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Langmuir 2000, 16, 9410-9413
Lipid Lateral Diffusion in Multi-bilayers, and in Monolayers at the Air/Water and Heptane/Water Interfaces Thorsteinn Adalsteinsson and Hyuk Yu* Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706 Received May 22, 2000. In Final Form: August 17, 2000 Lipid lateral diffusion in multilamellar stacked bilayers and in monolayers at the interfaces of air/water (A/W) and oil/water (O/W) have been examined. The technique for the diffusion measurement is fluorescence recovery after photobleaching, in conjunction with the Wilhelmy plate method for equilibrium surface pressure. The oil used is heptane, and the lipid is L-R-dilauroylphosphatidylcholine (DLPC). The diffusion in the multi-bilayer system is found to be slower than that in monolayers at the A/W interface by a factor of 2-3. On the other hand, the diffusion at the O/W interface is established to be constant at lower lipid surface density, while that at the A/W interface is faster in the same range of lipid surface density. The difference at the O/W and A/W interfaces, however, diminishes as the surface density increases and eventually disappears altogether at a high enough surface density that is equivalent to 40 Å2 per lipid molecule. The observed different diffusion coefficient profile with respect to the lipid surface density is interpreted as the hydrocarbon molecules coming to the surface at the low lipid density, through intermediate stages of interdigitation with lipid hydrocarbon chains, to eventual squeezing out of the monolayer at the high density. Once a fully packed monolayer is formed at the interface, the diffusion is primarily controlled by the in-plane viscosity and scarcely affected by those of upper and lower phases. The difference in the diffusion coefficients in multi-bilayers and monolayers is tentatively attributed to an artifact of the multiple stacking of bilayers, not to an additional frictional resistance exerted by the apposing hydrocarbon tails within the bilayers.
Introduction The objective of this study arises from a noteworthy observation that there exists a small but significant difference, by a factor of 2-3, between the diffusion coefficients in two model systems of biomembranes, that is, phospholipids in multilamellar stacked bilayers (called multi-bilayer for short) and in monolayers at the air/water interface (A/W). The difference could be ascribed to the intrinsic character of bilayer structure wherein apposing hydrocarbon tails produce extra frictional resistance absent in the monolayers. Alternatively, it could be attributed to an artifact of the multi-lamellar stacking, whereby polar heads of phospholipids apposing in the stacks exert the additional viscous resistance. We focus in this report on the observed difference and whether this casts doubt on the use of monolayers as a model for biomembrane bilayers. Thus, we choose to examine whether the additional frictional resistance could be induced to the A/W monolayers by placing an oil as the upper phase in place of air. Plasma membrane structure has gone through several refinement stages1 since 1925 when Gorter and Grendel2 first proposed lipid bilayers. The principal matrix being lipid bilayers, however, remains the same, whereas a great deal has changed relative to the structural homogeneity and dynamic processes within the bilayers. We know today that the membranes are a complex system with differing compositions, structure, and dynamics, laterally within a plane and transversly between exoplasmic and cytoplasmic hemileaflets. Focusing on the dynamics of the principal matrix while suppressing a multitude of structural and dynamic complexities of the biomembranes, the lipid diffusions in the multi-bilayers and monolayers have been examined extensively. Curiously enough, however, (1) Gennis, R. B. Biomembranes, Molecular Structure and Function; Springer-Verlag: New York, 1989; p 36-84. (2) Gorter E.; Grendel, F. Blood. J. Exp. Med. 1925, 41, 439.
