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Kinetic Studies of Spreading DMPC Vesicles at the Air-Solution Interface Using Film Pressure Measurements Ming Li, Utz Retter,† and Jacek Lipkowski* Department of Chemistry and Biochemistry, University of Guelph, N1G 2W1, ON, Canada, and Federal Institute for Materials Research and Testing, Richard-Willsta¨ tter-Strasse 11, D-12489, Berlin, Germany Received December 23, 2004. In Final Form: February 25, 2005 DMPC vesicles were injected into a 50 mM NaF solution in water, and the kinetics of a monolayer formation at the air-solution interface was investigated by measuring changes of the film pressure, π, as a function of time. The studies were carried out in temperature range from 5 to 35 °C. The solutions were stirred in order to eliminate the mass transport limitations. Under these conditions, the monolayer formation was controlled by the surface processes only. At temperatures above the critical temperature Tc, compression isotherms were measured and used to convert the π-t curves into the Γ-t plots, with Γ being the surface concentration of DMPC. These kinetic data indicate that the monolayer formation involves fast rupture of vesicles and formation of a constant number of disk-shaped monolayer islands at the air-solution interface that grow with a constant radial rate. At higher coverages, the growth is restricted by the availability of the monolayer-free surface area. At temperatures below Tc, the π-t curves cannot be converted into the Γ-t plots. Here the kinetics can be discussed only qualitatively. The data indicate that the kinetics of the monolayer formation involves two steps. Initially, an expanded film is formed. At higher film pressures, the expanded film is slowly transformed into a liquid condensed state.
Introduction The spreading of small unilamellar vesicles at the airsolution interface constitutes an attractive method to produce monomolecular films of phospholipids that can be later transferred onto a solid support using the Langmuir-Blodgett technique. The formation of monomolecular films of phospholipids by fusion of vesicles was first observed by Verger et al.1,2 Schindler3 provided the first theoretical model of the monolayer formation from vesicles that assumes an exchange of lipids between the film spread at the interface and vesicles in the subsurface region. A pictorial description of Schindler’s model was given by Obladen et al.4 The mechanism of vesicle spreading has been later simplified assuming that it involves only two steps: diffusion of vesicles from the bulk into the subsurface region and their disintegration at the interface.5-10 Several other research groups investigated kinetics of the monolayer film formation from solutions of vesicles. Heyn et al.11 postulated that fusion of vesicles requires a * Author to whom correspondence should be addressed. † Federal Institute for Materials Research and Testing. (1) Verger, R.; Pattus, F. Chem. Phys. Lipids 1976, 16, 285. (2) Pattus, F.; Desnuelle, P.;Verger, R. Biochim. Biophys. Acta 1978, 507, 62. (3) Schindler, H. Biochim. Biophys. Acta 1979, 555, 316. (4) Obladen, M, Popp, D.; Scholl, C.; Schwartz, H.; Jaenig, F. Biochim. Biophys. Acta 1983, 735, 215. (5) Ivanova, T.; Georgiev, G.; Panaiotov, I.; Ivanova, M.; LaunoisSurpas, M. A.; Proust, J. E.; Puisieux, F. Prog. Colloid Polym. Sci. 1989, 79, 24. (6) Launois-Surpas, M.; Ivanova, T.; Panaiotov, I.; Proust, J.; Puisieux, F.; Georgiev, G. Colloid Polym. Sci. 1992, 270, 901. (7) Ivanova, T.; Raneva, V.; Panaiotov, I.; Verger, R. Colloid Polym. Sci. 1993, 271, 290. (8) Panaiotov, I.; Ivanova, T.; Balashev, K.; Proust, J. Colloid Surf., A 1995, 102, 159. (9) Panaiotov, I.; Proust, J.E.; Raneva, V.; Ivanova, T. Thin Solid Films 1994, 244, 845. (10) Mitev, D. J.; Ivanova, Tz.; Vassulieff, C. S. Colloids Surf., B. 2002, 24, 185.
