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Kinetics of the Unrolling of Small Unilamellar Phospholipid Vesicles onto Self-Assembled Monolayers Louise M. Williams, Stephen D. Evans,* Thomas M. Flynn, Andrew Marsh, Peter F. Knowles, Richard J. Bushby, and Neville Boden Centre for Self-Organising Molecular Systems, University of Leeds, Leeds, LS2 9JT, U.K. Received August 13, 1996. In Final Form: November 26, 1996X Self-assembled monolayers (SAMs) comprised of a mixture of a cholesterol functionalized thiol derivative and a short chain ethyleneoxythiol derivative have been used to attach phospholipid bilayers to gold surfaces. The cholesterol derivatives serve as “anchoring units” and are inserted into the lower leaflet of the attached bilayer. The short chain ethyleneoxy derivatives are present to promote a disordered hydrophilic region beneath the bilayer. The bilayers were formed by incubation of the SAMs with small unilamellar vesicles. On single component hydrophobic surfaces a single lipid layer was adsorbed, while on mixed SAMs containing the cholesterol anchoring units, and single component hydrophilic surfaces, a lipid bilayer was adsorbed. The kinetics of bilayer formation was followed using surface plasmon resonance spectroscopy, and showed dramatic differences depending on the SAM composition.
Introduction Interest in planar supported single phospholipid bilayers is 2-fold. Firstly, they provide simple models of biological membranes which can be studied using a wide variety of spectroscopic and microscopic techniques.1 Secondly, the incorporation of functionally active peptide, or protein, ion-channels into these single supported bilayers has potential for biosensor applications.2 The “gating” of such channels is often dependent on the presence of some external stimulus to the system. For example, there are many natural ion channels which react to the presence of specific molecules, thus triggering the opening of the channels and allowing a cascade of ions to flow through them. Thus, by incorporating such proteins into a single lipid bilayer, supported on the gate of an ion-sensitive field-effect transistor (ISFET), the presence of the stimulus molecule should be detectable to very low concentrations. There has been considerable interest in the manufacture of these planar bilayer systems via the exposure of suitable surfaces to small unilamellar vesicles (SUVs).1-11 Stelzle et al. have used charged self-assembled monolayer (SAM) surfaces, having a charge opposite to that of the phospholipid headgroups, to promote bilayer formation and adhesion.5 Spinke et al., on the other hand, have used a polymeric SAM with phospholipid analogues incorporated into side-chains so that they will insert into the bilayer, thus tethering it to the surface.10 Yet another approach has been that of Lang et al., who created a microstructured * Author to whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, February 1, 1997. (1) Sackmann, E. Science 1996, 271, 43-48. (2) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229-1237. (3) Kalb, E.; Frey,S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316. (4) Kalb, E.; Tamm, L. K. Thin Solid Films 1992, 210/211, 763-765. (5) Stelzle, M.; Weissmuller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (6) Brink, G.; Schmitt, R.; Tampe, R.; Sackmann, E. Biochim. Biophys. Acta 1994, 1196, 227-230. (7) Plant, A. L. Langmuir 1993, 9, 2764-2767. (8) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126-1133. (9) Ohlsson, P.-A.; Tjarnhage, T.; Herbai, E.; Lofas, S.; Puu, G. Bioelectrochem. Bioenerg. 1995, 38, 137-148. (10) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667-1671. (11) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210.
