Organization of β-Cyclodextrin under Pure Cholesterol, DMPC, or

9616

Langmuir 2008, 24, 9616-9622

Organization of β-Cyclodextrin under Pure Cholesterol, DMPC, or DMPG and Mixed Cholesterol/Phospholipid Monolayers J. Mascetti,† S. Castano,‡ D. Cavagnat,† and B. Desbat*,‡ ISM, Institut des Sciences Mole´culaires (UMR 5255 CNRS), UniVersite´ Bordeaux 1, 351, cours de la Libe´ration, 33405 Talence Cedex, France, and CBMN, Chimie et Biochimie des Membranes et Nano-objets (UMR5248 CNRS), UniVersite´ Bordeaux 1, ENITAB, 2 rue Robert Escarpit, 33607 Pessac, France ReceiVed February 8, 2008. ReVised Manuscript ReceiVed June 4, 2008 The complexation of β-cyclodextrin with monolayers of cholesterol, DMPC, DMPG, and mixtures of those lipids has been studied using Brewster microscopy, PMIRRAS, and ab initio calculations. An oriented channel-like structure of β-cyclodextrin, perpendicular to the air/water interface, was observed when some cholesterol molecules were present at the interface. This channel structure formation is the first step in the cholesterol dissolution in the subphase. With pure DMPC and DMPG monolayers, weaker, less organized complexes are formed, but they disappear almost completely at high surface pressure, and only a small amount of phospholipid is dissolved in the subphase.

Introduction Betacyclodextrin (β-CD) is a cyclic oligosaccharide molecule comprising seven R-(1,4)-glucopyranose units. This conical molecule has an external polar surface and an internal hydrophobic cavity, which allows it to complex and solubilize hydrophobic molecules in aqueous solution. This property has been used in many applications and theoretical work including biomimetic reactions, mediated organic reactions,1 drug carriers,2 and the extraction of lipids.3–5 An extensive review of the properties and applications of cyclodextrins has recently been published.6 β-CD (and its derivatives) is the most used cyclodextrin because it has the capacity to extract cholesterol from lipid membranes very efficiently.7 Many studies have been performed to elucidate the conditions of cholesterol extraction by β-CD in cultures of cell membranes8–11 and in related membrane models.12–16 They showed that cholesterol can be extracted from both cell and monolayer membranes, but some questions remain on the mechanism of extraction and on the ability of β-CD to extract other compounds from membranes. The Langmuir method is able to give some information on this problem because the physicochemical * To whom correspondence should be addressed. Phone: (33) 5 40 00 30 46. Fax: (33) 5 40 00 30 73. E-mail: [email protected]. † ISM. ‡ CBMN.

(1) Breslow, R.; Dong, S. D. Chem. ReV. 1998, 98, 1997–2011. (2) Uekama, K.; Hirayama, F.; Irie, T. Chem. ReV. 1998, 98, 2045–2076. (3) Courelongue, J.; Maffrand, J. P. U.S. Patent 4,880,573, 1989. (4) Cully, J.; Vollbrecht, R. R. U.S. Patent 3,342,663, 1994. (5) Shieh, W.; Hedges, A. U.S. Patent 5,371,209, 1994. (6) Chem. ReV. 1998, 98(5). (7) Christian, A. E.; Haynes, M. P.; Phillips, M. C.; Rothblat, G. H. J. Lipid Res. 1997, 38, 2264–2272. (8) Parpal, S.; Karlsson, M.; Thorn, H.; Stralfors, P. J. Biol. Chem. 2001, 276, 9670–8. (9) Rothberg, K. G.; Heuser, J. E.; Donzell, W. C.; Ying, Y.-S.; Glenney, J. R.; Anderson, R. G. W. Cell 1992, 68, 673–682. (10) Fujimoto, T.; Fujimoto, K. J. Histochem. Cytochem. 1997, 45, 595–598. (11) Levitana, I.; Christian, A. E.; Tulenkoc, T. N.; Rothblatb, G. H. J. Gen. Phyiol. 2000, 115, 405–416. (12) Giocondi, M. C.; Milhiet, P. E.; Dosset, P.; Le Grimellec, C. Biophys. J. 2004, 86, 861–869. (13) Dietrich, C., Z. N.; Volovyk, M.; Levi, N. L.; Thompson, K.; Jacobson, Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10642–10647. (14) Lawrence, J. C.; Saslowsky, D. E. ; Edwardson, J. M.; Henderson, R. M. Biophys. J. 2003, 84, 1827–1832. (15) Leventis, R., J. R.; Silvius, Biophys. J. 2001, 81, 2257–2267. (16) Niu, S.-L.; Litman, B. J. Biophys. J. 2002, 83, 3408–3415.