flip-flop of molecules from the inner to the outer leaflet of vesicles. Gugliotti et al.12 studied the influence of the polar headgroup, the salt concentration, and the vesicle size on the fusion of vesicles at the air/water interface. There are also several papers that describe kinetics of insertion of collagen,13 an enzyme,14 and an immunoglobuline15,16 into the monolayer formed by vesicles fused at the air-solution interface. In parallel, a lot of efforts have been made to describe the mechanism and kinetics of supported phospholipid bilayer (SPB) formation, by fusion of small unilamellar vesicles (SUVs) at a solid-solution interface.17-21 A theory describing adhesion, fusion, and rupture of vesicles at solid surfaces has been developed by Seifert and Lipowsky.22 The experimental verification of this model has been provided by Reviakine and co-workers.23 More recent theoretical modeling is described by Zhdanov and Kasemo.24 An interesting study of vesicle fusion at a mercury electrode was published by Scholz et al.25 In the past, the kinetics of vesicle fusion was investigated in unstirred solutions, and hence, it was predomi(11) Heyn, S. P.; Egger, M.; Gaub, H. E. J. Phys. Chem. 1990, 94, 5073. (12) Gugliotti, M.; Chaimovich, H.; Politi, M. Biochim. Biophys. Acta 2000, 1463, 301. (13) Ghannam, M. M.; Mady, M. M.; Khalil, W. A. Biophys. Chem. 1999, 80, 31. (14) Marron-Brignone, L.; Morelis, R. M.; Chauvet, J.-P.; Coulet, P. R.; Langmuir 2000, 16, 498. (15) Girard-Egrot, A. P.; Morelis, R. M.; Boullanger, P.; Coulet, P. R. Colloids Surf., B 2000, 18, 125. (16) Girard-Egrot, A. P.; Chauvet, J.-P.; Boullanger, P.; Coulet, P. R. Langmuir 2001, 17, 1200. (17) Tamm, L.; McConnell, H. Biophys. J. 1985, 47, 105. (18) Stelzle, M.; Weissmuller, G.; Sackman, E. J. Phys. Chem. 1993, 97, 2974. (19) Wenzl, P.; Fringeli, M.; Goette, J.; Fringeli, U. P. Langmuir 1994, 10, 4253. (20) Koenig, B.; Krueger, S.; Orts, W. J.; Majkrzak, C. F.; Berk, N.F.; Silverton, J. V.; Gawrisch, K. Langmuir 1996, 12, 1343. (21) Raedler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539. (22) (a) Seifert, U. Adv. Phys. 1997, 46, 13. (b) Lipowsky, R.; Seifert, U. Mol. Cryst. Liq. Cryst. 1991, 202, 17. (23) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806.
10.1021/la046796n CCC: $30.25 © 2005 American Chemical Society Published on Web 03/26/2005
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nantly controlled by a slow diffusion of vesicles from the bulk to the interface. The objective of this work was to investigate spreading of SUVs of DMPC at the airsolution interface under conditions of a forced convection. Using this approach, we were able to eliminate the mass transport limitation and to measure the rate of the monolayer formation controlled by two-dimensional processes. We succeeded to demonstrate that the kinetics of the film formation involves two stages that correspond to the transformation of the monolayer from a liquidexpanded to a liquid-condensed phase. Experimental Section Small, unilamellar vesicles were prepared using the Barenholz procedure.26 A 10 mg/mL chloroform (Aldrich, ACS HPLC grade, Milwaukee, WI) solution of DMPC was used as stock solution. The stock solution (0.2 mL) was dried by vortexing in a test tube under a flow of argon. To remove the residue of the solvent, dried DMPC film was placed in a vacuum desiccator for at least 2 h. Next, 2 mL of 50 mM NaF electrolyte was added to the dry lipid and the mixture was sonicated using a sonicator (Aquasonic Model50D) at ∼35 °C for at least 1 h. (Usually the mixture became translucent after 20-30 min.) The dynamic light scattering measurements performed on these solutions showed that the diameter of 96.5% (wt/wt) vesicles ranged from 16 to 30 nm; ∼3.5% from 72 to 154 nm; and ∼0.02% from 328 to 601 nm. The kinetics of the monolayer formation was studied by measuring the film pressure at the air-solution interface as a function of time, using a Wilhelmy plate attached to a microbalance (KSV Instruments, Finland). The Wilhelmy plate was a piece of clean filter paper as recommended by the KSV 5000 user manual. The Wilhelmy plate made of filter paper