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surface of hydrophilic island “wells” within a thiolipid matrix, so that bilayers could be formed within the “wells”.2,11 Our approach has been to use mixed alkanethiol based self-assembled monolayers (SAMs).12,13 By using solutions containing two thiol derivatives, one can adsorb mixed monolayers; variation of the solution composition enables one to create SAMs having a range of relative surfactant concentrations.14-20 This approach gives a high degree of control over the composition of the supporting SAM. We have used a cholesterol derivative, which should insert into the bilayer, mixed with shorter “packing” molecules. Both components have ethyleneoxy chains close to the substrate, thus providing a disordered hydrophilic environment. This is desirable in order to preserve the bilayer in a biologically natural environment. To our knowledge this is the first report on the use of cholesterol functionalized SAMs, giving an added dimension for controlling bilayer fluidity, since cholesterol is known to increase the fluidity of gel phase bilayers or the rigidity of liquidcrystalline phase bilayers.21,22 The surfactants used in this study were substantially different, both structurally and chemically, and thus not expected to exhibit similar kinetics of adsorption. The “anchoring” units, cholesterol derivatives, were found to be adsorbed preferentially over the shorter, hydrophilic, “packing” molecules (Figure 1). Furthermore, it is likely that there will be some phase segregation of the components into islands.20 The surface wettability can be used (12) Ulman, A. An Introduction to Ultrathin Organic Films from Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (13) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (14) Folkers, J. P.; Laibinis, P. E.; Whitesdies, G. M. Langmuir 1992, 8, 1330-1341. (15) Bertilsson, L.; Liedberg, B. Langmuir 1993, 9, 141-149. (16) Offord, D. A.; Griffin, J. H. Langmuir 1993, 9, 3015-3025. (17) Offord, D. A.; John, C. M.; Linford, M. R.; Griffin, J. H. Langmuir 1994, 10, 883-889. (18) Offord, D. A.; John, C. M.; Griffin, J. H. Langmuir 1994, 10, 761-766. (19) Stranick, S. J.; Parikh, A. N.; Tao, Y.-T.; Allara, D. L.; Weiss, P. S. J. Phys. Chem. 1994, 98, 7636-7646. (20) Sagiv, J. J. Am. Chem. Soc. 1980, 102, 92-98. (21) Casal, H. L.; Mantsch, H. H. Biochim. Biophys. Acta 1984, 779, 381-401. (22) Ringsdorf et al. have synthesized polymeric thiolipid derivatives containing the cholesteryl moiety, which we used for some preliminary experiments in our laboratory. To the best of our knowledge no data have been published concerning these compounds.
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Figure 2. Schematic of the SPR equipment in our laboratory. An enlarged, in-situ, view of the process of vesicle unrolling is shown.
Figure 1. Schematic illustration of a single-supported phospholipid bilayer.
to estimate the composition of the mixed SAMs.14,15,17 However, while contact angle measurements provide a useful and convenient technique, for the determination of surface composition to be reliable they need to be calibrated using an independent technique. One of the most reliable and surface sensitive techniques for determining the chemical composition of the surface is that of X-ray photoelectron spectroscopy (XPS).14,15,17 Thus, we have used XPS data to calibrate our wetting results. In this paper we present the results of studies on the kinetics of lipid layer formation via vesicle unrolling onto a variety of surfaces using the surface plasmon resonance (SPR) technique.2,10 The experimental arrangement used is shown schematically in Figure 2. The incident laser light excites surface plasmons, i.e., electron density waves propagating at the metal-dielectric interface. At a certain angle of incidence there exists a resonance condition for the excitation of surface plasmons, which causes the energy of the incident laser light to be absorbed by the surface plasmon modes. The angular position of this minimum is critically dependent on the thickness of any layer adsorbed at the gold surface. Thus, by monitoring the relative intensity of the internally reflected laser light at a particular angle of incidence close to resonance, the kinetics of adsorption at the interface can be monitored. The final lipid layer thickness is determined via Fresnel fits to surface plasmon curves obtained before and after the lipid adsorption. Experimental Details Synthesis. The “anchoring” cholesterol molecule was synthesized according to Scheme 1. The product was verified by thin-layer chromatography, 1H NMR, and mass spectrometry. Further details of this synthesis, together with the synthesis of related compounds, is to be published separately.23 (23) Manuscript submitted to Perkin Lett.
