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Micellization of Hydrophobically Modified Cyclodextrins: 2. Inclusion of Guest Molecules R. Auze´ly-Velty,*,† C. Pe´an,‡ F. Djedaı¨ni-Pilard,§ Th. Zemb,| and B. Perly| Institut de Recherches SERVIER, Service de Physico-Chimie Analytique, 11 rue des Moulineaux, 92150 Suresnes, France, Laboratoire de Chimie Organique, Universite´ de Picardie Jules Verne, 80039 Amiens Cedex, France, and Service de Chimie Mole´ culaire, DRECAM, CEA Saclay, 91191 Gif sur Yvette, France Received July 26, 2000. In Final Form: October 23, 2000 The association of guest molecules in aggregates of a modified cyclodextrin, 6I-(cholest-5-en-3R-ylamido)succinylamido-6I-deoxy-per(2,6-di-O-methyl)cyclomaltoheptaose, was investigated for four different sparingly water-soluble molecules and an anionic surfactant (sodium dodecyl sulfate). The binding and spatial proximities were demonstrated for these different guests by NMR (nuclear Overhauser effect pumping). By use of small-angle X-ray and neutron scattering, the microstructure at the supramolecular scale of the modified cyclodextrin micelle, i.e., aggregation number, charge, and volume, in the presence of guest molecules could be defined. From the analysis of the variations in terms of aggregation number and charge induced by the presence of the guest molecule, the cavity of the cyclodextrin was shown to remain available for solubilization and transport. The stability and specificity of the mixed micelle involving target molecules demonstrated here make these hydrophobically modified cyclodextrins good candidates as molecular carriers.

Introduction In a previous paper, we have described the formation of monodisperse spherical micelles from a new hydrophobically modified cyclodextrin (CD), namely, 6I-(cholest5-en-3R-ylamido)succinylamido-6I-deoxy-per(2,6-di-O-methyl)-cyclomaltoheptaose (chol-DIMEB, 1) (Figure 1).1 These highly water soluble micelles have been shown to be twoshell objects, the cyclodextrin moieties being exposed toward the aqueous medium making them prone to include guest molecules in the cavities. These original aggregates may open new ways for the targeting of biologically active molecules and especially for the delivery of drugs. The aim of the experiments described in the present paper is to estimate the inclusion and solubilization properties of these CD aggregates. To adress these questions, we have used a combination of small-angle X-ray scattering (SAXS), small angle neutron scattering (SANS), and dedicated NMR experiments to determine the type of associations encountered with the different guests and to provide evidence of the location of the guest molecule and its influence on the stability of the micelle. Inclusion of hydrophobic guest molecules in the cholDIMEB micelle may indeed occur at different sites, i.e., in the CD cavities, between the CD cavities, between the chol-DIMEB molecules in the palisade layer, or in the inner core of the micelle. The exact location in the micelle of the hydrophobic guest depends on the nature of the latter and is of importance since it reflects the type of interaction occurring between the chol-DIMEB molecule and the hydrophobic guest. * Corresponding author. † Present address: Centre de Recherche sur les Macromole ´ cules Ve´ge´tales, BP53, 38041 Grenoble cedex 9, France. Tel: + 33 4 76 03 76 70. Fax: +33 4 76 54 72 03. E-mail: rachel.auzely@ cermav.cnrs.fr. ‡ Institut de Recherches SERVIER, Service de Physico-Chimie Analytique. § Laboratoire de Chimie Organique, Universite ´ de Picardie Jules Verne. | Service de Chimie Mole ´ culaire, DRECAM, CEA Saclay. (1) Auze´ly-Velty, R.; Djedaı¨ni-Pilard, F.; De´sert, S.; Perly, B.; Zemb, Th. Langmuir 2000, 16, 3727.

Figure 1. Structure of chol-DIMEB 1.

