Langmuir 2000, 16, 5029-5035
5029
Structure of Freestanding Phospholipidic Bilayer Films Nicolas Cuvillier, Fre´de´ric Millet, Vera Petkova,2 Mickael Nedyalkov,2 and Jean-Jacques Benattar*,1 Service de Physique de l’Etat Condense´ , CEA/Saclay, F-91191 Gif sur Yvette Cedex, France Received June 9, 1999. In Final Form: February 8, 2000 We have determined the molecular structure of black films of various phospholipids using X-ray reflectivity. This technique measures the electronic density profile perpendicular to the film. It allows us to accurately determine the structure of the lipidic film, i.e., to measure the density and the thickness of the zone occupied by either the aliphatic chains or the hydrophilic groups and to quantify the amount of water in the center of the film. We have studied the relations between the chemical nature of the lipids and the structural properties of the film. In particular, for zwitterionic lipids, we show that the presence of water is controlled mainly by the chemical nature of the lipid headgroup and not by the electrostatic interactions.
Introduction A lipid foam film is formed by two monolayers of lipids with the hydrophobic parts facing the air and the hydrophilic headgroups of lipids in contact with a central water core. When this film is supported by a solid frame, this water layer remains connected to the bulk solution via the meniscus of the film. Its thickness, which could ultimately be reduced to almost zero, when only hydration water remains in the film (i.e. the Newton black film), depends both on the nature of the lipids that stabilize the film and on the physicochemical state of the bulk. Due to their well-defined geometry,3 such films have been widely used as model system for the study of foam. Within this context, many studies have focused on the measurement of interaction forces within the film, both for insoluble4,5 and soluble6-8 phospholipidic compounds. For these studies, the forces are derived from the measurement of the film thickness by the microinterferometric method of Scheludko and Exerowa.9,10 In this technique, the intensity of a monochromatic light reflected by the film is used to calculate its thickness. Unfortunately, with this optical method, it is not possible to obtain direct information on the internal structure of the film, as the incident wavelength is too large. Even when a more complex model was used for the film structure (3-layer model),11,12 most of the model parameters were assessed from other experiments,13 which were made on different systems. (1) Corresponding author. (2) Permanent address: Department of Physical Chemistry of the University of Sofia, 1, bul. “James Bourchier”, Sofia, Bulgaria. (3) Exerowa, D.; Krugliakov, P. M. Foam and Foam Films: Theory, Experiment, Application; Studies in Interface Science, Volume 5; Elsevier: Amsterdam, 1998. (4) Cohen, R.; Koynova, R.; Tenchov, B.; Exerowa, D. Eur. Biophys. J. 1991, 203, 208. (5) Toca-Herrera, J. L.; Mu¨ller, H.-J.; Krustev, R.; Exerowa, D.; Mo¨hwald, H. Colloids Surf. A 1998, 144, 319. (6) Cohen, R.; Exerowa, D.; Kolarov, T.; Yamanaka, T.; Muller, V. M. Colloid Surf. 1992, 65, 201. (7) Cohen, R.; Exerowa, D. Colloid Surf. A 1994, 85, 271. (8) Cohen, R.; Exerowa, D.; Yamanaka, T. Langmuir 1996, 12, 5419. (9) Sheludko, A. Adv. Colloid Interface Sci. 1967, 1, 391. (10) Exerowa, D.; Kashchiev, D.; Platikanov, D.; Toshev, B. Adv. Colloid Interface Sci. 1994, 49, 303. (11) Cohen, R.; Exerowa, D.; Kolarov, T.; Yamanaka, T.; Tano, T. Langmuir 1997, 13, 3172. (12) Frankel, S. P.; Mysels, K. J. J. Appl. Phys. 1966, 37, 3725. (13) Donners, W. A. B.; Rijnbout, J. B.; Vrij, A. J. Colloid Interface Sci. 1977, 61, 249.