no direct comparison of the lateral diffusions in the two systems with the same lipid has been reported thus far, to the best of our knowledge. Once we made such a comparison, the diffusion coefficients are found to be different by a factor of 2-3. This brings us to the objective on hand, namely, where the difference should arise. It behooves us to establish whether the lateral dynamics that extract in monolayers is indeed related to those in bilayers. To start, we choose L-R-dilauroylphosphatidylcholine (DLPC), which is the phospholipid first studied as monolayers at the A/W by Peters and Beck3 in 1983, for it shows no complex phase behavior over a wide range of surface pressure at room temperature. No diffusion study of DLPC in multi-bilayer is reported; hence, that is the starting point of this report. On the other hand, multi-bilayers with other lipids have been reported extensively.4-8 Fluorescence recovery after photobleaching (FRAP) is the method for determining the lateral diffusion coefficient in both systems. With the multi-bilayers, the dependence of the diffusion coefficient on temperature5 probe and molecular weight,6,7 as well as cholesterol effects,6-8 have been studied using FRAP. By placing a hydrocarbon in the upper phase in lieu of air, we attempt to delineate additional frictional resistance exerted on the lipid molecules in the monolayers if indeed such resistance exists; perhaps a more appropriate biomembrane mimic can also be constructed with lipid monolayer at the oil/ water interface (O/W).9 We choose heptane as the oil phase, (3) Peters, R.; Beck, L. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 7183. (4) Peters, R. C. Biol. Int. Rep. 1981, 5, 733. (5) Wu, E. S.; Jacobson, K.; Papahadjopoulos, D. Biochemistry 1977, 16, 6, 3936. (6) Smith, B. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 2759. (7) Johnson, M. E.; Berk, D. A.; Blankschtein, D.; Golan, D. E.; Jain, R. K.; Langer, R. S. Biophys. J. 1996, 71, 2656. (8) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31, 6739.
10.1021/la0007022 CCC: $19.00 © 2000 American Chemical Society Published on Web 10/11/2000
Monolayer Diffusion at Interfaces
since a considerable body of thermodynamics work exists on the interface of heptane/water with the monolayers of phosphatidylcholine.10-14 The same interface has also received previous attention from this laboratory, using dipalmitoylphosphatidylcholine (DPPC).15 More recent studies with fluorescence microscopy and X-ray reflectivity of DPPC monolayers16,17 and dipalmitoylphosphatidylethanolamine (DPPE) at the O/W have also provided some insight into the molecular behavior of the lipids.18 Experimental Section Materials and Monolayer Preparation. 1-Acyl-2-[12-[(7nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) and L-R-dilauroylphosphatidylcholine (DLPC) were purchased from Avanti Polar Lipids and used without further purification. HPLC grade chloroform (Sigma) was used as the spreading solvent for DLPC. House-deionized water further purified by a Milli-Q system to reach an initial resistivity >17 MΩ was used in the aqueous phase. For the oil phase, HPLC grade n-heptane (Sigma) was used after further extensive purification; it was eluted through a column of active alumina1919 until a stable interfacial tension of 50.8 mN/m was obtained, as measured by the Wilhelmy plate technique. The interfacial tension of the purified heptane was constant for a period of over 4 h, which proved a sufficient time to complete one measurement. Preparation of DLPC Monolayer at the Air/Water Interface. Solutions with concentrations ∼0.08-0.12 mM with 0.5-1.0 mole % NBD-PC were used to prepare the monolayers. A phosphate buffer at pH 7.0, consisting of 9.13 × 10-2 M Na2HPO4, 3.87 × 10-2 M NaH2PO4, and 0.01 M NaCl, was used as the subphase. The monolayers were prepared as follows. A Teflon trough 128.14 cm2 or 126.68 cm2 was filled with the buffer solution. The top layer of the buffer was siphoned off after 10-15 min, using a pipet connected to an aspirator, to remove surfaceactive impurities. Final volumes varying from 10 to 200 µL of the DLPC solution were spread in small aliquots (1-10 µL), using a Hamilton microsyringe. A monolayer was spread on the surface and allowed to reach a stable interfacial tension between additions, as probed by the Wilhelmy plate method, which provides a reliable and