was used to study similar systems.11,27 The balance was mounted on an arm of an elevator and could be moved up and down in the vertical direction with a high precision. The vesicles were injected into a 25 mL beaker containing 50 mM NaF solution. The beaker had a water jacket that allowed for temperature control. It was placed in a chamber with a laminar flow of argon. The Wilhelmy plate was immersed to one-third of its height, and the balance was zeroed. Next, with the help of the elevator, the balance was moved up and the Wilhelmy plate was emerged. The solution was then vigorously stirred with a Teflon-coated stirring bar (at the rate 200-500 rpm). After 10 min, the stirring was interrupted, the elevator was moved down to its previous position, and the Wilhelmy plate was immersed again into the solution. After about 1 min of equilibration time, the film pressure was measured again. Preliminary tests showed that the Wilhelmy plate can be moved out and into the solution giving reproducible reading of the film pressure with precision better than 1 mN/m. The film pressure versus time curves were recorded using the KSV LB5000 controls and software and were stored in a computer. In addition, a few drops of DMPC solution in a mixed solvent consisting of hexane (Fisher 99.9%) and ethanol (Commercial Alcohols, Inc.) in a 9:1 (v/v) ratio were injected onto the water surface of a Langmuir trough (KSV LB5000, Finland) equipped with a moving barrier and a Wilhelmy plate (KSV, Finland) and compression isotherms were recorded. The trough was controlled by a computer using KSV LB5000 software. It was equipped with a water jacket to allow temperature-controlled experiments. The surface temperature was measured using a thermocouple (K-type 1 F0) calibrated with a help of a thermometer with 0.1 °C precision. The temperature was controlled with the help of a water circulating bath.
Results and Discussion Preliminary Experiments. The goal of initial experiments was to determine the optimal stirring rate and (24) Zhdanov, V. P.; Kasemo, B. Langmuir 2001, 17, 3518. (25) (a) Hellberg, D.; Scholz, F.; Schauer, F.; Weitschies, W. Electrochem. Comm. 2002, 4, 305. (b) Scholz, F.; Hellberg, D.; Harnisch, F.; Hummel, A.; Hasse, U. Electrochem. Comm. 2004, 6, 929. (c) Hellberg, D.; Scholz, F.; Schubert, F.; Lovric, M.; Omanovic, D.; Hernandez, V. A.; Thede, R. J. Phys. Chem., submitted for publication. (26) Barenholz, Y.; Gibbes, D.; Litman, B. J.; Goll, J.; Thompson, T. E.; Carlson, R. D. Biochemistry 1977, 16, 2806. (27) Vollhardt, D.; Melzer, V. J. Phys. Chem. B 1997, 101, 3370.
Figure 1. The influence of the mass transport measured as a number of rotations per minute of the stirring bar on the rate of DMPC monolayer formation from a 0.02 mg/mL DMPC vesicle solution in 50 mM NaF at a temperature of 22 °C.
Figure 2. Film pressure vs time plots for DMPC monolayer formation at 22 °C from 50 mM NaF solution with the following concentration of DMPC vesicles: (a) 0.1; (b) 0.03; (c) 0.02; and (d) 0.01 mg/mL. Solutions were stirred at the stirring rate of 400 rpm.
vesicle concentration for further kinetic studies. Figure 1 shows the film pressure versus time plots determined at 22 °C for a 0.02 mg/mL DMPC vesicle solution without stirring and with stirring at two rotation rates. The results show that the effect of stirring is dramatic. In unstirred solutions, the monolayer formation is very slow, and its formation is clearly controlled by a slow mass transport. In the stirred solutions, the film pressure attains the limiting value of ∼47 mN/m (which is the value of the equilibrium spreading pressure) after ∼70 min. The change of the stirring rate from ∼200 to ∼500 rpm has a relatively small influence on the spreading rate. A turbulent flow was observed at higher stirring rates, and hence, 400 rpm was used in the rest of the experiments described in this paper. The data in Figure 1 show that at this stirring rate the spreading of DMPC vesicles may be considered as being approximately stirring-rate independent. Figure 2 shows the effect of vesicle concentration on the kinetics of monolayer formation. The effect is very strong.