Experimental Section Materials. Chol-DIMEB has been synthesized in the laboratory as described previously.1 Methyl R-D-mannopyranoside, 4-tert-butyl benzoic acid, chloramphenicol, and sodium dodecyl sulfate (SDS) were purchased from Fluka (Buchs, Switzerland). Dosulepine was a generous gift from BASF Pharma (Valenciennes, France). D2O (99.9% D) was obtained from Euriso-Top (Saclay, France). Preparation of the Samples. To an aqueous solution of cholDIMEB (5 × 10-3 M) at room temperature, a solution of the pertinent guest molecule in a suitable organic solvent (acetone or methanol) was added. The mixture was stirred, and the organic solvent was allowed to evaporate slowly under a stream of nitrogen. The resulting solution was freeze-dried. After redissolution in deuterium oxide, it was freeze-dried again and the resulting powder was weighed and rehydrated at fixed water contents. NMR Techniques. 1H NMR experiments were performed at 500.13 MHz using a Bruker DRX500 spectrometer with a 5 mm reverse broad-band z-axis gradient probe. In all cases, the samples were prepared in deuterium oxide and measurements were performed at 25 °C under careful temperature regulation. NMR spectra were collected using 16 384 data points. Chemical shifts are given relative to external tetramethylsilane (TMS ) 0 ppm) and calibration was performed using the signal of the residual protons of the solvent as a secondary reference. Details concerning

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Figure 2. (a) BPP-STE pulse sequence. (b) Diffusion-assisted NOE pumping pulse sequence. (c) Modified diffusion-assisted NOE pumping pulse sequence.

Figure 4. Partial 1H NMR spectrum of DIMEB 8 (5 × 10-3 M in D2O) at a temperature of 25 °C in the absence (a) and in the presence of 1 mol equiv of guest 2 (b), 3 (c), 5 (d), 6 (e), and 4 (f).

Figure 3. Structure of the guest molecules investigated in this study. experimental conditions are given in the figure captions. Three pulsed magnetic field gradient NMR (PFG-NMR) sequences were used in addition to the normal proton NMR sequence. The standard bipolar pulse stimulated echo (BPP-STE) experiment2,3 was used in order to evaluate the type of interaction which occurs between the chol-DIMEB micelle and the guest molecules. This sequence (see Figure 2a) indeed allows discrimination of molecules according to their apparent diffusion coefficient. Small guest molecules which are in fast exchange between the cholDIMEB micelle and the aqueous medium may have an apparent diffusion coefficient close to their diffusion rate in the free state and, therefore, their NMR signals may be suppressed if a large enough gradient is applied. Conversely, if the potential guests are in very slow exchange between the chol-DIMEB micelle and the aqueous medium, their apparent diffusion coefficient is closer to that of the chol-DIMEB micelles and, therefore, their NMR signals may not be suppressed under identical conditions. This BPP-STE experiment may therefore indicate if the hydrophobic guest molecules are located in the CD cavities exposed to external aqueous medium, in the inner core of the micelle, or between the individual chol-DIMEB molecules in the palisade layer. Since however the BPP-STE experiment does not allow distinguishing between bound and free guest molecules, in the case where the latter are in fast exchange (see above), diffusion-assisted nuclear (2) Tanner, J. E. J. Chem. Phys. 1970, 52, 2523. (3) Wu, D.; Chen, A.; Johnson, C. S., Jr. J. Magn. Reson. 1995, A115, 260.

Overhauser effect (NOE) pumping experiments4 were performed in a second step in order to evidence interaction between guests and the chol-DIMEB micelles. The 1D NOE pumping method allows a fast and direct detection of ligand binding to target macromolecules without regard to the size of the latter. This NMR diffusion experiment relies on NOE to transfer the signal from the receptor to potential ligands. The pulse sequence is displayed in Figure 2b. Finally, to determine the main site of interaction in the micelle, a third pulse sequence shown in Figure 2c was used. A selective 180° pulse is inserted before the third 90° pulse in order to deduce the location of the interactions from the sign of the observed NOE signals. All NMR data were further processed and plotted using the XWINNMR program (Bruker Analytische Messtechnik) on an INDY workstation (Silicon graphics). Scattering Techniques. SANS measurements were performed at ILL-Grenoble (France) using the beamline D11. The neutron wavelengths used in these experiments were 4.5 and 10 Å. The range of scattering vector q (Å-1) ) 4π/λ sin(θ/2) (λ being the incident neutron wavelength and θ the scattering angle) covered is 0.012 Å-1 < q < 0.44 Å-1. Scattering spectra were normalized using water as a standard.5 SAXS measurements were obtained on a pinhole collimation Huxley-Holmes type SAXS camera6 using a two-dimensional gas detector. The range of scattering vector covered is 0.015 Å-1 < q < 0.35 Å-1. The experiments were performed at 25 °C on the same samples used for SANS measurements. Data were normalized vs water. The scattering produced by pure water can be theoretically obtained (4) Chen, A.; Shapiro, M. J. J. Am. Chem. Soc. 1998, 120, 10258. (5) Cotton, J. P. In Neutron, X-ray and light scattering: introduction to an investigation tool for colloidal and polymeric systems; Lindner, P., Zemb, Th., Eds.; Elsevier Science Publishers B.V.: Amsterdam, The Netherlands, 1990; p 19. (6) Le Flanchec, V.; Gazeau, D.; Tabony, J.; Zemb, Th. J. Appl. Crystallogr. 1996, 29, 1.