reproducible routine to the surface pressure-area (per molecule) isotherms (Π-A). Monolayers of DLPC at the Heptane/Water Interface. DLPC does not spread spontaneously at the heptane/water interface, and heptane does not wet the DLPC monolayer. Addition of DLPC solution to the interface increases the surface pressure by 4-5 mN/m, where it stays constant for all volumes of DLPC solution added. To avoid this problem, a monolayer at the A/W was prepared and then heptane was carefully layered with a pipet onto the interface. At high initial surface densities of DLPC (∼0.7 mg/m2), heptane formed large drops that finally interlocked and formed a film of minimum thickness ∼0.1-0.2 mm. If heptane was layered too rapidly, the spreading behavior was different and the minimum oil film thickness was larger or roughly 1 mm. Surface Pressure. The surface pressure of the monolayer is determined by the Wilhelmy method, using a sandblasted platinum plate (22.1 mm × 0.1 mm × 5.1 mm). The surface (9) Philips, M. C.; Chapman, D. Biohcim. Biophys. Acta 1968, 163, 301. (10) Brooks, J. H.; Pethica, B. A. Trans. Faraday Soc. 1964, 60, 208. (11) Taylor, A. G.; Mingins, J.; Pethica, B. A.; Tan, B. Y. J.; Jackson, C. M. Biochim. Biophys. Acta 1973, 323, 157. (12) Taylor, A. G.; Mingins, J.; Pethica, B. A. J. Chem. Soc., Faraday Trans. 1 1976, 72, 2694. (13) Yue, B. Y.; Jackson, C. M.; Taylor, J. A. G.; Mingins, J. M.; Pethica, B. A. J. Chem. Soc., Faraday Trans. 1 1976, 72, 2685. (14) Mingins, J. M.; Taylor, J. A. G.; Pethica, B. A.; Jackson, C. M.; Yue, B. Y. J. Chem. Soc., Faraday Trans. 1 1982, 78, 323. (15) Sauer, B. B.; Chen, Y. L.; Zografi, G.; Yu, H. Langmuir 1988, 4, 111. (16) Thoma, M.; Pfohl, T.; Mo¨hwald, H. Langmuir 1995, 11, 2881. (17) Brezesinski, G.; Thoma, M.; Struth, B.; Mo¨hwald, H. J. Phys. Chem. 1996, 100, 326. (18) Thoma, M.; Mo¨hwald, H. J. Colloid Interface Sci. 1994, 162, 340. (19) Goebel, A.; Lunkeinheimer, K. Langmuir 1997, 13, 369.
Langmuir, Vol. 16, No. 24, 2000 9411 tension, γ, was observed as a function of time by a Cahn electrobalance until the time dependence, (dγ/dt), reached ∼10-4 mN/m‚s-1. The experiment is carried out under high relative humidity (above 85%) in a poly(methylmethacrylate) box at room temperature (296.0 ( 0.5 K). In measurements of the interfacial tension at the O/W, the oil layer thickness was ∼1 cm, completely covering the platinum plate. Fluorescence Recovery after Photobleaching. The instrumental setup and the data analysis method are reported earlier in detail.20 A set of recent modifications of the signal acquisition step is described elsewhere.21 Briefly, a laser beam of 1 W power is used to bleach a pattern of Ronchi ruling into the monolayer. The decay of fluorescence in the unbleached regions and the recovery of fluorescence in the bleached regions were monitored over time. The time profile of the fluorescence signal envelope is commonly found to be a single exponential after truncating away the first part. Thus, the time profile is represented by
V(t) ) V(0)e-t/τ
(1)
1/τ ) D(2)q2
(2)
with
where V(t) is the difference signal amplitude, V(0) is the amplitude immediately following the bleaching pulse, τ is the relaxation time of the difference signal, and q is the spatial wave vector of the Ronchi ruling fringe. The q2 dependence of 1/τ is thoroughly examined and reported separately;21 hence, we rely on the measurements of τ from a single fringe spacing at 34 µm. Under these conditions, we can deduce the lateral diffusion coefficient in a range of 5 × 10-10 and 5 × 10-14 m2/s. Diffusion at the Air/Water Interface. Once a given monolayer reached an apparent equilibrium state, as specified earlier, a 2 mm diameter cylinder made out of sandblasted platinum with a sharp circular edge is used to grab a small portion of the surface to arrest the surface convection problem. The cylinder is raised until it just touches the surface but does not penetrate. The first diffusion measurement is taken 20 min after the initial raising of the cylinder. Once the cylinder is raised, 12 decay curves were collected with a 2-min interval in between. If more profiles are desired, the cylinder is lowered, the surface allowed to re-equilibriate, the cylinder raised again to capture a fresh portion of the surface, and the process repeated. Diffusion at the Heptane/Water Interface. Once the diffusion measurements at the