adsorbing surface (TiO2).58 In Figure 11, the change in dissipation, ∆D, is plotted versus the corresponding change in frequency, ∆f, which removes time as an explicit parameter. This, in turn, makes it possible to compare the relation between changes in rigidity and added/lost mass for different systems. Comparing the monotonically increasing TiO2 curve with the SiO2 curve, showing a cusp, and noting their different slopes, it was previously concluded58 (i) that there is a phase transformation from intact vesicles to a bilayer on SiO2 but not on TiO2 and (ii) that the rigidity and deformation of the initially adsorbed intact vesicles (at low coverage) is considerably larger on SiO2 than on TiO2. This implies that vesicle-surface interaction is important for the transition and that this interaction is stronger on SiO2. The fact that a critical coverage is required for vesicle rupture even on SiO2 signals that vesicle-vesicle interaction is also important. Since the transition does not occur on Pt or TiO2 under otherwise identical conditions, we conclude that the transition is caused by a combination of vesicle-surface and vesicle-vesicle interaction, the former being stronger on SiO2. The vesicle-vesicle interaction increases when the population of vesicles grows on the surface, leading to rupture at a critical surface coverage, probably promoted by vesicles approaching a densely packed monolayer of vesicles on the surface. At this point, it is appropriate to compare the present results with one important result by Reviakine and Brisson found on mica. The latter can be summarized in the following way: the bilayer formation on the mica surface proceeds by smaller vesicles first fusing to larger ones before they rupture and form a SPB.7 If this were the dominant route also in our case on SiO2, there should be a more pronounced difference than we observe in the kinetics for vesicles of different diameters; the larger vesicles would then not show a pronounced maximum in D and a minimum in f since they would rupture immediately at low coverage. (The latter is, for example, the case when spontaneous rupture is stimulated using for example charged lipids and Ca2+.) Whether this difference between the two studies is due to the different chemical properties of SiO2 and mica and/or if it originates from other factors, for example, the difference in surface roughness, is still an open question.

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Figure 12. (a) A plot of the time, tc, to reach the minimum in ∆f (cf. Figure 6) versus temperature for 40 nm EUVs. (b) Arrhenius plot of ln(1/tc) vs T-1. The slope yields an estimate of the activation energy, Ea, for the onset of bilayer formation on SiO2 of 63-78 kJ/mol.

Temperature Dependence. The results from the experiments at different temperatures demonstrate a thermally activated phase transition from surface-bound vesicles to a SPB on SiO2. Because the vesicle-rupture process is thermally activated on this surface and since vesicle-vesicle interaction at higher coverage promotes rupture (see above and refs 8, 58, and 61), the required critical surface coverage for the phase transition becomes higher and takes a longer time to reach the lower the temperature (Figure 6). The time, tc ≈ t1, to reach the critical coverage is displayed as a function of temperature in Figure 12a. Extrapolation by the eye suggests that spontaneous rupture in the zero-coverage limit might occur at 310320 K. (However, a least-squares fit with a second-order polynomial places this point at considerably higher temperatures.) The same experiment performed with pure POPC vesicles at 273 K (Figure 7) reveals that the temperature limit where intact vesicle adsorption occurs on SiO2 at all coverages up to saturation (i.e., the behavior seen on TiO2) lies around 273 K. All higher temperatures show bilayer formation. Since SPB formation on SiO2 is thermally activated, we might expect that sufficiently high temperature would cause bilayer formation also on TiO2 or oxidized gold. However, when vesicle adsorption was made on Au at 303 K no bilayer formation was observed. We have previously attempted an Arrhenius type analysis of the data points in Figure 12a to obtain an estimate of the (apparent) activation energy, Ea, for the coverage-dependent vesicle rupture61 using a first-order rate equation. The actual order of the kinetics is not readily obtained from the data, and we assumed the simplest case of one rate-limiting step, yielding a first-order rate equation (thus neglecting vesicle-vesicle interaction), assuming (i) mass-transport-limited adsorption of vesicles (demonstrated by SPR)8 and (ii) a rate-limiting step of rupture of adsorbed vesicles to form bilayer patches. This analysis gave a value for Ea in the range 0.65-0.78 eV/ molecule (63-78 kJ/mol), with a slight difference between EUVs and SUVs (the apparent activation energy for the latter is a little higher). An important remaining question is the physics behind the critical coverage. Several possible mechanisms can be identified. A common denominator in a mechanistic model has to be a combination of vesicle-surface and vesiclevesicle interactions. This can take place in a mean field type model of uniform coverage (requiring mobile vesicles on the surface), or through local clustering of mobile vesicles due to attractive vesicle-vesicle interaction, or by vesicle clustering due to the statistical nature of the