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Figure 5. SANS spectrum obtained at 25 °C for a solution of chol-DIMEB 1 (5 × 10-3 M in D2O) in the absence (R ) 0) and in the presence of dosulepine 3 (5 × 10-3 M, R ) 1). The dotted line represents the calculated model curve for independent homogeneous spherical micelles (P(q) only, S(q) ) 1). The solid line represents the simulation of charged interacting spheres of identical radius and number per unit volume, but with an effective charge Z ) 4 (S(0) ) 0.65).

Figure 7. chol-DIMEB 1 (6 × 10-3 M in D2O) in the presence of methyl R-D-mannopyranoside 2 (18 × 10-3 M) and dosulepine 3 (18 × 10-4 M) at a temperature of 25 °C. (a) Normal 1D 1H NMR spectrum. Arrows indicate the signals corresponding to the protons of 2. (b) 1D 1H NMR spectrum with BPPSTE, where g ) 35 G‚cm-1, δ ) 3 ms, Tdiff ) 50 ms, and 3000 scans. (c) 1D 1H NMR spectrum with NOE-pumping sequence, where g ) 35 G‚cm-1, δ ) 3 ms, Tdiff ) 50 ms, Tm ) 100 ms, and 3000 scans. is the number of electrons per water molecule. Finally, the scattering produced by water is found to be Iwater ) 1.62 × 10-2 cm-1. The modeling of the scattering was performed using molecularly constrained fitting as reviewed by Pedersen.9

Results and Discussion

Figure 6. chol-DIMEB 1 (6 × 10-3 M in D2O) in the presence of dosulepine 3 (18 × 10-3 M) at a temperature of 25 °C. (a) Normal 1D 1H NMR spectrum. (b) 1D 1H NMR spectrum with BPP-STE, where g ) 35 G‚cm-1, δ (gradient pulse duration) ) 3 ms, Tdiff ) 60 ms, and 3000 scans. (c) 1D 1H NMR spectrum with NOE-pumping sequence, where g ) 35 G‚cm-1, δ ) 3 ms, Tdiff ) 60 ms, Tm ) 100 ms, and 3000 scans. from the isothermal compressibility χT.7 For q ) 0

S(0) ) χT/χT° ) χT/(FkT)-1 and

I(0) ) F fe2 ne2 S(0) where χT ) 45 × 10-11 Pa,8 F ) 55 mol‚L-1 is the density for pure water, the Thomson factor fe ) 0.282 × 10-12 cm, and ne ) 10

The inclusion properties of the chol-DIMEB micelles in water have been investigated using different types of guests: hydrophilic molecules which are not expected to interact with the micelle (methyl R-D-mannopyranoside (2)); sparingly water-soluble aromatic compounds (dosulepine, 4-tert-butyl benzoic acid sodium salt, chloramphenicol); a surfactant molecule (sodium dodecyl sulfate (SDS)). The structures of the model guests are shown in Figure 3. The neurotropic molecule dosulepine (3),10 the bacteriostatic agent chloramphenicol (5),11 SDS (6),12 and 4-tertbutyl benzoic acid sodium salt (4)13 have been shown to form inclusion complexes with the natural β-cyclodextrin. However, no data were found in the literature concerning the inclusion in the selectively methylated β-cyclodextrin per(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) (8), which mimics the polar headgroup of chol-DIMEB. To better (7) Levelut, A. M., PhD Thesis, Paris XI, France, 1968. (8) Handbook of Chemistry and Physics, 72nd ed.; CRC Press: Boca Raton, Fl, 1991-1992. (9) Pedersen, J. S. Curr. Opin. Colloid Interface Sci. 1999, 4, 190. (10) Djedaı¨ni-Pilard, F.; De´salos, J.; Perly, B. Tetrahedron Lett. 1993, 34, 2457. (11) Djedaı¨ni, F., PhD Thesis, Paris XI, France, 1991. (12) Lin, J.; Djedaı¨ni-Pilard, F.; Guenot, P.; Perly, B. Supramol. Chem. 1996, 7, 175. (13) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803.