A/W are completed, the cylinder is lowered and heptane is carefully layered onto the top of the monolayer. The system is allowed to come to a stable interfacial tension, as determined by the Wilhelmy plate method. The time interval for stabilization varies, depending on how rapidly the oil is layered on the monolayer as well as on the monolayer surface density. The equilibration time is longest when the oil is layered rapidly at high surface densities. The oil layer thickness is >1 cm to avoid the cylinder causing a rupture in the oil layer. Small impurities in the oil seem to have a negligible effect on the diffusion coefficient measurements. Preparation of Multi-bilayer Samples for Diffusion Measurements. We followed the established procedure reported by Almeida et al.8 Briefly, a 10 mg/mL stock solution of lipid with 0.1 mol % NBD-PC is prepared. A solution volume containing ∼2-3 mg of the mixture is deposited on an ∼1 cm2 area on a warm microscope slide, precoated with trichlorododecylsilane. The solvent is removed by placing the slide in a vacuum desiccator over anhydrous calcium chloride for 4-5 h. The slide is then placed in a 70 °C oven for 10 min. A 70-80 µL drop of the phosphate buffer is added to the lipid deposit. The plate is allowed to stand and cool to room temperature in a high-humidity environment and then covered with a pretreated thin cover glass slide. Excess water is removed by incubating the sample for 2 h at 70 °C. The slide is then allowed to cool slowly to room temperature and finally the edges are sealed with silicone paste. The samples are stored in a high-humidity container for 3 days (20) Kim, S.; Yu, H. J. Phys. Chem. 1992, 96, 4034. (21) Ma, J. Ph.D. Thesis, University of Wisconsin-Madison, 1998.
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Figure 2. Left side: O, D(2) in DLPC monolayer at the airwater interface at 23 °C as a function of surface area. The line is merely to guide the eye and exptrapolates down to A ) 0.4 nm2/molecule, which is the physical minimum due to molecular size of the phospholipid. Right side: b, D(2) in DLPC multibilayers at different temperatures. Error bars represent 90% confidence intervals. The arrow indicates 23 °C.
Figure 1. (A) O, Π-A isotherm of DLPC at the air-water interface at 23 °C; b, D(2) as a function of surface area per molecule A. Error bars represent 90% confidence intervals. (B) D(2) in DLPC at the A/W plotted against inverse free area, 1/af ) [A - ao]-1, with ao ) 0.41 nm2. The solid line represents the free area model prediction. Different symbols indicate totally independent measurements with different solutions to show the replicability of the determinations. at room temperature prior to use. Our procedure and diffusion measurements in the multi-bilayers were checked against the results of Almeida et al.8 with DMPC multi-bilayers We were able to reproduce their results completely. Thus, we took this as evidence for validation of our procedure and proceeded to examine DLPC multi-bilayers.
Results and Discussion We first show the results for monolayers at the A/W to establish the starting point. They are in complete accord with those reported by Tamada et al.22 The Π-A isotherm is shown in Figure 1A. The monolayer is homogeneous at surface pressures between 3 and 45 mN/m. Hence, the coexistence region of gas-liquid expanded (LE) states must occur at lower surface pressures than 3 mN/m. Overlaid on the figure is a plot of D(2) versus area per molecule, which we call the D(2)-A isotherm, obtained at the same temperature. In the region of A between 0.5 and 0.9 nm2 molecule-1, the diffusion coefficient is represented by the free area model of Tra¨uble, Sackmann, and coworkers23,24 which has its origin in the free volume model in the bulk state;25 it depends exponentially on the inverse free area, D(2) ∝ e-β/af, where β is a constant and the free area is defined as af ≡ A - ao, with ao being the occupied area per molecule. The line over the points in Figure 1B is drawn with ao ) 41 Å2. The applicability range of the model is indicated by two arrows in the figure. The upper (22) Tamada, K.; Kim, S.; Yu, H. Langmuir 1993, 9, 1545. (23) Tra¨ube, H.; Sackmann, E. J. Am. Chem. Soc. 1972, 94, 4499. (24) Gala, H. J.; Hartmann, W.; Theilen, U.; Sackmann, E. J. Membr. Biol. 1979, 48, 215. (25) Cohen, M. H.; Turnbull, D. J. Chem. Phys. 1959, 31, 1164.