properties of the monolayer (temperature, surface pressure, lipids proportions, subphase, etc.) can be controlled at will. In this regard, Ohvo et al.17 use compression curves at constant pressure and measurements of scintillation to determine the essential parameters that drive the desorption of cholesterol from a monolayer at the air/water interface. They thus showed that at 30 °C and 20 mN/m the monolayer of cholesterol loses around 20% of its molecules within 10 min. This rate of cholesterol desorption increases exponentially with the surface pressure and decreases when the temperature drops. Moreover, the desorption of cholesterol strongly decreases when it is mixed with a phospholipid and also depends on the nature of the phospholipid. The results obtained by Ohvo et al.17 are not fully consistent, and the authors explain this discrepancy by the absorption of the cholesterol/β-CD complex on the walls of the Teflon trough. Previous work18 showed that the ability of β-CD to form a soluble complex depends not only on the host molecules but also on its environment. In the case of cholesterol extraction from cell membranes, the composition of the membrane will play a key role not only in the ability of these two molecules to form a stable complex but also in the accessibility of the cholesterol. To date, there is no structural data on these complexes and even less on their organization at the interface comprising phospholipidembedded cholesterol. Therefore, the goal of this article is to determine, using polarization modulation infrared absorption spectroscopy (PMIRRAS) and Brewster angle microscopy (BAM), the organization and the orientation of the complexes of β-CD with cholesterol in the presence and absence of neutral and charged lipids.

Experimental Procedures Materials. Cholesterol, β-CD, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DMPG (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)] sodium salt) were purchased from Sigma, France. Lipids and β-CD were used without further purification. Chloroform (CHCl3) and methanol (CH3OH) were purchased from Sigma-Aldrich, Steinheim, Germany. The ultrapure water subphase was obtained from a Milli-Q (Millipore, Molsheim, France) system with a nominal resistivity of 18.2 MΩ · cm. Saturated β-CD solutions (5 mg/mL) were prepared in water at 20 °C. Cholesterol, DMPC, DMPG, or mixed cholesterol/DMPC or (17) Ohvo, H.;.; Slotte, J. P. Biochemistry 1996, 35, 8018–8024. (18) Harata, K. Chem. ReV. 1998, 98, 1803–1827.

10.1021/la8004294 CCC: $40.75  2008 American Chemical Society Published on Web 08/02/2008

Organization of β-Cyclodextrin cholesterol/DMPG stock solutions at ∼1 mg/mL were prepared by dissolution of the powders in CHCl3 (for neutral lipids) or CHCl3/ CH3OH (5/1 v/v) (for negatively charged lipids), and a few microliters were deposited at the interface of either the β-CD-saturated or water subphase of the Langmuir trough. Isotherm Measurement. The compression of monolayers was recorded on two Nima Langmuir troughs (Coventry, England) at 20 °C with a compression rate of 5 Å2/molecule/mn. The surface pressure (π) was monitored by a Wilhelmy surface balance (Nima) using a filter paper plate (Whatman), and the accuracy was checked by comparing with the previously published isotherm of cholesterol. Brewster Angle Microscopy. The morphology of the layers at the air-water interface was observed using a Brewster angle microscope (NFT BAM2plus, Go¨ttingen, Germany) mounted on two Teflon Langmuir troughs (Nima). The microscope was equipped with a frequency-doubled Nd:Yag laser (532 nm, 20 mW), ×10 lens, polarizer, analyzer, and CCD camera. The spatial resolution of the BAM was about 2 µm, and the image size was 600 × 450 µm. The reflectivity calibration obtained from the gray level and the layer thickness estimate, using the refractive index of water (nwater ) 1.33) and of the monolayer (nmonolayer ) 1.49 and 1.45 for cholesterol and an aqueous solution of β-CD, respectively) was calculated with the BAM2plus software package (I-Elli2000). For thin films, the reflected intensity is proportional to (nd),2 where n is the refractive index and d is the film thickness. Assuming a 10% relative error in the gray level (which is an overestimate of the error), the relative error in the thickness is ca 5%. PM-IRRAS. The Fourier transform infrared spectra were recorded on a Nicolet 870 spectrometer equipped with an external HgCdTe photovoltaı¨c detector (SAT, France) cooled to 77 K. A detailed description of the PM-IRRAS setup as well as the experimental procedure has already been published.19 A previous report20 has shown that the normalized PM-IRRAS signal is proportional to two parameters: the number of molecules per unit area and an orientation function f(θ):