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Figure 9. chol-DIMEB 1 (6 × 10-3 M in D2O) in the presence of 4-tert-butyl benzoic acid sodium salt 4 (6 × 10-3 M) at a temperature of 25 °C. (a) Normal 1D 1H NMR spectrum. (b)(d) 1D 1H NMR spectra with modified NOE-pumping sequence (selective inversion of the CD protons), where g ) 35 G‚cm-1, δ ) 3 ms, Tdiff ) 60 ms, and Tm ) 3 µs, 50 ms, and 100 ms, respectively. 3000 scans were collected for the modified NOEpumping experiments. Figure 8. (a) SANS spectrum and (b) SAXS spectrum obtained at 25 °C for a solution of chol-DIMEB 1 5 × 10-3 M in D2O in the absence (R ) 0) and in the presence of chloramphenicol 5 (5 × 10-3 M, R ) 1). The solid lines represent the calculated model curve for independent homogeneous spherical micelles.

understand the behavior of the model guests toward the chol-DIMEB derivative, we have investigated in a first step the inclusion of the former in DIMEB. Figure 4 shows the partial 500 MHz 1H NMR spectra of DIMEB in the absence and in the presence of the different guests. Inclusion in DIMEB is evidenced by modifications of the 1H NMR spectrum of the host molecule.14 Under these conditions, only shifts of the signals were observed. In all cases, no new peak appeared which could be assigned to the pure complex. This observation implies that complexation, if it occurs, is a dynamic process; i.e., the included molecule is in fast exchange (relative to the NMR time scale) between the “free” and “bound” states. It is observed here that compounds 3-6 induce large shifts in the NMR signals of the protons located in the cavity (H-3 and H-5) of DIMEB indicating the formation of inclusion complexes. Conversely, no detectable shifts are evidenced with compound 2 under the same conditions (Figure 4b), indicating that DIMEB does not complex 2. These preliminary results will be of importance for the following study. Interaction of Methyl r-D-Mannopyranoside (2) and Hydrosoluble Aromatic Molecules (3-5) with the chol-DIMEB Micelles. Inclusion of charged dosul(14) Djedaı¨ni, F.; Perly, B. Magn. Reson. Chem. 1990, 28, 372.

epine 3 in the chol-DIMEB micelle is expected to mainly induce electrostatic repulsion between positively charged micelles, provided that inclusion occurs. In Figure 5, we compare the scattering pattern of chol-DIMEB alone and in the presence of 3 (1 mol equiv). At small q, the low-q depletion in the scattering patterns along with the invariance at high-q clearly indicate a change in the intermicellar interactions between micelles of similar volume with and without guests. The decrease of the scattered intensity at small q values for the chol-DIMEB/ dosulepine mixture is typical of the scattering of strongly repulsive micelles.15 The scattering is modeled as a product of a form factor and a structure factor. The latter was calculated according to the RMSA approximation introduced and discussed by Hayter.16 The only unknown parameter is the effective charge Z of the micelle, i.e., the charge felt by the neighboring micelles. The best fit was found for Z ) 4 (see Figure 5). The structural charge of the micelle in the presence of guests is lower or equal to 24. If the micelle in the presence of guests had obeyed to the electrostatic equilibrium, according to the charge regulation model,17 we would have found an effective charge Z ) 8. From these data, two main conclusions can be derived. First, there are enough guest molecules at any time to confer four dissociated species to the micelle. Second, the equilibrium effective charge is half the value which would have been observed for a micelle having the (15) Zemb, Th.; Charpin, P. J. Phys. 1985, 46, 249. (16) Hayter, J. B. In Physics of amphiphiles: micelles, vesicles and microemulsions; Corti, M., Degiorgio, V., Eds.; NATO ASI series 242; North-Holland: Amsterdam, 1985; p 59. (17) Hayter, J. B. Langmuir 1992, 8, 2873.

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Figure 10. chol-DIMEB 1 (6 × 10-3 M in D2O) in the presence of 4-tert-butyl benzoic acid sodium salt 4 (6 × 10-3 M) at a temperature of 25 °C. (a) Normal 1D 1H NMR spectrum. (b)(d) 1D 1H NMR spectra with modified NOE-pumping sequence (selective inversion of the cholesterol protons), where g ) 35 G‚cm-1, δ ) 3 ms, Tdiff ) 60 ms, and Tm ) 3 µs, 50 ms, and 100 ms, respectively. 3000 scans were collected for the modified NOE-pumping experiments.