one represents the uniphasic boundary of the LE and the lower one stands for the probable end of the monolayer state when the Π-A isotherm exhibits an inflection. The diffusion coefficient in the biphasic region of LE-G coexistence is established by Tamada et al. to follow Saxton’s prediction,22,26 which is a two-dimensional analogue of the effective medium model of Bruggeman and Landauer.27,28 Furthermore, a computer simulation study by Schlicht and Ilgenfritz29 validates the general scheme of the two-dimensional analogy. Having established the starting point with the monolayers at the A/W, we now turn to the comparison of the diffusion coefficients obtained in the multi-bilayers and monolayers. The results are displayed in Figure 2. Contrast is exhibited by plotting the diffusion coefficient against A (D(2)-A isotherm at 23 °C) for the A/W monolayers (left-hand frame) and against temperature for the multi-bilayers (right-hand frame). The difference in the independent variables arises from the fact that we cannot vary the lateral pressure with the multi-bilayers as with the monolayers, whereas the lateral thermal expansion would serve as the controlling variable of a similar nature. For the monolayers, D(2) varies by a factor of 8 for a range of A between 0.4 and 0.8 nm2/molecule. For the multi-bilayers, on the other hand, D(2) varies by a factor of 3 for a range of 292-323 K. For the monolayers, the limiting diffusion coefficient of ∼1 × 10-11 m2/s at A ) ao (0.41 nm2/molecule) was deduced by extrapolation. The corresponding value for the multi-bilayer is 0.4-0.5 × 10-11 m2/s. Thus, we conclude that the difference is only by a factor of 2, although it could be larger if the lipid packing density in the multi-bilayer is larger than 1/ao. In any event, the difference would be about a factor of 2 to 3, significant and well outside of our precision limit for the determination of D(2). Deferring the discussion of possible origins of the difference, we come to the diffusion at the oil/water interface (O/W). Figure 3 shows the plots of D(2) versus A for the A/W and the O/W, where A at the O/W is assumed to be the same as on the A/W, since each O/W monolayer was started from the corresponding one at the A/W. We make three specific points by comparing the two sets of results: (1) The diffusion coefficients, D(2), on the O/W (26) Saxton M. J. Biophys. J. 1989, 56, 3. (27) Bruggeman, D. G. A. Ann. Phys. (Leipzig) 1935, 24, 636. (28) Landauer, R. J. Appl. Phys. 1952, 23, 779. (29) Schlicht, L.; Ilgenfritz, G. Physica A 1996, 227, 269.
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≡ (a/h)[(η′ + η′′)/η]
Figure 3. O, D(2) in the DLPC monolayer at the air-water interface; b, D(2) at the heptane-water interface at 23 °C. Error bars represent 90% confidence intervals. The line and the curve are merely to guide the eye.