Sample Preparation. Gold-Coated Substrates. Glass slides (for contact angle measurements) were cleaned by ultrasonication in a dilute solution of Decon-90 in Milli-Q ultrapure water (20:1), followed by a thorough rinsing in copious amounts of Milli-Q water. The slides were then dried in a stream of nitrogen and plasma cleaned in an argon plasma for 5 min on the highest power setting. The slides were then placed directly into an Edwards Auto 306 Turbo evaporator. Approximately 75 Å of chromium was evaporated first, to promote good adhesion, followed by ∼1500 Å of gold. These were deposited at a rate of 1-2 Å/s and a pressure of ∼2 × 10-6 mbar. For the XPS measurements silicon wafers were employed. These were cleaned by ultrasonication in ethanol. The evaporation was as described above, but at a temperature of 150 °C.24 High refractive index glass substrates (TiH53; n ) 1.85) for the SPR experiments were also cleaned by ultrasonication in ethanol. Approximately 500 Å of gold was evaporated, with no chromium underlayer, at a rate of 1-2 Å/s and a pressure of ∼2 × 10-6 mbar. The slides were placed in optical contact with a prism made of the same glass using an appropriate index matching fluid. SAM Formation. Single component SAMs were prepared by adsorption from1 mM solutions in dichloromethane (HPLC grade, obtained from Aldrich). Immersion times varied between 1 and 12 h; there was no discernible difference in the quality of the SAMs thus formed. For the mixed monolayers, the solutions were prepared having an overall concentration of 1 mM, in dichloromethane. The immersion time was kept to exactly 1 h. This time was chosen to give reproducible monolayer formation and to minimize preferential adsorption5,8 and phase segregation.6,10 Vesicle Formation. Small unilamellar vesicles were prepared as follows: (i) A quantity of grade 1 egg-lecithin (egg-PC) or dimyristoyllecithin (DMPC) (obtained from Lipid Products, Inc., Nutfield Nurseries, England), was dried under nitrogen. (ii) The dried lipid was vortexed in 2 mL of 0.1 M sodium chloride solution (99.99% pure sodium chloride obtained from Aldrich) for ∼5 min, until a milky dispersion was obtained. (iii) The resultant milky dispersion was tip sonicated for ∼40 min, until the mixture became opalescent. (iv) The vesicle solution was then diluted, typically to 0.5 mg/ mL, in 0.1 M sodium chloride solution, stored at -4 °C, and used within 24 h. (24) The use of elevated temperatures during evaporation has been shown to yield smoother gold surfaces, presumably due to an annealing process which occurs on account of the increased surface mobility of the gold atoms upon contact.
Kinetics of Lipid Layer Formation Scheme 1
Note that although vesicle dimensions were not measured, this method is known to produce vesicles having diameters in the range 20-50 nm.19 Physical Characterization. XPS Measurements. For the XPS measurements a Scienta ESCA-300 instrument (RUSTI facility, Daresbury laboratory, U.K.) was used to acquire the spectra. A monochromatic Al Ka X-ray source, at 1464.6 eV, was used, with a power level of 2.8 kW and a slit width of 0.8 mm. The pass energy was 150 eV, and the electron take-off angle was 90°. The data presented in this paper primarily involve the C(1s) peak area normalized with respect to the combined Au(4f) peak areas. This parameter directly relates to the quantity of carbon in the SAM and therefore to the proportion of the cholesterol component. Contact Angle Measurements. For the contact angle measurements a microsyringe was used to advance a droplet of ultrapure Millipore water across the surface. The drop profile was captured using a Hamamatsu C3077 CCD camera and was analyzed using Accuware software. The angle at both sides of the drop was measured, at several positions on each sample, and the average is reported. Contact angle measurements were made on the samples used for the XPS analysis to allow the XPS data to be used as a calibration of the contact angle measurements for the determination of chemical composition. SPR Measurements: Bilayer Thickness and Kinetic Determination. Surface plasmon resonance experiments were performed using the Kretschmann geometry, on a home-built apparatus, as illustrated schematically in Figure 2. The cell was equipped with an inlet port, into which fluid could be injected using a syringe, and an outlet port, from which excess fluid could be released. SPR spectroscopy measurements were made, before and after vesicle adsorption. The curves were obtained by recording the reflected intensity, R, normalized with respect to the incident intensity, Ro, as a function of angle of incidence.25 The curves obtained from such before and after measurements enable the determination of the adsorbed phospholipid film thickness using Fresnel reflectivity calculations. From the initial SPR curve, a suitable choice of incidence angle could be made to give maximum response (i.e., change in reflectance) when observing the kinetics of the adsorption of the lipid layer. This is typically ∼ 0.5° below the minimum of the reflectance curve. (25) The incident laser beam is split by a 50/50 beam splitter (Figure 2). One beam is then directed straight to a photodetector, which gives the incident intensity onto the prism, Ro. The other beam is totally internally reflected by the prism and then directed to a second photodetector, yielding the reflected intensity, R. It is the intensity of the reflected laser light relative to the incident laser light, i.e., R/Ro, which is recorded.