same volume and made of 24 cationic surfactants. The previous paper1 demonstrated tight packing of the cyclodextrin headgroups (340 Å2 per molecule). This condition is still met in the presence of guest 3. Dosulepine has no “cosurfactant” effect, because this would have been correlated with an increase of area per molecule. The interaction of dosulepine 3 with the chol-DIMEB micelles was also supported by NMR. Figure 6a shows the normal 1H NMR spectrum of the chol-DIMEB/dosulepine mixture in D2O. It is noteworthy that the peaks corresponding to the aromatic protons of 3 are broad compared with 3 alone in water (data not shown) suggesting interaction between 3 and chol-DIMEB. The BPP-STE experiment performed on the same mixture with a storage period Tdiff ) 60 ms (see Figure 2a) suppressed the signals corresponding to 3 as shown by Figure 6b. This indicates that 3 has a diffusion coefficient close to the diffusion rate of the molecule in the free state and is located in the outer polar layer of the micelle, in fast exchange between the latter and the aqueous bulk medium. The interaction of 3 with chol-DIMEB must however be clearly evidenced. Diffusion-assisted NOE pumping experiments were therefore performed using increasing mixing times (Tm ) 25, 100, 150, 200, 300 ms). The dosulepine proton signals which could be hardly seen before NOE transfer reappeared after the NOE experiment even after a mixing time of 25 ms. A 1D NOE-pumping spectrum obtained from the mixture (Tm ) 100 ms) is displayed in Figure 6c. To validate the NMR results, we checked to ensure that hydrophilic methyl R-D-mannopyranoside 2 does not include into the chol-DIMEB micelle under identical NMR conditions. As demonstrated by the preliminary studies

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Figure 11. chol-DIMEB 1 (6 × 10-3 M in D2O) in the presence of sodium dodecyl sulfate 6 (6 × 10-3 M) at a temperature of 25 °C. (a) Normal 1D 1H NMR spectrum. (b)-(g) 1D 1H NMR spectra with BPP-STE as the storage period Tdiff increases (from 50 to 100 ms in intervals of 10 ms), where g ) 35 G‚cm-1, δ ) 3 ms. 3000 scans were collected for each BPP-STE experiment.

performed on DIMEB, compound 2 cannot interact with chol-DIMEB. Figure 7a displays the 1H NMR spectrum of the chol-DIMEB/2/3 mixture. The BPP-STE diffusion filtered experiment performed on this system with a storage period Tdiff ) 50 ms led to the complete suppression of the proton signals of 2 and 3 as shown by Figure 7b. However, after the NOE transfer, only signals from the hydrophobic guest 3 are restored (Figure 7c). These NMR observations confirm that 3 interacts with the chol-DIMEB micelle whereas the hydrophilic sugar 2 remains in the external aqueous medium. Similar SANS and NMR studies were performed using tert-butyl benzoic acid sodium salt (4) as a second example of hydrophobic guest. Comparison of the SANS spectra of chol-DIMEB alone and in the presence of 4 led to similar results as in the case of 3. The BPP-STE diffusion filtered and NOE pumping experiments demonstrated that guest 4 interacts with the chol-DIMEB micelles as in the previous case. In both pulse sequences the value of the interval Tdiff which slightly depends on the observed diffusion coefficient of the guest molecule was optimized. In the case of 4, it was found that a storage period of 40 ms in the BPP-STE sequence was sufficient to lead to the complete disappearance of the proton signals of the guest molecule. In contrast to compounds 3 and 4, no modification of the chol-DIMEB micelles in the presence of the neutral guest molecule 5 was observed by SANS (Figure 8a). However SAXS shows an increase in the high-q oscillation in the presence of chloramphenicol 5 (Figure 8b). As can be seen from the molecular modeling of the form factor P(q) for the chol-DIMEB micelle described in part 1 of

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Figure 12. Schematic representation of a chol-DIMEB micelle with dosulepine 3 in the bulk and bound state in the CD cavities.