N IPMIRRAS ∝ f(θ) A I is the normalized PMIRRAS signal intensity, N is the number of molecules on the surface, A is the area of the trough at a given pressure, f(θ) is an orientation function, and θ is the average tilt angle of the vibrational transition moment with respect to the normal surface. The expression of the orientation function f(θ) for a single transition moment19 is f(θ) ) sin2 θ - sin2 θb, with θb˜ ) 38°, the “magic angle” for which no PM-IRRAS signal is observed at the air/water interface. When the transition moment is parallel to the interface, the absorption is a strong positive band whereas the band is negative when the transition moment is perpendicular. Ab Initio Calculations and Simulation of PMIRRAS Spectra. The minimum-energy structure of the β-CD molecule was calculated at the DFT level (B3LYP functional, 6-31G* basis sets) with Gaussian 9821 on four processors on a SGI IRIX64. Owing to the large number of atoms in this system (147 atoms), this calculation needs 1295 basis functions and 2436 Gaussian primitives. Vibrational frequencies and IR intensities were calculated at the same level. For comparison to experimental data, the calculated frequencies were scaled by a factor of 0.951. The components of the dipole moments corresponding (19) Blaudez, D.; Turlet, J. M.; Dufourcq, J.; Bard, D.; Buffeteau, T.; Desbat, B. J. Chem. Soc., Faraday Trans. 1996, 92, 525. (20) Mao, L.; Ritcey, A. M.; Desbat, B. Langmuir 1996, 12, 4754–4759. (21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, Gaussian, Inc.: Pittsburgh, PA, 1998.

Langmuir, Vol. 24, No. 17, 2008 9617

Figure 1. Isotherm of cholesterol at the air/water interface with inset Brewster pictures taken during compression.

to each vibration were calculated from the ab initio-computed atomic polar tensors.22 Previously described23 general software has been used to obtain the optical indexes of β-CD and to simulate its PM-IRRAS spectrum on water. In the first step, the IR transmittance spectra of β-CD powder in a diamond cell have been recorded under an infrared microscope. The interferences of the cell in the near-IR have been used to determine the thickness of the sample. In the second step, the IR spectrum and the thickness give us the possibility to calculate the complex isotropic indexes of β-CD. Finally, assuming that the isotropic absorption index (k) is related to the uniaxial anisotropic indexes by the expression k ) (kx + ky + kz)/3, with kx ) ky, and taking into account the ratio intensity (kz/kx) of the main absorption bands given by the ab initio calculation, we have built the anisotropic optical file of β-CD in the infrared.