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adsorption process. We are currently working with Monte Carlo simulations, and with additional experiments to obtain microscopic data as input into the computer models, to improve the mechanistic understanding. Influence of Osmotic Pressure. The very similar frequency and dissipation shifts at different osmotic stresses, shown in Figure 8 for vesicle adsorption on gold, indicate that the vesicles have fairly similar deformation after adsorption, regardless of osmotic stress. Due to the osmotic stress, the vesicles are expected to have different sizes and rigidities in the bulk solution. Equilibration of the osmotic stress already in the bulk is not likely, since the membrane efficiency as a barrier to diffusion of Na+ and Cl- (the permeability coefficients are ∼10-14 for Na+ and ∼10-11 for Cl-)65 suggests that relaxation of the osmotic gradient would take at least an order of magnitude longer than the time scale of the experiment. The fact that there is no or little difference in deformation at different osmotic stresses (on gold) might mean that the vesicles are very leaky (exchange of ions with the bulk) and equilibrate fast on the time scale of the experiment, once they have attached and been deformed by the surface. Alternatively, the resulting changes in ∆f and ∆D for the actual volume changes caused by osmotic stress are within the error margins of the experiment. (The latter is less likely in view of the results by for example Fu et al.,66 demonstrating a substantial volume change of vesicles under similar stress.) The most prominent effect of osmotic stress is its effect on the vesicle to bilayer transformation kinetics on SiO2 shown in Figure 9. The time to reach the critical surface coverage for rupture is significantly shorter at some values of the osmotic stress. All deviations from the equilibrium (i.e., when there is no osmotic stress) tend to decrease the critical coverage. Thus, the barrier to rupture is clearly lowered by osmotic stress. The possible occurrence of vesicle rupture far below the “normal” critical coverage, when there is osmotic stress, was investigated by interrupting the adsorption below the critical coverage. No evidence of very early spontaneous rupture was observed. The critical coverage is reduced the most when the osmotic stress is created by a higher NaCl concentration outside the vesicle. This salt concentration gradient should decrease the vesicle volume, which would in turn lead to a lower mass load per added vesicle. Using a given bulk concentration of vesicles, the surface coverage can be estimated in two ways: (i) by the exposure time (as in the temperature-dependent measurements above) or (ii) by the mass load (shifts in ∆f and ∆D). For the same exposure time (a measure that is unaffected by a change in NaCl concentration), a high bulk NaCl concentration yields lower shifts in ∆f and ∆D, compared to experiments without an osmotic gradient. This indicates experimentally a lower mass load, that is, smaller or more compressed vesicles, when there is a high salt concentration outside the vesicles, which is also supported by Figure 13, displaying ∆D as a function of ∆f (added mass) for various osmotic stress cases. Adsorption of vesicles with the highest salt concentration outside the vesicles results in a lower dissipation per added mass compared to vesicles which have the equilibrium volume or higher. This may be interpreted as a higher spreading of the membrane of vesicles that due to osmotic stress have a tendency to reduce their size; flatter, more deformed vesicles would show lower dissipation per unit added mass. A lower mass load due to the volume is less likely in view of the results on gold. (66) Fu, D.; Libson, A.; Miercke, L. J. W.; et al. Science 2000, 290, 481-486.


Figure 13. D-f plot for the adsorption process on SiO2 at different osmotic stresses (same data as in Figure 9).