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Figure 3. Reciprocal of the half time (time at half of the maximum film pressure) plotted versus the vesicle concentration; 50 mM NaF solution, stirring rate 400 rpm.
Figure 4. Film pressure versus time plots for 0.02 mg/mL DMPC vesicle solution in 50mM NaF at different temperatures; stirring rate 400 rpm.
The reciprocal of the half time is plotted in Figure 3 as a function of the vesicle concentration. The plot is linear, indicating that the monolayer formation is a first-order reaction with respect to vesicles concentration. For a 0.02 mg/mL DMPC solution, the rate of the monolayer formation is neither too low nor too fast for our method. Therefore, all further experiments were performed using this concentration. Temperature Dependence. The effect of temperature on the monolayer formation rate was investigated in a temperature range from 5 to 35 °C. This range encompasses both the gel-to-liquid crystalline phase transition for vesicles29a at 24 °C and the critical point for monolayer29b observed at 21 °C. Figure 4 shows the film pressure versus time plots for selected temperatures. Each curve is the average of at least two independent measurements. The result was considered to be satisfactory when the differences between independently measured points were less than 1 mN/m. The results show a significant effect of temperature on the monolayer formation rate. Further, the data show that the film pressure versus time curves display two steps at temperatures less than 22 °C (28) Melzer, V.; Vollhardt, D.; Brezesinski, G.; Moehwald H. J. Phys. Chem. B 1998, 102, 591. (29) (a) Heerklotz, H.; Seeling, J. Biophys. J. 2002, 82, 1445. (b) Albrecht, O.; Gruler, H.; Sackman, E. J. Phys. (Paris) 1978, 39, 301.
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Figure 5. Compression isotherms for DMPC monolayer at different temperatures. Insets: (a) compression isotherms for temperatures close to the critical point Tc ) 21 °C; (b) compression isotherm at 5 °C with the descriptions of various physical states of the film.
and one step at higher temperatures. Significantly, the change in the shape of the film pressure versus time curves is observed at the critical point of the monolayer and below the main transition temperature for the bilayer in vesicles. This behavior suggests that the rate of the monolayer formation is determined by the phase properties of the monolayer rather than the bilayer in vesicles. The rates of the monolayer formation were calculated using the method by Ponaiotov et al.8 It is based on the assumption that, at an identical film pressure, an adsorbed DMPC molecule occupies the same area regardless of whether it is adsorbed from a solution of vesicles or it is spread as a Langmuir film at the surface of a Langmuir trough. Recently, Vollhardt et al.27,28 compared Gibbs isotherms and compression isotherms for surfactants with a moderate solubility and demonstrated that indeed the morphological features of the film are independent of the process of the monolayer formation for monolayers in the gaseous and expanded states. Therefore, we have employed the Langmuir trough to determine the compression isotherms (film pressure versus area per molecule plots). The film pressure versus time plots were then converted into the surface concentration versus time plots with the help of compression isotherms. Figure 5 shows the compression isotherms for different temperatures. These results are in very good agreement with similar measurements performed earlier by Albrecht et al.29b Insets to Figure 5 show that at temperatures lower than 21 °C the monolayer of DMPC may exist in several phases such as gaseous, liquid expanded (LE), liquid condensed (LC), and solid (S). The LE-LC phase transition has the first-order character, while the LC-S transition is continuous (higher order). With the help of X-ray diffraction and neutron reflectivity measurements, it has been established that the LE phase is formed by molecules with melted chains, while the LC, or more correctly tilted condensed phase, is formed by molecules with chains in the all-trans conformation.30 Above 21 °C, only the gaseous and LE states are observed. The coordinates of the critical point are Tc ) 21 °C and πc ) 47.5 mN/m. These numbers agree well with the literature.29b Analysis of Kinetic Data. We will first analyze the kinetic data for temperatures above Tc. Figure 6 illustrates how the compression isotherms and π-t curves were (30) Kaganer, V. M.; Moehwald, H.; Dutta, P. Rev. Mod. Phys. 1999, 71, 779.