this work,1 the high-q oscillation is due to the external polar part of the micelle. The enhancement of the high-q oscillation thus reflects an increase in the electronic density of the outer polar layer of the chol-DIMEB micelles which may be due to the presence of 5 in or between the cyclodextrin moieties. The modification of the high-q oscillation is about 10 times the uncertainty in the level of background, which is of the order of 10-3 cm-1 as can be seen from the figure. Since the wide-angle asymptotic limit of the SANS spectra does not change whereas the SAXS pattern is modified, we can assume that the guest molecule is not located between the cyclodextrin cavities. Indeed, this would have led to a decrease of the area per headgroup. This type of complexation was confirmed by NMR. In the 1H NMR spectrum of chol-DIMEB/5 mixture with BPP-STE (Tdiff ) 50 ms), signals arising only from chol-DIMEB are observed. The reappearance of signals from chloramphenicol after the NOE transfer proved the interaction of 5 with the chol-DIMEB micelles. Suppression of the signals arising from 5 in the NMR experiment with BPP-STE indicates that guest 5 has a diffusion coefficient close to the diffusion rate of the free state and thus mainly binds to the CD moieties exposed to the aqueous medium as suggested by SAXS. In this example, it is shown that indication concerning the main site of interaction in the micelle may be achieved by the combination of SAXS and NMR experiments. We then investigated if such information could be derived from NMR experiments only using the modified pulse sequence displayed in Figure 2c. Since the guest signals that reappear after the NOE experiment are transferred from chol-DIMEB polarization, detailed binding site information is expected to be obtained by the selective inversion of the spin population of the cyclodextrin or the cholesterol protons followed by examination of the sign of the recovered NOE signals. A typical example is displayed in Figures 9 and 10 for the chol-DIMEB/t-BuPhCOONa 4 mixture. When the proton signals of the CD moiety are selectively inverted using a 180° pulse, signals arising from guest 4 are shown to reappear after the NOE transfer

with the same sign as the CD ones (Figure 9d). Conversely, as the proton signals of the cholesterol nucleus are selectively inverted, the signals of 4 can be observed after the NOE transfer but they do not have the same sign as those of cholesterol (Figure 10c,d). These results suggest that interaction of 4 with the chol-DIMEB micelle takes place close to the CD cavities. As can be seen in Figure 9, the signal intensity of 4 substantially grows as the mixing time increases. However the sign inversion of the H-1 proton signals of the CD moiety as Tm increases shows the existence of spin-diffusion effects. This implies that the experiments must be performed using short mixing times (Tm e 100 ms). Moreover the loss of the proton signals of the cholesterol nucleus followed by their sign inversion as Tm increases due to transverse relaxation effects (short T2 values) is a second reason for using short mixing times in such experiments. The study of the exact location in the micelle at which interaction occurs for guest 4 was extended to dosulepine 3 and chloramphenicol 5. In both cases, the guest molecule was shown to selectively bind to the CD cavities. Interaction of Sodium Dodecyl Sulfate (SDS, 6) with the chol-DIMEB Micelles. Since the chol-DIMEB micelle is an uncharged assembly of 24 molecules, the strong tendency of mixed micelle formation in the presence of another surfactant is expected. This assumption was supported by the BPP-STE experiments using increasing storage periods Tdiff. As shown by Figure 11, whatever the storage period is, the SDS signals are not suppressed. This suggests that SDS is tightly bound to the chol-DIMEB micelle and must form a mixed micelle. Moreover, since we have evidenced a strong affinity between 6 and the cavity of DIMEB (see Figure 4e), it is reasonable to assume that 6 is also included in the cavity of chol-DIMEB. These results are in good agreement with the literature.18 (18) Bellanger, N.; Perly, B. J. Mol. Struct. 1992, 273, 215.

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Conclusion Using a combination of SAXS, SANS and NMR experiments, we have evidence of several types of interaction of guest molecules with the monodisperse and spherical micellar aggregates of hydrophobically modified cyclodextrin 1 (chol-DIMEB). The original micelles have a coreshell structure with the CD moieties exposed to the aqueous medium making them prone to include guest molecules in the cavities. Upon inclusion of the different guest molecules, the aggregation number of the micelle was not shown to change noticeably. This observation has two implications: The well-defined packing of the cyclodextrin moieties around a hydrophobic core made of the cholesterol residues is retained after inclusion of the guest molecule. When electrical charges are added to the micellar aggregate through the hydrophobic guest, the variation of the aggregation number is negligible. This means that even strong lateral electrical interactions are not able to

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increase the area per molecule from its initial value of 340 Å2. Only complex glycolipids show this behavior. The packing of the chol-DIMEB into spherical micelles is exceptionally robust. This study clearly demonstrates that the inclusion properties of the CD cavities of chol-DIMEB aggregated into micellar objects are retained. A micelle of chol-DIMEB with guests included in the CD cavities is schematized in Figure 12. Acknowledgment. This work was supported by the European Commission (DG XII) under the FAIR Program CT 95-0300. We thank Dr. B. Deme´ for the SANS measurements. We gratefully acknowledge the assistance of O. Tache´ (CEA Saclay) with the SAXS measurements. We also thank Dr. H. Desvaux for helpful discussions on NMR data. LA001063Y