The strongly facilitated rupture for osmotically compressed vesicles compared to osmotically expanded vesicles under equal stress indicates that compression enhances the rupture step more than is predicted by a mere increase in stress. The latter can be obtained also by a decrease in the external salt concentration, which, however, has less influence on the rupture kinetics. The difference between high and low bulk salt concentrations (outside the vesicles) is that a vesicle at higher bulk concentration can equilibrate with its surroundings by decreasing its volume on the surface (i.e., without steric hindrance), which facilitates spreading of the membrane, while a vesicle at low bulk NaCl concentration needs to expand to equilibrate, which does not facilitate spreading. In principle, the change in salt concentration could also have an effect on the surface interaction. However, the strongest promoting effect (on SPB formation) was observed for the highest salt concentration, which is expected to increase screening of the electrostatic surface interactions, thus impairing rupture. The results and discussion above are consistent with a mechanistic picture, where vesicle rupture occurs at the point of highest deformation of the vesicle membrane (smallest radius of curvature). The latter is most likely the region where the membrane curves away from the surface. More spreading of the vesicle membrane increases the vesicle-surface interaction and induces a stronger curvature at this point, due to mass conservation and geometric constraints. How perfect is the planar bilayer? In Figure 10, a weak increase is observed of the asymptotic values of |∆f| and ∆D with increasing vesicle size on SiO2. |∆f∞| and ∆D∞ are in these data always somewhat larger than the theoretically expected values for a bilayer. In the temperature-dependent data of Figure 6, a similar increase in |∆f∞| and ∆D∞ is observed as the temperature is decreased. At 303 K, the value of ∆f∞ is -25 Hz, exactly corresponding to the theoretically expected frequency shift of a coherent, fully covering bilayer, while at 278 K the value is -29 Hz, that is, >15% higher mass load. We also observe that high salt concentration outside the vesicles leads to saturation values closer to the theoretically expected result. These variations in the asymptotic mass load per unit area are attributed to a fraction of trapped, nonruptured vesicles that cannot fuse into the bilayer (probably due to steric hindrance).67 The exact amount of such trapped vesicles depends on the experimental conditions. From the size dependence of ∆f∞, we estimate that a fraction of ∼1% of the surface area is covered by trapped vesicles at 295 K (and the rest by a SPB). With the same estimate, (67) Note that the Sauerbrey relation is a very good approximation for the planar bilayer, since the bilayer is much thinner than the extinction depth of the probing evanescent acoustic wave in the liquid, and it is also sufficiently rigid to cause very low dissipation.

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the “extra” 4 Hz at 278 K corresponds to about 5% surface coverage of nonruptured vesicles. Thus, an increasing temperature increases the ideality of the SPB in the investigated T range. Nonruptured vesicles after bilayer formation have been observed directly with atomic force microscopy (AFM) by Reviakine and Brisson on mica, where they estimated that ∼2% of the area was covered with vesicles.7 Interestingly, SPB formation seems to include an important steric component in addition to the thermodynamic and kinetic effects illustrated and discussed above. Already in the osmotic stress experiments we observed that vesicles that could relax by shrinking (higher external salt concentration) were more easily transformed to a bilayer, compared to when the same salt gradient had the opposite direction. A steric effect is even more obvious in the experiment shown in Figure 7, where the surface was first saturated with vesicles at 273 K (where they do not rupture and form a bilayer). The saturation coverage of intact vesicles was then beyond the coverage required for complete bilayer formation at 283 K. (The surface was covered to at least 55% with intact vesicles, considerably more than needed to form a complete planar bilayer.) When the so-prepared system was heated to 283 K, some bilayer formation was observed (decrease in mass load) but only partially. A large fraction of intact vesicles remained on the surface, as deduced from the final high ∆f and ∆D values. The interpretation is that when the surface is oversaturated by vesicles (i.e., there is no lack, but rather a surplus, of lipid material for SPB formation), it leads to stabilization of the intact vesicle state, which sterically prevents transformation to a bilayer. (Desorption could remove this steric hindrance, but we have not seen any evidence of vesicle desorption in any of the experiments.) Consequently, the best way of forming SPBs seems to be through a continuous supply of vesicles that begin to transform to bilayers at coverages that are as low as possible, either by keeping the temperature sufficiently high or by destabilizing the vesicles by other means, such as osmotic pressure, chemical destabilization, or surface chemical modification. Current Picture of Vesicle Adsorption and Vesicle f SPB Formation and Open Questions. The mechanistic picture that we suggest to explain the whole set of data above is an extension of the one proposed by Keller et al.8 and Zhdanov et al.68 and also includes important elements proposed and discussed earlier by, for example, Seifert et al.69 For references, see the just-quoted references and references therein. Our results suggest that the larger vesicle deformation observed on SiO2 compared to TiO2 (and the other metal oxides), after the vesicles have adsorbed, causes a higher stress/strain in the individual vesicles on SiO2, which in turn makes them less stable and more apt to rupture and fusion to a SPB. In other words, one key component to explain the difference in the vesicle adsorption behavior on these two surfaces is a difference in the strength of vesicle-surface interaction, inducing rupture on SiO2 but not on TiO2. Another observation related to this point is obtained from the osmotic experiments, where osmotic stress was deduced to decrease the barrier to rupture by increasing membrane spreading and decreasing the radius of curvature at the point where the vesicle curves away from the surface. Even on SiO2, the deformation of vesicles due to surface interaction only is apparently not strong enough to cause (68) Zhdanov, V. P.; Keller, C. A.; Glasma¨star, K.; et al. J. Chem. Phys. 2000, 112, 900-909. (69) Seifert, U. Adv. Phys. 1997, 46, 13-137.