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Figure 7. (a) Surface concentration versus time plots, (b) monolayer formation rate, for several temperatures above Tc; 0.02 mg/mL DMPC vesicle solution in 50mM NaF at rotation rate 400 rpm.
Figure 6. Transformation of t-π into Γ-t plot; (a) t versus π plot; (b) Γ versus π plot; (c) resultant Γ versus t plot, for 0.02 mg/mL DMPC vesicle solution in 50mM NaF at T ) 26 °C, rotation rate 400 rpm.
combined to calculate the change of the surface concentration of DMPC (Γ) with time. Panel a plots time versus film pressure, determined from the vesicle spreading experiment (rotated curve from Figure 4). The points are the experimental data, and the line is a polynomial fit used for interpolation. The compression isotherm is shown as Γ ) 1/A plotted versus the film pressure in the middle panel b. The bottom panel c shows the dependence of the surface concentration on time. Each point on this curve corresponds to t taken from the top panel and Γ from the middle panel at a given value of π. In this procedure, it is implied that the mean molecular areas determined from an equilibrium state (compression isotherm at a slow compression rate) can be used to describe the nonequi-
librium state. For an LE state, the validity of this approach was demonstrated by Vollhardt et al.27,28 However, the curve in panel c shows that the coverage has already quite a significant value at zero time. This is an artifact of the conversion procedure. Shortly after addition of vesicles to the solution, the film pressure jumps to a value of ∼2 mN/m. This initial fast increase is most likely caused by adsorption of nonfractured vesicles at the interface. The formation of the monolayer is the next, much slower step. However, when the t-π curve (panel a) is combined with the Γ-π (panel b), the nonzero value of π at t ≈ 0 gives a nonzero value, Γo, for the coverage. This initial coverage will have to be subtracted in the further data analysis. Figure 7a plots the Γ-t curves for several temperatures above Tc. They have a regular sigmoidal shape, and they all display the initial nonzero Γo. These curves can be dΓ differentiated to give the monolayer formation rates dt shown in Figure 7b. The monolayer formation rates display a characteristic bell shape with a shoulder at a descending section of the plot. With increasing temperature, they become narrower and taller and their maximum shifts toward shorter times. In the absence of mass transport limitations, the monolayer of DMPC grows from vesicles accumulated in the subsurface region. The rupture of a vesicle creates a growth center. There must be a large number of such growth centers that initially expand, but later their growth is restricted because the area uncovered by the phospholipids diminishes. The kinetics of such
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Figure 8. Avrami analyses for temperatures above critical points. Inset: ln K vs 1/T to calculate activation energy, where K ) ΠNk2.
growth may be described by the generalized equation:31,32
Γ(t) - Γ0 ) 1 - exp(- kxtx) Γ∞ - Γ0
(1)
where Γ(t) and Γ∞ are the surface concentrations of the phospholipids at time t and tf∞ and kx is a constant whose value depends on the growth mechanism. The exponent x has a numerical value that is characteristic for the specific growth mechanism and the growth geometry. Thus, from the experimental value of the exponent x, the mechanism of the monolayer formation may be determined. Figure 8 shows double logarithmic plot of (Γ∞ - Γ(t)/Γ∞ - Γ0) versus time. In the middle section, the plots are fairly linear and the slopes of this segment give exponents, x, ranging from 1.85 to 2.05. These numbers are close to 2, which is a characteristic value of the exponent for the growth of a monolayer from a fixed number of instantaneously formed growth centers.32,33 That result suggests that the monolayer formation involves fast rupture of a constant number of vesicles that form disk-shaped monolayer islands at the air-solution interface. The radial growth of the island is constant and hence dr/dt ) constant, where r is the radius of the island. Under these conditions, the unrestricted growth of the area, A, of the island with time is described by A ) πk2t2, where k is the islandgrowth rate constant. The real growth however is slowed by the mutual contact of the adsorbed monolayer islands. Using Avrami law,31 one can demonstrate that in this case:32,33
Γ(t) - Γ0 ) 1 - exp(-ΠNk2t2) Γ∞ - Γ0
(2)
where N is the number of growth centers and Π ≈ 3.14. The double logarithmic plot crosses the ordinate at ln(ΠNk2). The activation energy of the radial growth can therefore be determined by plotting this intercept versus T-1. This plot is shown in the inset to Figure 8. Consistent (31) Avrami, M. J. Chem. Phys. 1939, 7, 1103; J. Chem. Phys. 1940, 8, 212; J. Chem. Phys. 1941, 9, 177. (32) Vollhardt, D.; Retter, U. J. Phys. Chem. 1991, 95, 3723. (33) Buess-Herman, C. In Adsorption of Molecules at Metal Electrodes; Lipkowski, J, Ross, P. N., Eds.; VCH: New York, 1992; Chapter 2, p 77.