Results Cholesterol Film on Water. It was previously reported that cholesterol molecules form monolayers and multilayers at the water surface.24 This is confirmed through the pressure isotherm and Brewster picture analysis (Figure 1). At low surface pressures, the cholesterol molecules form circular domains, typical of fluid phases. Around 35 mN/m and 38 Å2/molecule, the monolayer is homogeneous. Beyond the collapse point, at 47mN/m, some multilayers appear for which the thickness can be estimated through the calibration of gray intensities of BAM pictures. In Figure 1, pictures at 0 and 35 mN/m have been recorded with a shutter opening time of 20 ms, whereas the latest one has been recorded within 2 ms. This difference indicates roughly that the gray level on the last picture is 10 times more intense than on the first two. More quantitatively, the calibrated reflected intensities allow us to evaluate a thickness of 18 ( 1 Å for the monolayer and 54 ( 3 Å for the multilayer film. Thus, after the collapse, the cholesterol molecules form essentially three-layer films. Incidently, the hypothesis of a bilayer film in equilibrium with the trilayer one after collapse, which was suggested in a previous work,24 seems to be incorrect. Furthermore, the physical aspect of these domains shows that we are no longer dealing with fluid or crystalline phases but more likely with a liquidcrystal mesophase. (22) (a) Biarge, J. F.; Herrantz, J.; Morcillo, J. J. Annu. ReV. Soc. Esp. Fis. Quim. 1961, A57, 81. (b) Person, W. B.; Newton, J. H. J. Chem. Phys. 1974, 61, 1040. (23) Buffeteau, T.; Blaudez, D.; Pe´re´, E.; Desbat, B. J. Phys. Chem. B 1999, 103, 5020–5027. (24) Lafont, S.; Rapaport, H.; So1mjen, G. J.; Renault, A.; Howes, P. B.; Kjaer, K.; Als-Nielsen, J.; Leiserowitz, L.; Lahav, M. J. Phys. Chem. B 1998, 102, 761–765.

9618 Langmuir, Vol. 24, No. 17, 2008

Mascetti et al.

Figure 2. Isotherms of cholesterol on a β-CD aqueous solution (s) and pure water (---) with inset Brewster pictures taken during compression and decompression.

Cholesterol Film on Saturated Aqueous Solutions of β-Cyclodextrin. First, the purity of the β-CD solution was checked by performing BAM, isotherm, and PMIRRAS measurements. No difference was observed with respect to pure water, including the surface pressure. As shown in Figure 2, the compression isotherm of cholesterol molecules deposited on a saturated aqueous solution of β-CD is very different from that obtained on pure or salted water (dotted-line graph). A nonzero pressure (5 mN/m) is measured over a molecular area of 60 Å2, indicating a larger steric hindrance in the presence of β-CD. Also, the pressure does not reach high values, even for lower molecular areas, compared to the molecular area limit of cholesterol (38 Å2). Moreover, the isotherm exhibits irreversible hysteresis during the decompression. Later, we will see the influence of the formation of aggregated domains at the interface and/or of the diving of cholesterol molecules into the subphase for this hysteresis. The Brewster pictures (Figure 2) allow us to understand the cholesterol behavior better. At 60 Å2/molecule, the cholesterol layer exhibits a more intense and heterogeneous luminosity in the presence of β-CD than on pure water over the same molecular area. This behavior is a sign of a greater concentration of matter at the interface. For very low areas such as 25 Å2, domains with large, variable luminosity appear. Their shapes are totally different from those observed for three-layer cholesterol films and are more characteristic of polycrystalline material. Using a refractive index of 1.45 for the β-CD aqueous solution, different thicknesses of 40 ( 2, 60 ( 3, 80 ( 4, and 100 ( 5 Å have been calculated from calibrated gray intensities of Brewster pictures. During decompression, the thickest domains disappear, and the film becomes more homogeneous (∼60 Å), even if a few aggregated domains remain at the surface. To obtain molecular information on this system, PMIRRAS spectroscopy was employed. The PMIRRAS spectrum of cholesterol on a saturated aqueous β-CD solution obtained at a large compression value (25 Å2) is shown in Figure 3a, together with that obtained for a cholesterol monolayer on pure water (Figure 3b) and the absorption spectrum of solid β-CD (Figure 3c). The spectrum obtained with β-CD solution is very intense (for instance, the most intense band is greater than the PMIRRAS bands observed for a monolayer of a large protein system such as PSII25) and essentially exhibits β-CD lines. This implies that β-CD molecules gather under the (25) Gallant, J.; Desbat, B.; Vaknin, D.; Salesse, C. Biophys. J. 1998, 75, 2888–2899.