Recently, infrared spectroscopy (FTIR) was also used to measure the absorbance at 3400 cm-1 (OH stretching vibration band) for thin lipid films.9,14 If one knows the molar absorption coefficient , this absorbance could be converted into the thickness of the central water core by using the Lambert-Beer law. Unfortunately, this coefficient could only be estimated for foam films and values as different as ) 116 15 and ) 150 16 have been put forward (25% discrepancy on the thickness). Despite this problem, FTIR did provide new information such as for example the molecular orientation17 (by measuring the absorption corresponding to the surfactant stretching bands). Up to now, most of the studies have examined the response of the film to the variation of a control parameter (e.g. ionic force of the solution, presence of divalent ions, ...). The study of the relation between the film structure and the chemical nature of the surfactants has however been neglected. To address this problem, we have used X-ray reflectivity to determine the internal structure of lipid films. This technique, which has been successfully used in the case of soluble surfactants,18,19 measures the electronic density profile perpendicular to the surface of the film. In this study, we have measured in detail the X-ray reflectivity of films made with various phospholipids and we describe the respective roles of the aliphatic chains and of the headgroup on the film structure. The effect of electrical charges, which sometimes occurred, was also thoroughly studied. Experimental Section Materials. The chemical formulas of the lipids used in this study are summarized in Figure 1. The PC lipids are zwitterionic and share the same headgroup (R3 ) C), but the aliphatic chains differ: R1 ) R2 ) M for DMPC (dimyristoyl phosphatidylcholine), R1 ) R2 ) P for DPPC (dipalmitoyl phosphatidylcholine), and R1 ) R2 ) O for DOPC (dioleoyl phosphatidylcholine). The PE lipids are also zwitterionic but have a smaller headgroup (R3 ) E). For these lipids, we have selected the same types of chains as in the (14) Yamanaka, T.; Tano, T.; Kamegaya, O.; Exerowa, D.; Cohen, R. D. Langmuir 1994, 10, 1871. (15) Smart, C.; Senior, W. A. Trans. Faraday Soc. 1966, 62, 3253. (16) Umemura, J.; Matsumoto, M.; Kawai, T.; Takenaka, T. Can. J. Chem. 1985, 63, 1713. (17) Tano, T.; Umemura, J. Langmuir 1997, 13, 5718. (18) Be´lorgey, O.; Benattar, J.-J. Phys. Rev. Lett. 1991, 66, 313. (19) Sentenac, D.; Benattar, J.-J. Phys. Rev. Lett. 1998, 81, 160.
10.1021/la990745m CCC: $19.00 © 2000 American Chemical Society Published on Web 04/21/2000
5030
Langmuir, Vol. 16, No. 11, 2000
Figure 1. Chemical formula of the lipids. All the phospholipids (except DOTAP) share the same central function but could differ by their aliphatic chains (R1 and R2) and their hydrophilic group R3. In this study, we used three chains of different lengths: myristoyl (M) with 14 carbons, palmitoyl (P) with 16 carbons, and oleoyl (O) with 18 carbons and a double bond CdC. We also used three different functions for R3: choline (C), ethanolamine (E), and glycerol (G). case of PC lipids: R1 ) R2 ) M for DMPE (dimyristoyl phosphatidylethanolamine), R1 ) R2 ) P for DPPE (dipalmitoyl phosphatidylethanolamine), and R1 ) R2 ) O for DOPE (dioleoy phosphatidylethanolamine). DMPG (dipalmitoyl phosphatidylglycerol), characterized by R1 ) R2 ) M and R3 ) G is negatively charged whereas DOTAP (N-[1-(2,3-dioleoyloxy)propyl]trimethylammonium salt) is positively charged. In this last case, the phosphate group is not present and the headgroup (R3 ) E) is directly connected to the aliphatic chains via the glycerol group. The chains are R1 ) R2 ) O. All of these chemicals and the NaCl were purchased from Sigma and used without further purification. Dispersion in ultrapure water (Millipore Alpha-Q) was obtained by vortex mixing of these lipids at a concentration of 0.5 mg/mL followed by sonication for 10 min at a temperature higher than the transition temperature of the considered lipid.20 This method is known to form mostly small unilamellar vesicles of phospholipids.4 The suspension was then kept at 27 °C and was used as soon as possible (maximum conservation time: 3 days). All the experiments were also performed at 27 ( 1 °C. X-ray Reflectivity Experiments. Macroscopic vertical foam films were drawn using a rectangular frame (40 × 6 mm) inside a sealed box to maintain water vapor saturation (geometry shown in Figure 2). These films stayed connected to the bulk reservoir via the meniscus. The vertical dimension was adjusted to optimize the film stability and the observable surface. Typically, we adjusted this parameter to near 4 mm. This allowed both longtime stability (at least several hours) and the later measurement of well-defined reflectivity curves. The X-ray reflectivity measurements were performed using a high-resolution diffractometer (Micro-Controle Optix), described elsewhere,21,22 at a wavelength of λ ) 1.5405 Å (Cu KR1 line). Our setup allowed us to measure the ratio R(θ) ) I(θ)/I0, where I0 is the intensity of the incident beam.23 I(θ) is the intensity of the specular beam reflected by the film at an angle θ in the range 6-70 mrad (Qz ) 0.03-0.5 Å-1) and with a low 0.1 mrad divergence. For each incidence angle, the diffusion curve around the specular position was recorded in 10 points (“double-scan” (20) Yamanaka, T.; Hayashi, M.; Matuura, R. J. Colloid Interface Sci. 1982, 88, 458. (21) Schalchli, A.; Sentenac, D.; Benattar, J.-J. J. Chem. Soc., Faraday Trans. 1996, 92, 553. (22) Benattar, J.-J.; Schalchli, A.; Sentenac, D.; Rieutord, F. Prog. Colloid Polym. Sci. 1997, 105, 11113. (23) I0 is measured by placing the detector directly in the incident beam, in absence of the frame. To avoid the destruction of the detector, a calibrated aluminum absorber is intercalated in the beam path.