Figure 9. (a) Time vs film pressure; (b) surface concentrated vs film pressure at 5 °C; 0.02 mg/mL DMPC vesicle solution in 50 mM NaF, rotation rate 400 rpm.
with the Arrhenius equation, the plot is linear and its slope gives the activation energy of the radial growth equal to15 kJ/mol. This number is somewhat less than the energy gain due to lowering of the interfacial tension of water estimated to be ∼20 kJ/mol, and hence, it is a reasonable number. The analysis of the kinetic data below the critical point is much more difficult. Figure 9 shows a comparison of the t-π curve representing the kinetics of the monolayer formation and the Γ-π curve calculated from the compression isotherm for a temperature of 5 °C. The two curves display some similarity. One can assign the first step on the t-π curve to the formation of the LE film and the second step to the kinetics of the LE-LC phase transition. However, the LE-LC transition is observed at much lower film pressures on the compression isotherm than on the t-π curve. Hence, the Γ-π curve cannot be used to transform the t-π curve into the Γ-t plot and consequently to calculate the rate of the film formation. Similar difficulties with comparison of the properties of Gibbs and Langmuir films in the presence of the first-order LE-LC phase transition were reported by Melzer et al.28 Nevertheless, our data show that below the Tc the kinetics of the monolayer formation involve two steps. Initially, at low film pressures, an LE film is formed. At higher π, the monolayer is transformed into the LC film. The kinetics of the LC film formation are slower than the kinetics of formation of the LE film. Summary and Conclusions Several studies of the kinetics of monolayer formation by fusion of DMPC vesicles at the air-solution interface have been described in the literature.1-11 The experiments performed in the past were carried out in unstirred solutions, and hence, the kinetics of the monolayer formation was to a large extent controlled by diffusion.
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We have described first studies of the monolayer formation under conditions of forced convection. In this way, we were able to eliminate the mass transport limitations and to observe the kinetics controlled by surface processes only. The measurements were carried out in broad range of temperatures below and above the critical point of the monolayer. Significant qualitative differences were observed between the rates of the monolayer formation at temperatures below and above the critical point of the monolayer. The temperature of the critical point, 21 °C, is lower than the gel-liquid crystalline phase transition temperature in the vesicles, 24 °C. We observed that the change in the kinetics of the monolayer formation correlates better with the phase properties of the monolayer at air-solution interface than with the phase properties of the bilayer in vesicles. This behavior suggests that the kinetics of the monolayer formation are controlled by a two-dimensional surface process rather than by the rate of vesicles rupture.
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For temperatures above the critical point, the kinetic data are described well by a model that assumes a fast rupture of a constant number of vesicles and formation of disk-shaped monolayer islands at the air-solution interface, which grow with a constant radial rate. At higher coverages, the growth is hindered by the decrease of the monolayer-free surface area. Below the critical temperature, the kinetics of monolayer formation has a two step character. Initially, an LE film is formed that at higher film pressures is transformed into the LC state. Acknowledgment. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. J.L. acknowledges Canada Foundation for Innovation for the Canada Research Chair Award LA046796N