Figure 3. (a) PMIRRAS spectrum of cholesterol on an aqueous β-CD solution (s). (b) PMIRRAS spectrum of cholesterol on water (---). (c) Absorbance IR spectrum of β-CD powder.

Figure 4. Isotherms of DMPC on a β-CD aqueous solution (s) and on pure water (---), with inset Brewster pictures taken during compression.

cholesterol layer with a significant thickness. However, the β-CD molecules are oriented under the cholesterol layer because the relative intensities of the strongest β-CD infrared absorptions are different from those obtained for solid disordered β-CD. DMPC on β-CD Solution. The DMPC monolayer isotherms on pure water and on saturated β-CD solution are shown in Figure 4. In the presence of β-CD, the beginning of the pressure increase occurs over larger molecular areas. Then, both isotherms converge at an area value of 60 Å2. The decompression curve exhibits irreversible hysteresis, which can be associated with the loss of molecules at the interface. Depending on the subphase, water or β-CD solution, the Brewster pictures of DMPC monolayer are very different. On pure water, DMPC is in a homogeneous fluid phase at 24 °C whatever the surface pressure, whereas on β-CD solution, aggregated bright domains are observed at low and medium pressure (5 and 15 mN/m in Figure 4). Those domains partially disappear when the pressure increases (33 mN/m in Figure 4). Thanks to the PMIRRAS spectra, the presence of β-CD is observed near the interface over the full range of pressure (Figure 5b,d), but with a much weaker intensity than in the case of cholesterol.

Organization of β-Cyclodextrin

Figure 5. PMIRRAS spectrum of DMPC (a) on water (π ) 5 mN/m), (b) on an aqueous β-CD solution (π ) 5 mN/m), (c) on water (π ) 30 mN/m), and (d) on an aqueous β-CD solution (π ) 30 mN/m).

Langmuir, Vol. 24, No. 17, 2008 9619

Figure 7. PMIRRAS spectrum of DMPG on an aqueous β-CD solution (a) (π ) 5 mN/m) and (b) (π ) 30 mN/m).

Figure 8. Isotherms of the DMPC/cholesterol 3/1 molar mixture on the β-CD aqueous solution (s) and on pure water (---), with inset Brewster pictures taken during the compression. Figure 6. Isotherms of DMPG on a β-CD aqueous solution (s) and on pure water (---), with inset Brewster pictures taken during compression.

A comparison of these spectra with those of DMPC on pure water at the same surface pressure (Figure 5a,c) shows a broadening of the phospholipid bands at 1730 and 1450 cm-1 and β-CD absorption at around 1050 cm-1. Carbonyl groups are well known to be sensitive to hydrogen bond interaction, so the broadening of the ester vibration at 1730 cm-1 suggests the presence of hydrogen-bonded β-CD with the phospholipids. Moreover, the β-CD absorption band does not have any clearly defined structure, whatever the pressure, and the maximum observed at 1060 cm-1 no longer corresponds to the most intense band of β-CD but to weaker modes of the bulk β-CD spectrum (Figure 3c). DMPG on β-CD Solution. The behavior of β-CD molecules under a monolayer of DMPG is different from that observed under a DMPC monolayer. Indeed, if the Brewster pictures show some thicker domains of β-CD at low surface pressure, then those domains disappear almost completely at higher surface pressure (Figure 6). This behavior is confirmed by the PMIRRAS spectra where the absorption bands of β-CD appear weakly at low surface pressure (π ) 5 mN/m, Figure 7a) but disappear at higher surface pressure (π ) 30 mN/m, Figure 7b). DMPC/Cholesterol Mixtures on a β-CD Solution. We have run experiments with two different mixtures (9/1 and 3/1 DMPC/

cholesterol molar ratios), and we present here the results obtained for the 3/1 mixture because the effects are the same but the amplitudes are greater. In Figure 8, the isotherms of DMPC on pure water and of the 3/1 DMPC/cholesterol mixture on the β-CD solution are represented as a function of the molecular area of DMPC. The major effect occurs over large areas (80 Å2 < A 4 mN/m). The main difference with the DMPC/cholesterol system comes from the quantity of β-CD at the interface with respect to the surface pressure.