Cuvillier et al.
Figure 2. Schematic of the setup installed at the center of the diffractometer. A metallic frame is immersed in the phospholipid solution and is then drawn up to form the film suspended on the central part of the frame. The film is then illuminated by the X-ray beam. procedure) in a 1 mrad angular range. This method led to a precise determination of the background level as well as the shape of the reflected beam. The experimental reflectivity was then calculated by integration of this diffusion curve after subtraction of the background. Due to the large number of measurement points, 10 h was needed to acquire a full reflectivity curve so that the method could only be applied to very stable films. However, the method greatly increased both the accuracy and the sensitivity of the measurement: reflectivity as low as 10-7 could be correctly handled. In the other cases (stability limited to a short period of time of between 10 and 60 min), only the specular reflectivity was recorded (“simple-scan” procedure). This leads to shorter measuring times but also to a lower accuracy, especially for the largest incidence angles, where the reflected intensity is low. This method does not provide a accurate determination of the internal film structure. Fortunately, some properties of the film (such as the overall thickness or the average density) could still be determined by the analysis of the low-angle part of the reflectivity curve. In this angular range, the reflectivity is high and the differences between the two measuring procedures are insignificant. In our experiments, the wave-vector transfer Qz is perpendicular to the surface (Qz ) 4π sin θ/λ). Reflectivity provides information about F(z), the electron density profile perpendicular to the surface of the film:24
∫e
R(Qz) ∝ |
iQzz
∂F(z) dz|2 ∂z
(1)
This relation cannot be directly inverted to yield the electron density F(z). The easiest inversion method is to postulate a lamellar model25 for the interface, according to Parrat’s formalism,26 and to adjust its parameters using a least-squares fit.27 But, the accuracy of the reconstructed profile really depends on the agreement between the slab model and the chemical structure of the film. In most cases, our films were described using a symmetrical 5-box model (see Figure 3). This model combines several assumptions that are described here. We will discuss the validity of this model in the discussion paragraph below. First, we assumed that the phospholipid molecule was in a “quasi-extended” state28 (such as the molecules presented in the Figure 3) and that it could be arbitrarily divided into three regions of distinct electron densities. The first zone coincides with the hydrophobic tails of the lipids and contains neext electrons. As the tails contain only C and H atoms, the electron density will be relatively low. The second zone accounts for the central part of the lipid, i.e., around the phosphate group. This part contains (24) Braslau, A.; Pershan, P. S.; Swislow, G.; Ocko, B. M.; Als-Nielsen, J. Phys. Rev. A 1988, 38, 2457. (25) Born, F.; Wolf, E. Principles of optics, 6th ed.; Pergamon: London, 1984; p 51. (26) Parrat, L. G. Phys. Rev. E 1954, 95, 359. (27) We have used SPEEDO (Mike Knewtson, Robert Suter) as the fitting software. (28) Wiener, M. C.; White, S. H. Biophys. J. 1992, 61, 434.