Langmuir, Vol. 13, No. 4, 1997 753 The initial curve was obtained for the SAM, on gold, maintained in contact with 0.1 M NaCl solution via the cell. We did not look at the bare gold substrate and then the substrate plus the SAM separately because we wished to avoid the possibility of contaminating the thiol solution with index matching fluid (which contains sulfurous species). Thus we use a composite “gold + SAM” substrate in which the SAM is treated as a perturbation of the refractive index of the gold. This approach reduces the number of fitting parameters required but does make the assumption that there is no significant change in the SAM conformation during lipid adsorption. While for the mixed SAMs this cannot always be assumed, for single component monolayers and monolayers with significant phase segregation this approximation should be valid. We found that the results obtained on these surfaces are consistent with those obtained on the mixed surfaces, thus validating the assumption in the latter case. The reflectivity curve was corrected to account for the reflective losses inside the prism by recording R/Ro25 as a function of angle of incidence for the bare prism and normalizing the data accordingly. The data were then fitted using standard Fresnel theory. A three-layer model was assumed. Layer 1 was the prism, with n ) 1.85 (where n is the real part of the refractive index) and k ) 0 (where k is the imaginary part of the refractive index). Layer 2 was the gold plus the SAM, for which n was found to vary between 0.2 and 0.4, k was found to vary between -3.2 and -3.7, and the thickness was found to vary between 450 and 550 Å, to obtain the best fit to the data. Layer 3 was the aqueous ambient, for which n was assumed to be that of pure water, i.e., n ) 1.33 and k ) 0. From this initial curve a suitable angle at which to record the kinetics of adsorption was chosen. An excess (∼5 times the volume of the cell) of a 0.5 mg/mL dispersion of phospholipid vesicles in 0.1 M NaCl was injected via the inlet port into the cell. The injection was performed manually using a Luer syringe which could be attached directly to the inlet port of the cell, and the excess was collected from the outlet port. Changes in the reflectivity were monitored as a function of time, until no further change could be observed. The cell was then flushed with an excess (∼5 times the volume of the cell) of 0.1 M NaCl to assess the stability of the supported lipid layer and to remove any adventitiously bound lipid material. A second curve was then obtained similarly to the initial curve. To fit the data a four-layer model was assumed, with layer 1 being the prism, layer 2 being the gold plus the SAM, layer 3 being the lipid bilayer, and layer 4 being the aqueous ambient. For the lipid layer n was found to vary between 1.44 and 1.54, k ) 0, and the thickness was found to vary between 10 and 60 Å, to obtain the best fit to the data. Thus, the thickness for the adsorbed lipid layer was evaluated and subsequently used to calibrate the kinetics data (i.e., convert the increase in R/Ro with time to an increase in thickness with time).
Results and Discussion Composition of the Mixed SAMs. XPS measurements were carried out to calibrate the relative concentrations of the anchoring cholesterol and packing molecules on a series of surfaces. The C(1s) peak was used as a measure of the amount of cholesterol component present and was normalized with respect to the Au(4f) integrated intensity to account for varying sample sizes. Figure 3 shows the variation in the normalized carbon peak intensity with solution composition (circles). Assuming that the amount of carbon is linearly related to the cholesterol content in the SAM, the relationship between the solution composition and the SAM composition was derived and is also shown in Figure 3 (solid line).5 Figure 3 shows that the ratio of cholesterol to packing molecules adsorbed onto a gold substrate is highly nonlinear and that there is a strong preferential adsorption of the cholesterol groups. Effects of solvent type, or adsorption times, were not monitored, but the conditions for film formation were kept constant to permit the XPS data to be used for calibration purposes. The variation in the cosine of the advancing water contact angle as a function of the surface composition, as determined from XPS (Figure 3), is shown in Figure 4.
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Figure 3. Normalized C(1s) peak area as a function of solution composition (circles, left hand axis) and relationship between SAM composition and solution composition (solid line, right hand axis).
Figure 4. Relationship between SAM composition, obtained from XPS data, and the advancing water contact angle measured on the SAM.
The linearity of this plot confirms that the cosine of the advancing water contact angle is also directly proportional to the cholesterol content in the SAM. That is, the Cassie equation is applicable to these surfaces; i.e. cos θ ) f1 cos θ1 + f2 cos θ2, where θ, θ1, and θ2 are the advancing contact angles on the mixed surface and the two single component surfaces, respectively, and f1 and f2 are the fractions of each component present in the mixed monolayer.3 Hence we are able to use water contact angle measurements to routinely establish the composition of our mixed monolayers. Yang et al. have recently suggested that the bulkiness of the cholesterol group restricts surface coverage so that only 65% of all the potential (x3×x3)R30° gold lattice sites are occupied.26 However, from our XPS data, a comparison of the normalized O(1s) peaks for the 100% “packing” and the 100% “cholesterol” SAMs indicates identical surface coverages, to within ∼5%. This result either is in disagreement with the work of Yang et al. or suggests that the short ethyleneoxythiol derivatives contain a high degree of conformational disorder giving a limiting coverage of