The gap of pressure observed over large molecular areas for the cholesterol monolayer in presence or absence of β-CD is characteristic of complex formation between cholesterol and β-CD. The Brewster pictures are in line with this conclusion because unlike the cholesterol monolayer on pure water, the pictures obtained on β-CD solutions have brighter domains. During the compression cycle, the instability of the surface pressure can be explained by the partial solubility of the complex in the subphase. The Brewster pictures give more information because they show that one monolayer still exists at the interface with very bright domains of large and variable thicknesses. The observation of only β-CD bands on PMIRRAS spectra allows us to conclude that these domains mainly contain this molecule. It is also noticeable that, in these domains, the β-CD molecules are oriented relative to the surface plane. The selection rules of PMIRRAS spectroscopy at the air/water interface allow us to determine the orientation of the transition moments of vibrational modes relative to the interface,23 but it is necessary to know the directions of these moments relative to the axes of the molecule in order to determine the orientation of the whole molecule. These data are not known yet for β-CD, so we have calculated them using the Gaussian 98 program21. The β-CD molecule contains many atoms (147) and has few symmetry elements, so a long calculation was required to obtain the energy minimum and the vibrational modes. The final data are available in Supporting Information. They contain normal mode wavenumbers and the directions of the transition moments relative to the molecule axes (x, y, z). For β-CD, two axes (x and y) are located in the molecular plane, and the z axis is defined as being perpendicular to this plane. Among all modes, the most important for our study are located in the 1300-900 cm-1 range. Scheme 1 gathers the calculated wavenumbers and the orientation of the transition moments for the two most intense bands of β-CD. (The complete assignment is available as Supporting Information.) These two bands are located at 1030 and 1172 cm-1, the first one being quasi-degenerate with two components, one along the x axis and the other one along y. Our calculation shows that the mode at 1030 cm-1 is essentially located in the x,y plane, whereas the other one at 1172 cm-1 has an important contribution along

Organization of β-Cyclodextrin

Langmuir, Vol. 24, No. 17, 2008 9621

Scheme 1

Scheme 2

the z axis. Because our spectra exhibit an intense low-wavenumber mode, we deduce from the general rules of PMIRRAS spectroscopy that the cycles of β-CD molecules are almost parallel to the interface plane. In this position, the transition moment of the 1172 cm-1 mode is more perpendicular to the surface, and its intensity is lowered compared to that of the 1030 cm-1 mode. To confirm this hypothesis and to evaluate the average thickness of the β-CD layer, we have simulated the PMIRRAS spectrum of β-CD using calculated anisotropic indices of β-CD oriented in the x, y plane (Experimental Procedures). The result is reported on Figure 12 for a thickness of 80 Å, which leads to the best agreement between calculated and observed spectra. Bearing in mind that one spectrum is purely experimental and the other is built from ab initio calculations, this agreement is fairly good. We can now assess that β-CD molecules form organized and oriented multilayers under the cholesterol layer with an average thickness close to that observed in Brewster pictures. We can then propose two types of complexes (Scheme 2) because the β-CD molecule has a truncated cone shape. In the first one, type a, the β-CD molecules are stacked as a pile of bowls, head-to-tail, with all z axes being aligned in the same direction. In the second one, type b, they are piled up head-tohead/tail-to-tail.