Phospholipidic Black Films
Langmuir, Vol. 16, No. 11, 2000 5031
Figure 3. Modelization of the phospholipidic bilayer. On the basis of its supposed molecular structure, three regions could be distinguished in a lipid molecule. Taking into account the structure of a foam film, this corresponds to a 5-box model for the electron density. The final profile is then calculated by taking into account the roughness of the interfaces. neint electrons. Due to the presence of several heavier atoms (mainly P and O), it should have a higher electron density. The last part accounts for the headgroup of the lipid, namely the choline or the ethanolamine group. The number of electrons is necen, and the electron density is lower than in the central part of the lipid. In the real molecule, the boundaries (and therefore the density gradients) between the different groups are most likely not as clear, but we believe that our model satisfactorily takes into account the electronic properties of the lipid. In a second stage, the layered model is constructed, assuming that the lipids stay side-by-side and form a bilayer. In this bilayer, the hydrophobic tails face the air and the lipid axis is approximately perpendicular to the film surface (actually, our model is also valid for tilted lipids). Therefore, the 3-part model used for the lipid molecule will naturally correspond to a 5-box model for the whole film. The outer boxes (1 and 5) account for the hydrophobic tails and have a thickness of text that will be adjusted during the fitting procedure. The intermediate boxes (2 and 4) of thickness tint (variable) account for the central part of the molecule. As this part is highly hydrophilic, we allowed for the presence of additional water molecules (nwater molecules, adjusted during the fitting), which represent the hydration water. Finally, the central box (3) accounts for the headgroup of the lipids and for any liquid water zone, which might appear. Its thickness is tcen (variable parameter). In the choline case, the headgroup is bulky and hydrophobic: we expected the water to be expelled from this zone, according to the null hydration number given by Israelachvili.29 For the ethanolamine, the headgroup is hydrophilic and of small size. Therefore, we made the assumption that this box contains many water molecules and that the lipid contribution to the electron density is insignificant. The last parameter of the model is the lipid density, given as a mean area per lipid molecule (A). It is then possible to calculate the electronic density in the different layers:
boxes 1 and 5
Fext ) neext/(Atext)
(2)
boxes 2 and 4
Fint ) (neint + 10nwater)/(Atint)
(3)
box 3
cen
F
cen
F
cen
) 2ne
water
)F
cen
/(At
) (for PC lipids) 3
) 0.3 e/Å (for PE lipids)
(4)
Finally, the density discontinuities between the different boxes were smoothened using an interfacial roughness (variable but identical for all the interfaces). This roughness accounts both for the thermally excited collective motions and for the local disorder of the lipid molecules. In addition to this detailed model, we also used simpler 1- or 3-box models to analyze the reflectivity data. This was done when the experimental accuracy was lower (e.g. short lifetime of the film). In each case, the error bars for the whole set of parameters were automatically determined by the fitting software using the following algorithm. It assumes that the best parameters for the layered model correspond to a fit quality characterized by its χ2min. Each of the variable parameters is then slightly modified (one by one), and a new optimum for the other parameters is then recalculated using the same fitting procedure. We have arbitrarily decided that the range of acceptable values corresponds to the following situation: the recalculated χ2 should be lower than χ2min + 1. Actually, this estimate could not take into account all the correlation between the parameters, and it surely underestimates the resulting errors. In general, one can say that the error on the overall thickness is negligible. On the other side, the parameters for the central layers (3, 4, 5) are strongly coupled and the real incertitude on the parameters could be several times higher than the calculated error. For the aliphatic chains, we are in an intermediate situation as these layer parameters are decoupled from the others but do not have a strong influence on the reflectivity curve.
Results PC Films. Black films of DMPC, DPPC, and DOPC were obtained at 27 °C from suspensions of these lipids without added salt. The stability of the DPPC films was limited to a few minutes, and no X-ray reflectivity experiments could be performed. By contrast, the DMPC and the DOPC films were stable for days and we obtained highly accurate measurements of the reflectivity curves. Figure 4 shows the reflectivity for a film of DMPC, recorded using the “double-scan” method. We were able to measure this reflectivity curve for scattering wave vector Qz between 0.06 and 0.5 Å-1. The accuracy of this method combined with the high angular resolution of the experimental setup enabled us to extract much information from this curve. In this case, only the five-layer model previously described in the Experimental Section was able to reproduce the main characteristics of the experimental reflectivity curve (solid line). After least-squares fitting, the calculated density profile, shown in the inset of Figure 4, presents large contrasts between the different parts of the film. The total thickness30 of the film is 55 Å. The hydrophobic tails of the phospholipids (14 carbon atoms), with a thickness of text ) 12.9 Å and an electron density Fext ) 0.8Fwater, are in contact with the air. These parameters also correspond to a mean area per molecule of A ) 57 Å2. The hydrophilic part of the molecule has an extension of 11.4 Å with an electronic density that indicates the presence of 11 molecules of water associated with each lipid. The central part of the film has a thickness of 6.6 Å, i.e., 3.3 Å for each single choline group. The low electron density in this part (0.8Fwater) evidences the absence of any water molecule. The roughness of the interfaces between these sublayers is 3.4 Å. These results are summarized in the Table 1. (29) Israelachvili, J. N. Intermolecular and surface forces; Academic Press: Orlando, FL, 1991. (30) The calculated error bars will not be presented in the text but only in the recapitulative table (Table 1) in order to make the text easier to read.
5032
Langmuir, Vol. 16, No. 11, 2000
Figure 4. X-ray reflectivity of DMPC foam films (no added salt). Thanks to the high stability of these lipid films, the measure was performed using the “double-scan” method over a large range of scattering wavevector (0.06 < Qz < 0.5 Å-1). Therefore, the accuracy of the recorded curve is high. In the case of a homogeneous film, the reflectivity curve presents strong and regular modulations (due to the interference between the X-ray reflected at the two air/film interfaces and called “Kiessig fringes”), shown by the dashed line. In the DMPC case, these fringes are modified, with characteristic structures such as the small intensity of the second fringe. This pattern could only be explained by the density model shown in the inset, corresponding to a 5-layer model. In contact with the air, the hydrophobic layer has a low electron density. The hydrophilic part of the molecule is characterized by its higher density, largely superior to that of the central core of the film. The high accuracy of the experimental curve provides an unambiguous determination of the various parameters used in this model, as revealed by the agreement between the experimental data (plain circles) and the reflectivity calculated from the model (solid line).