and the A/W are the same in the limit of high surface density; (2) D(2) on the O/W in the low surface density region (high A region) flattens out to a constant value of 5 × 10-11 m2/s; (3) D(2) on the A/W, on the other hand, continues to be surface density dependent even in the low density region and is faster than on the O/W by a factor of about 8/5 at the largest A. These points of observation are interpreted as follows. The controlling variable of the lipid diffusion in the low density region is the viscosity of the heptane molecules at the interface, with only a slight contribution from DLPC. In the intermediate region, heptane molecules may interdigitate with DLPC, resulting in the retardation of the diffusion coefficient of the probe. Upon further increase in the surface density of DLPC, heptane molecules are less likely to interdigitate, since they are not surface active as the lipid molecules. Once such a state is reached, we have the monolayer of only DLPC molecules on the heptane/water interface. The lipid diffusion coefficient at high surface densities becomes invariant of upper phase viscosity because the diffusion coefficient in such a monolayer is inversely proportional to the in-plane viscosity of the monolayer, particularly in light of the fact that the viscosities of the upper and lower phases are on the order of centipoise while the monolayer in-plane viscosity is on the order of poise. The basis for the claim is derived from our earlier findings4 in terms of the theory of Hughes, Pailthorpe, and White.30 Briefly, we present the argument as follows. The HughesPailthorpe-White (HPW) model stipulates that the upper and lower fluids are of different viscosities, η′ and η′′, but both are still much smaller than η of the lipid film; hence, it is applicable to monolayer systems. For the HPW model, the following formulation for the lateral diffusion coefficient, D(2), of a cylinder with a radius a confined to an interlayer of height h between two fluids is given by
D(2) ) kT/[4π(η′ + η′′)aΛ()]
(3)
where Λ() is numerically evaluated, called a reduced friction coefficient, and it is a monotonically decreasing function of the parameter , which is defined as (30) Hughes, B. D.; Pailthorpe, B. A.; White, L. R. J. Fluid Mech. 1981, 110, 349.
(4)
Λ() reaches the asymptotic limit of 2/π when exceeds ∼40. In the case of the phospholipid monolayer at the A/W and the O/W, is on the order of 0.01, and Λ() decreases almost linearly with increasing in this region, resulting in Λ() ∝ η. Thus, the crucial point is extracted: D(2) is inversely proportional to the viscosity of the lipid film η when η′ and η′′ are on the order of 1/100 of η, as in our case.31 We now come to the difference in D(2) between the multibilayers and monolayers. This small but significant difference needs to be understood. We offer the following hypothesis for the origin of the additional viscous resistance in multi-bilayers. Given the mass density gap in the tail-to-tail region of a bilayer,32,33 any viscous drag of one monolayer upon the apposing monolayer in the context of HPW is not likely. Hence, we postulate that the apposing monolayers are hydrodynamically uncoupled. If this were true, then the additional viscous drag could be attributed to an artifact of multiple stacking of bilayers. According to a recent report by Chen et al.34 for the DLPC multibilayer swelling by X-ray diffraction, the head-to-head distance is estimated to be 15-18 Å, depending on relative humidity of the measurement chamber at 20-30 °C. Our condition for the diffusion in the multi-bilayers is in the fully hydrated state, because free water coexists with the membrane stacks in the sample cells; hence, the distance corresponds to 18 Å. Thus, the hydration layer within such a narrow region may have a very different viscosity than that of free water such that η/η′ ) 100/1 may not hold. In such an event, the monolayer and multi-bilayer diffusion coefficients could be different, although its magnitude is difficult to estimate. On the other hand, studies with planar single bilayers have indicated that the diffusion coefficient is greater than that found in the multi-bilayer system. This effect was, however, attributed to residual solvent in the bilayer.9 We thus come to a tentative conclusion that the difference in the diffusion coefficients arises from the multiple stacking of bilayers with the additional frictional drag attributable to the apposing polar heads confined in thin aqueous layers of 18 Å and not from intrinsic differences in the dynamics between single planar bilayers sandwiched between two aqueous phases and monolayers at the A/W. It could have been more convincing if we had carried out the multibilayer diffusion studies under varying relative humidity conditions such that the additional viscous drag could be brought on by lowering the relative humidity. Such will be the subject of a future study. Acknowledgment. This work is partially supported by the Eastman Kodak Professorship and NSF grants (DMR-9711226 and -0084301) awarded to H.Y. We are most grateful to Dr. Keiji Tanaka for his assistance, and to Dr. Bryan B. Sauer and Prof. George Zografi for helpful discussion. LA0007022 (31) Saffman, P. G.; Delbru¨ck, M. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 3111. (32) Franks, N. P. J. Mol. Biol. 1976, 100, 599. (33) Davies, J. H. Biochim. Biophys. Acta 1983, 737, 117. (34) Chen, F. Y.; Hung, W. C.; Huang, H. W. Phys. Rev. Lett. 1997, 79, 4026.