The first type is rarely observed in crystallized β-CD complexes (the β-CD planes are then staggered), whereas the head-to-head/ tail-to-tail arrangement is quite common, forming parallel columns.2 Furthermore, the thickness of the layers measured via Brewster pictures in steps of 20 ( 1 Å, which is double the average thickness (10 Å) of the β-CD molecule itself, also seems to favor stacking type b. In the case of a pure DMPC monolayer on a β-CD solution, a slight increase in the surface pressure for large molecular areas is also an indication of the formation of one complex at the interface. As suggested by the PMIRRAS spectra, the hypothesis of the complex stabilization by hydrogen bond interactions seems to be the most probable. Indeed, the van der Waals interaction between the two molecules should be weak because the hydrophobic cavity of β-CD is too small to interact with the two DMPC acyl chains, and the electrostatic interaction should also be weak because β-CD has no charged groups. Over smaller molecular areas, the isotherm of DMPC on β-CD is close to that obtained on pure water, and there are only a few bright domains in the Brewster pictures. These two observations are in agreement with a decrease of the quantity of complexes at the interface with only a few DMPC molecules dissolved in the subphase. PMIRRAS spectra allow us to improve these conclusions. Indeed, β-CD bands, which are easily observed over large molecular areas, are still present at small ones. The β-CD molecules induce much disorder in the DMPC monolayer, observed through the presence of an enlarged CO stretching band. Furthermore, the relative intensities of the β-CD bands are different from those observed in the presence of cholesterol alone. Because these intensities are also different from those observed for the bulk molecule, we suggest that the β-CD molecules are oriented in a different way under the DMPC monolayer. Inplane vibrations are now less intense than the out-of-plane modes, indicating that the β-CD molecule plane is now oriented more perpendicular to the surface. Because this orientation is less favorable for the formation of channels, there are fewer β-CD molecules at the interface than in the presence of cholesterol. The interactions probably occur via the phosphate groups as we observe a shift of the antisymmetric mode ν(PO4) from 1223 to 1240 cm-1 (which is often associated with the dehydration of the phosphate group). The electric charge of the phospholipids seems to play an important role in the formation of one complex with β-CD because in the case of pure DMPG monolayers the detection of β-CD is more difficult and the complex appears almost exclusively at very low surface pressure. This is an indication that the negative and positive charges of the associated countercation are not conducive to the formation of a complex with β-CD, even by hydrogen bonds.

Figure 12. Calculated (s) and experimental spectra (---) of cholesterol on a saturated β-CD aqueous solution.

9622 Langmuir, Vol. 24, No. 17, 2008

For the DMPC/cholesterol mixture, the isotherm shows a phase transition over large molecular areas that can be related to the preferential complexation of β-CD with cholesterol, followed by its progressive transfer into the subphase during compression. The crossing of both isotherms shows that a small quantity of DMPC is also transferred into the subphase. Brewster pictures are in agreement with this conclusion because they show the presence of very bright aggregates as observed for cholesterol. However, these domains progressively disappear when compressing the monolayer. The shape of PMIRRAS spectra, similar to what is observed for pure cholesterol monolayers, proves that β-CD adopts a flat orientation at the interface with the formation of thinner channels. The DMPG/cholesterol mixture presents almost the same behavior as the DMPC/cholesterol mixture. At low surface pressure, β-CD lies flat at the interface and forms a complex with cholesterol. However, the concentration of the complex is weak, and it is quickly eliminated when the surface pressure increases. To summarize, cholesterol at the air/water interface forms a complex with β-CD even in a mixture with other phospholipids such as DMPC and DMPG. In all cases, before the complex dissolves in the subphase, β-CD molecules are parallel to the plane of the interface and form thick channel domains probably in a head-to-head/tail-to-tail fashion. DMPC and DMPG can

Mascetti et al.

also be associated with β-CD but in a different manner because the orientation of the resulting complex is different. This suggests that the complex with cholesterol is produced via hydrophobic interactions with the internal domain of β-CD, whereas the complex with phospholipids results from the formation of hydrogen bonds between their negative polar groups (carbonyl or phosphate) and the hydroxyl groups of β-CD. In agreement with our observations, such a weakly associated complex can be easily dissociated during the compression when the polar head groups are less accessible. We can postulate that the difference in the behavior of DMPC and DMPG is also due to the difference in accessibility for hydrogen bonds between the head groups of both phospholipids. Acknowledgment. We gratefully acknowledge ENSCPB (MASTER, Universite´ Bordeaux I) for allocating computing time and providing facilities. Supporting Information Available: Ab initio (B3LYP/6-31G*) calculated frequencies ν and ν′ ) ν*0.952 (cm-1), infrared intensities (KM/mol), and dipole derivative components µx, µy, and µz (D · Å-1) of β-CD. This material is available free of charge via the Internet at http://pubs.acs.org. LA8004294