Figure 5 shows the reflectivity of a film of DOPC (Qz between 0.05 and 0.5 Å-1), measured using the “doublescan” method. Again, only the same 5-box model can fit the observed reflectivity curve (solid line). After leastsquares fitting (inset of Figure 5), the overall thickness of the film is 59 Å and the internal structure is contrasted. The aliphatic chains (18 carbon length), on the outside of the film, are 14.2 Å thick and have a density of 0.9Fwater, corresponding to an area per molecule of A ) 64 Å2. The hydrophilic part of the DOPC is 11.8 Å thick and is associated with ca. 13 molecules of water. The measured size for the choline group is 3.3 Å, leading to a central layer thickness of 6.6 Å, with a density of 0.7Fwater. Again, no water could be present in the central part of the film. The interface roughness is ca. 3.6 Å. For these two PC lipids, an addition of salt (0.08 M NaCl) has no effect on the film structure. PE Films. Solid circles in Figure 6 show the “doublescan” reflectivity curve obtained for a film of DOPE, withdrawn from a solution without added salt. By using the 5-box model to fit this curve, a small modulation of the electronic density within the film could be detected. An area per lipid molecule of 74 Å2 and a thickness of 11.6 Å for the external layer (density: 0.9Fwater) were measured. The intermediate part of the film, which yields the hydrophilic zone of the lipid molecules, is 11.6 Å thick. Each lipid is hydrated by 12 water molecules. The central part of the film is mostly composed of liquid water. Its thickness is ca. 22 Å, and its density has been taken equal to Fwater. If one makes the assumption that the extent of an ethanolamine group is ca. 3 Å, the measured thickness indicates that a film of liquid water is present in the core of the film, with a thickness of ca. 16 Å. In the presence of small quantities of added salt (10-3 M of NaCl for the open circles in Figure 6), the lifetime
Cuvillier et al.
of the film is dramatically reduced to less than 1 h (in contrast to days for solutions of lipids in pure water). However, the position of the minimum of reflectivity (between the first and second Kiessig fringes) could be easily and accurately determined.31 Using a single-box model, a total thickness equal to 46 Å was measured, a value much smaller than that measured for the same film in the absence of salt (69 Å). DOTAP Films. The solid circles in Figure 7 show the reflectivity of DOTAP in the presence of 10-3 M of added NaCl. The gap between two consecutive minima (0.02 Å-1) corresponds to a thickness of several hundreds of angstroms. Moreover, the Kiessig fringes are regular, indicating the lack of electron density gradient within the film. Obviously, this situation could only be obtained if the central water core is the major part of the film and it could not correspond to the formation of a multilayer of DOTAP.32 This curve was fitted using a 3-box model, which gives an overall thickness of ca. 300 Å. Two thin layers (thickness: 20 Å) of low electronic density (0.8Fwater), corresponding to the lipids, surround a large central layer of water. This water, with a thickness of 260 Å, occupies almost all of the film. In the presence of large quantities of salt (0.15 M), as shown by the open circles in Figure 7, the reflectivity curve is different as it has much larger Kiessig fringes. Instead of 0.02 Å-1 between two minima, this gap is increased to 0.11 Å. With the salt, the lifetime of the film is decreased and the reflectivity curve was measured using the “singlescan” method. Qualitatively, the experimental curve should correspond to a film with an almost constant electron density along the film. This must be due to the lack of electronic density contrast between the hydrophobic and hydrophilic parts for the DOTAP molecule. We then used a single-box model for the fitting. The coincidence between the fitted and the experimental curves is limited to the Qz range 0.06-0.3 Å-1. With an overall thickness of 52 Å, the central water core was strongly reduced. Therefore, the mean electron density of 0.8Fwater should correspond to the lipids only, with a mean area per DOTAP molecule of about 76 Å2. PG Films. DMPG films were obtained only for a salt concentration of 0.15 M NaCl and with a limited lifetime. Squares in Figure 7 show the “single-scan” reflectivity for such a film. Regular and marked fringes in a Qz range of 0.06-0.23 Å-1 were observed. Due to this limited Qz range, the results obtained by using a 5-box model are not significant. However, by using a single-layer model, the precise overall thickness of the film could be obtained: 90 Å. In this model, the mean electron density is close to that of water, indicating that the central water core occupies an important part of the film. Due to this predominance of water, it is not possible to compute the mean area per DMPG molecule. Discussion Validity of the Model. X-ray reflectivity has a major advantage over most of the techniques previously used for the study of soap films: the internal structure of the film could be self-reliantly determined. However, the structure determination is biased by the method used for the data analysis. As a direct inversion of the reflectivity (31) In this case, the lack of time does not enable a precise setup of the system. Therefore, the measured reflectivity could be lower than the real one due to small misalignments. The position of the reflectivity minimum is however not influenced by such problems. (32) In this case, Bragg peaks would have appeared in the reflectivity curve.