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spontaneous rupture of single vesicles at low coverage. An additional promoting factor is needed. This is obtained in our experiments by accumulation of more and more intact vesicles on the surface (driven by the attractive surface interaction). The local or global increase in coverage eventually leads to direct vesicle-vesicle interaction and therefore further deformation beyond the one in the dilute adsorbed limit. This additional deformation causes more strain and a lowering of the activation barrier for rupture and fusion. Since the latter process is thermally activated, as demonstrated by the temperature-dependent results, the critical coverage becomes lower, the higher the temperature (Figure 12). The rupture point in the vesicle is probably where the radius of curvature is smallest, as discussed by Seifert.69 After initial rupture and fusion, the continued vesicle f SPB transition is enhanced in an autocatalytic manner by vesicle-SPB interaction as discussed earlier.8 The rupture step can also be facilitated by inducing extra stress, for example, through osmotic stress, on the vesicle membrane. Further articulation of this mechanistic picture will be given in forthcoming mean field and Monte Carlo simulations of the present results.70 The correct physical interpretation of the critical coverage is yet uncertain, since it is not known if the vesicle-vesicle interaction and critical coverage are (i) caused by a global effect of squeezing a new vesicle into an existing adlayer of mobile vesicles, which adjusts collectively, or (ii) due to a local clustering of vesicles caused by their mutual van der Waals attraction, or (iii) caused by statistical local (adsorptiondriven) high coverages of immobile vesicles. Although distinctly different physically, all three mechanisms are at the present level of detail compatible with the data. One can also not rule out that defect sites (chemical or topographic) influence the kinetics. Microscopic information, for example, by AFM and high-resolution fluorescence microscopy data, will help to refine the model. An indication that local clustering might be important is the following: The critical surface coverage, Θc, is estimated to be ∼30% of the total surface area covered by deformed vesicles at 295 K.71 This seems to be a number that is too low to force direct physical contact between mobile and uniformly spread vesicles. Less mobile vesicles could solve this apparent dilemma, since they would locally form high coverages, even at relatively small average coverage, due to the statistical nature of the adsorption process. Another possibility, with mobile vesicles, is local clustering by for example attractive van der Waals interaction between vesicles on the surface. It would lead to increased lateral stress in the vesicles through their attractive interaction and clustering. The latter could in cooperation with the surface-induced stress promote rupture and fusion. The speculative nature of these last comments is emphasized and underlines the need for more experimental information at the microscopic scale (e.g., by AFM) and better theoretical understanding of how vesiclesurface and vesicle-vesicle interaction cooperate to induce rupture and fusion. An appropriate comment in this context is that mobile vesicles have so far been observed by neither AFM nor fluorescence microscopy.72,73 (70) Dimitrievski, K.; Reimhult, E.; Zhdanov, V. P.; et al. To be published. (71) Estimated by comparing the position of the critical coverage peak on SiO2 at 295 K with the time to the corresponding magnitude compared to the saturated vesicle coverage at 273 K. This might underestimate the coverage if the deformation of the vesicles is substantially lower at 273 K. (72) Johnson, J. Personal communication. (73) Reviakine, I. Personal communication.