Phospholipidic Black Films
Langmuir, Vol. 16, No. 11, 2000 5033 Table 1. X-ray Reflectivity Results lipids DOPE + 0.001 M NaCl
DOTAP + 0.001 M NaCl
DOTAP + 0.15 M NaCl
DMPG + 0.15 M NaCl
69 ( 0.4 74 ( 0.4 4.8 ( 0.15
47 ( 0.2 NDa ND
300 ( 2 70-80 8 ( 0.2
52 ( 0.5 76 ( 2 2 ( 0.5
90 ( 0.3 ND 2 ( 0.7
0.9 ( 0.05 14.2 ( 0.07
0.9 ( 0.05 11.6 ( 0.03
ND ND
ND ND
ND ND
1.2 ( 0.04 11.4 ( 0.03 11.5 ( 0.2
1.1 ( 0.06 11.8 ( 0.04 13 ( 0.2
1.1 ( 0.04 10.1 ( 0.03 12 ( 0.5
ND ND ND
ND ND ND
ND ND ND
0.8 ( 0.05 choline 3.3 ( 0.02
0.7 ( 0.04 choline 3.3 ( 0.03
1 water (8 + 3)e ( 0.01
ND ND ND
ND ND ND
1 water ∼20 Å
params
DMPC
DOPC
DOPE
overall thickness (Å) area per lipid (Å2) roughness (Å) aliphatic chains densityb thickness (Å) hydrophilic part densityb thickness (Å) bound water terminal part densityb type thicknessd (Å)
55 ( 0.2 57 ( 0.2 3.4 ( 0.06
59 ( 0.3 64 ( 0.3 3.6 ( 0.1
0.8 ( 0.05 12.9 ( 0.05
1 water 130 ( 1
a Not determined. b The electronic density is normalized by the density of water (Fwater ) 0.3 e-/Å3). c Total lipid thickness: ∼20 Å. Mean density: 0.8. d In the 5-box model, the central layer is symmetric and groups lipids of both sides of the film. In this table, the given value is for one part of the film only. Then the thickness of the central layer is obtained by multiplying the given value by 2. e 3 Å estimated for the ethanolamine group + 8 Å of liquid water. f Estimated.
Figure 5. X-ray reflectivity of DOPC foam films (no added salt). This lipid only differs from that of Figure 4 by the nature of the aliphatic chains. The X-ray reflectivity curve is similar for these two compounds. The best fit is obtained by using the same 5-box model, leading to the same type of profile. The highest density coincides with the hydrophilic groups, whereas no water is present in the central core of the film. The overall thickness (60 Å) is slightly larger that of the DMPC film.
data is not possible, we had to resort to a modelization of the film and then to a least-squares fitting of the data. This procedure could strongly influence the determined structure if the model was not correctly chosen. In our case, the choice of the model was partly imposed by the shape of the reflectivity curve. Indeed, the second Kiessig fringe (between 0.25 and 0.32 Å-1) is strongly reduced (width and intensity) when compared to that for homogeneous films (see Figure 4). To reproduce this feature, we extensively tested free-constraint models with 1, 3, or 5 boxes. Our simulations indicate that the 1- and 3-box models do not correspond to the observed data: the simulated reflectivity curves are too regular. In the 5-box case, our tests indicate that the model should necessarily satisfy the following condition: the density of boxes 2 and 4 has to be higher than the density of the other boxes (1, 3, 5). Moreover, the shape of the second fringe depends mainly on the central part of the film (boxes 3, 4, 5). Taking these results into account, we used the model described above in the experimental paragraph, which also incorporates the chemical constraints created by the nature of the lipid. In addition to the quality of the fitting, the validity of our model could be verified by its internal
Figure 6. X-ray reflectivity of DOPE foam films (with and without added salt). Without added salt, the reflectivity curve (plain circles) presents very regular Kiessig fringes, corresponding to small density gradients within the film (see the profile in the inset). After addition of 10-3 M NaCl, the lifetime of the film is greatly reduced and we used the “single-scan” method to measure the reflectivity (open circles). However, only the Qz position of the first minimum could be easily determined. Therefore, only the overall thickness of the film (46 Å) could be accurately measured using the single-box model shown in the inset.
and external coherency. The internal coherency is tested by comparing the results for different lipids whereas the external coherency corresponds to the comparison of our results with those already published for lipid layers, e.g. in Langmuir monolayers,33 absorbed layers,34-36 or lamellar phases.26,37 All the values summarized in Table 1 are consistent. Moreover, the parameters corresponding to the constant part of the different lipids (i.e. their intermediate parts) are almost constant: they have the same thickness and the same number of hydration water molecules. This last value ((12 water molecules per lipid) might appear high, but it is compatible with that observed in Langmuir films31 and with the high hydrophilicity of this region. On the other hand, the measured thickness for this layer is larger than the given values obtained from a simple molecular model (8-9 Å) or those measured in other systems.32,33,38 (33) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (34) Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J. A.; Fu, J.; Conner, J. T.; Plant, A. L. Biophys. J. 1998, 74, 1388.
5034
Langmuir, Vol. 16, No. 11, 2000
Figure 7. Reflectivity curves (shifted for the visibility) corresponding to films of charged phospholipids. The points are the experimental data and the lines the reflectivity calculated from the best fitted electronic density profiles.
This could indicate an unusual conformation of this lipidic portion (elongated and perpendicular to the plane of the film) or a problem in the definition of the different zones used in the model. For example, for some authors,39 the headgroup is oriented parallel to the surface place. In our case, this could slightly change the results of our model and especially the number of water molecule in the different part of the film. However, thanks to the particular shape of the reflectivity curve, our model remains globally valid even in this unfavorable case. To be more specific, the results concerning the presence or the absence of liquid water in the central layer (box 3) will not be modified. Up to now, we have focused our discussion on the case of the 5-box model. However, even when only simpler models are used for the data analysis, X-ray reflectivity has a crucial advantage over optical techniques: the film thickness could be determined in absolute terms, with no influence from the internal composition of the film. Influence of the Tail. The stability of the film could be easily verified visually: DMPC, DOPC, and DOPE films are stable for several hours (even up to several days). The lifetime of the DOTAP and DMPG films is limited to a few hours. In the other cases, the films burst during the drainage. Actually, it is well-known that macroscopic Newton black films can be formed only if the surfactant forms a dense monolayer at the interface.1 In our case, the lipid surface concentration depends on the state of the lipid in the solution: below the gel/liquid crystal transition temperature, the surface film is in a gaseous state40 and both the surface pressure and the surface concentration are near zero. It is therefore expected that stable films will only be obtained for lipids with a transition temperature lower than room temperature. This is confirmed by our observations. For example, in the PC case, only the DMPC (Tc ) 23.5 °C) and the DOPC (Tc < 7 °C) form stable films in contrary to the DPPC (Tc ) 41 °C). Moreover, the X-ray reflectivity can directly probe the state of the aliphatic chains in the film. In every case, an area per molecule was measured well above the (35) Bayerl, T. M.; Thomas, R. K.; Penfold, J.; Rennie, A.; Sackmann, E. Biophys. J. 1990, 57, 1095. (36) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J. 1991, 59, 289. (37) Nagle, J. F.; Zhang, R.; Tristram-Nagle, S.; Sun, W.-J.; Petrache, H. I.; Suter, R. M. Biophys. J. 1996, 70, 1419. (38) Helm, C. A.; Mo¨hwald, H.; Kjaer, K.; Als-Nielsen, J. Europhys. Lett. 1987, 4, 697. (39) Seelin, J. Biophys. Acta 1978, 515, 105. (40) Tajima, K.; Gershfeld, N. L. Biophys. J. 1985, 47, 203.
Cuvillier et al.