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A few comments are appropriate about (the lack of understanding of) the nature of the various contributions to the vesicle-surface interaction. As proposed earlier,69 van der Waals interaction (vdW) is most likely a major component. vdW will cause the relatively large molecular weight vesicles to attract to the surface and deform. This attraction will in principle increase by an increase of the dynamic polarizability of the surface material. Why vesicles form supported bilayers on SiO2 but not on the other surfaces, which have higher polarizability (especially gold) and thus yield a stronger van der Waals interaction with the lipids, is still not clear. However, as discussed earlier in the context of protein adsorption74 an increase in surface polarizability may not have the effect just indicated; it also increases the binding strength and thickness of the hydration shell on the surface. (The presence of the hydration shell must be considered both for the surface and for the vesicles.) An increase of the water shell thickness due to increased surface polarizabilty could thus increase the vesicle-surface distance and reduce the vesicle-surface interaction. The thickness of the hydration shell is furthermore influenced by the hydroxylation of the surface (see below), which will be different for the different surfaces. It appears that silica, compared to titania, allows a thinner hydration shell and a stronger vesicle-surface interaction, as evidenced by the stronger deformation of vesicles on silica, which eventually leads to SPB formation on the latter surface. These questions become even more complex when the role of ions in the solution is considered. Surface topography could in principle also be an important factor, but since SPB formation takes place both on almost atomically flat mica and on very rough SiO2, surface topography is probably not the determining factor.75 More local interactions with the surface (than vdW) must also be considered. For example, the isoelectric points of TiO2 and SiO2 are ∼4-5 and 2, respectively, which will cause a significant presence of surface charges and hydroxyl groups on both surfaces, with a likely higher density on SiO2. This factor might be contributing to the SPB formation on SiO2, not seen on TiO2. Conclusions We found that vesicles adsorb irreversibly on all investigated surfaces: SiO2, Si3N4, TiO2, oxidized Pt, and oxidized Au. On the two former surfaces, the vesicles transform to a bilayer when a critical vesicle coverage is reached. On the remaining surfaces, vesicles stay intact but are deformed at all coverages. The qualitative features of the vesicle adsorption kinetics are not strongly dependent on vesicle size on SiO2 and TiO2. Larger vesicles are, however, more deformed by the surface interaction than smaller ones on both surfaces. The deformation on SiO2 is larger than on TiO2 for a given vesicle size, interpreted as a stronger attraction to the surface. This interpretation is consistent with the fact that SPBs are spontaneously formed on SiO2 but not on TiO2. We have also demonstrated that the vesicle to bilayer transition on SiO2 is thermally activated, with cooperating vesicle-surface and vesicle-vesicle interaction as central ingredients, manifested in a T-dependent critical coverage for the transition. The various contributions to the vesicle(74) Zhdanov, V. P.; Kasemo, B. Langmuir 2001, 17, 5407-5409. (75) However, the difference in topography might be the reason for the observed vesicle fusion and spontaneous rupture of vesicles on mica that do not occur on rough SiO2. It might also be a factor to consider reducing the number of nonruptured vesicles on the surface after SPB formation.


surface interaction are still quantitatively unknown and need theory input and further experiments for clarification. The bilayer formation can be completely prevented even on SiO2 by adsorption at a sufficiently low temperature. The SPB transition is facilitated by an outward osmotic gradient causing compressive stress on the vesicles. Analysis of the data for SiO2 at 293 K suggests that ∼1% of the surface remains covered by nonruptured vesicles, the rest being covered by bilayer. This number can be increased/decreased by lowering/increasing the temperature. From a practical viewpoint, we have learned

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that the highest temperatures and favorable osmotic stress produce the “best” SPBs in terms of residual nonruptured vesicles; there is, in contrast to the low-temperature data, no sign of trapped vesicles at 303 K. Acknowledgment. The authors thank V. P. Zhdanov for many valuable discussions. The Swedish Research Council (Dnr 621-2001-2649) and the Swedish Foundation for Strategic Research (program for Biocompatible Materials) are acknowledged for their financial support. LA0263920

Intact Vesicle Adsorption and Supported Biomembrane Formation

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