limiting area corresponding to the close-packing of the lipids (ca. 40 Å2). In the PC case, the areas measured in the film (DMPC, 57 Å2; DPPC, 64 Å2) largely correspond to those measured for DPPC in a liquid crystal phase: 60.8 Å2 for Meuse et al.34 in an absorbed bilayer; 62.9 Å2 for Nagle et al.37 or 59.4 Å2 for Wiener et al.28 in multilayers. The small difference between DOPC and DMPC is due to the presence of a double bond CdC in the aliphatic chains of DOPC that interferes with the stacking of the lipid chains. There is, therefore, a decrease in the surface density and an increase in the interfacial roughness (due to the disorder). Therefore, our X-ray reflectivity results confirm, at the molecular scale, that only lipids in their liquid crystal state are able to form stable films. Influence of the Headgroup. It is well-known that the thickness of lipid films is controlled by the force balance between the two lipid layers. In most cases and more specifically for thick film, the DLVO41,42 theory provides a good description of the variation of these forces with the modification of control parameters (e.g. disjoining pressure, ionic strength, or pH of the water solution). On the other hand, the relation between the chemical nature of the headgroup and the force scheme is not very well documented. Therefore, we have compared the thickness of lipid films for different headgroups. For charged lipids, the main forces in the film have an electrostatic origin.43,44 This is clearly visible in the case of the DOTAP, a positively charged lipid. In the absence of salt, the electrostatic forces are not screened and the film remains thick (300 Å) with a large quantity of water. By addition of a large amount of NaCl (0.15 M), the film becomes extremely thin (52 Å) due to the total screening of the electrostatic forces. The film structure has not been explicitly measured, but the thickness of the central water layer is most likely reduced to a few angstroms only: one has a Newton black film (NBF). For negatively charged lipids (DMPG), only a film of limited stability was obtained (with 0.15 M of NaCl). From our data, the overall thickness (91 Å) was measured. Although the internal structure has not been determined, the thickness of the water layer is estimated to be around 40 Å, corresponding to a common black film (CBF). The difference between the two charged lipids (DMPG vs DOTAP) is due to the metastability of the CBF made with DMPG. In this latter case, the film lifetime is short and we had to record the reflectivity rapidly. Therefore, the film was still in the CBF state and had not transited to the NBF state. If the transition leads to the bursting of the film, the NBF would not be observed as in the DOTAP case. We also studied the case of a neutral lipid using films of DMPE and DMPC. Both these lipids are zwiterionic, with a negative charge on the phosphatidyl group and a positive charge on the nitrogen atom of the headgroup. In such a situation, electrostatic interactions should be reduced to low dipole-dipole repulsions and should not play an important role in the structure of the film. This is mostly the case for the DMPC films, for which thickness does not depend on the ionic strength. In this case, the water is only present as hydration water, (41) Derjaguin, B. V.; Landau, L. Acta Physicochim. URSS 1941, 14, 633. (42) Verwey, E. J. W.; Overbeck, J. T. G. Theory of the stability of lyophobic colloids; Elsevier: Amsterdam, 1948. (43) Mysels, K. J.; Huisman, H. F.; Razouk, R. I. J. Phys. Chem. 1966, 70, 1339. (44) Exerowa, D.; Kolarov, T.; Kristov, K. Colloids and Surface 1987, 22, 171.
Phospholipidic Black Films
associated with the hydrophilic part of the lipids. Even in the middle of the film, no liquid water could be detected. Therefore, we have a NBF. In some films containing PC lipids (e.g. ref 6), the presence of a liquid water core was detected in contradiction with our results. Actually, the transition between a CBF and a NBF for this type of lipids appears to be highly dependent on the experimental setup, as shown by the large difference between the behavior of microscopic and macroscopic films. In our particular case, the use of X-ray reflectivity requires the formation of a film of large surface. Such a film has a higher probability of transiting to the NBF state, and this could be the reason we do not observe a CBF. A priori, one could expect the same behavior for PE and PC films if the structure is imposed mainly by the electrostatic interactions. However, this is not the case. We detected the presence of a 16 Å water core for DOPE film without salt. This thickness corresponds to a layer of free water. This could be due mainly to the hydrophilicity of the ethanolamine group. This chemical group could largely favor the presence of a large quantity of water, by its ability to form an H bond. Therefore, this film is in a CBF state in the absence of added salt. The electrostatic origin of the forces that prevent the formation of a NBF has been verified by adding some salt to the water. The 10-3 M of NaCl (to be compared to 0.15 M for DOTAP) is enough to totally screen the electrostatic repulsion and to obtain a NBF (of thickness 47 Å).
Langmuir, Vol. 16, No. 11, 2000 5035
These examples obviously indicate that, for films made from charged lipids, the electrostatic interactions regulate the state (and the thickness) of the film. In the case of zwiterrionnic lipids, the situation is much more subtle and the chemical nature of the headgroup has to be taken into account to understand the behavior of the films. Conclusion X-ray reflectivity is perfectly suited for the study of phospholipidic black films. In contrast with the other techniques already used in such investigations, the internal substructure can be determined with a molecular scale resolution. Therefore, we were able to accurately study the relation between the film structure and the molecular composition of the lipids. This enabled a fine-tuning of the film properties and especially the selection of the hydrophile/ hydrophobe balance for the central part of the film. The behavior of pure lipid films now being well established, we aim to study the interaction of these films with proteins, dissolved in the subphase. This situation could be a suitable tool for the formation of large bidimensional mixed films of proteins and lipids, following the scheme proposed by Benattar et al.45 for the insertion of BSA within black films of soluble surfactants. LA990745M (45) Benattar, J. J.; Nedyalkov, M.; Prost, J.; Verger, R.; Tiss, A.; Guibert, C. Phys. Rev. Lett. 1999, 82, 5097.
