Micellization of Hydrophobically Modified Cyclodextrins. 1. Micellar

Mar 14, 2000 - Moreover, it has been shown that “skirt-shaped cyclodextrins” bearing fatty acyl chains on their secondary hydroxyl groups can form...
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Langmuir 2000, 16, 3727-3734

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Micellization of Hydrophobically Modified Cyclodextrins. 1. Micellar Structure R. Auze´ly-Velty, F. Djedaı¨ni-Pilard, S. De´sert, B. Perly,* and Th. Zemb Service de Chimie Mole´ culaire DRECAM CEA Saclay, F-91191 Gif sur Yvette, France Received October 15, 1999. In Final Form: December 22, 1999 The grafting of a cholesterol derivative onto a methylated cyclodextrin through a spacer arm produces an amphiphilic compound exhibiting high solubility in water. This new molecule was fully characterized in terms of chemical and optical purities by high resolution NMR and mass spectrometry. An analysis of its behavior in aqueous solution using surface tension measurements and light, small-angle X-ray, and neutron scattering techniques proved that it self-assembles into monodisperse spherical micelles with an average aggregation number of 24. The micelles can be described as two-shell objects, the cyclodextrin moieties being exposed to the aqueous medium, making them prone to include guest molecules in the cavities. These objects can therefore be of high interest for the targeting of biologically important molecules and especially for the delivery of drugs.

Introduction A large number of chemically modified cyclodextrins (CDs) have been synthesized in order to improve or modify some of the properties of these host molecules.1 Our attention was drawn to amphiphilic cyclodextrins, which are obtained by grafting one or several hydrophobic group(s) on the cyclic oligosaccharide core. Such compounds may be inserted into lipid systems such as artificial or natural membranes through their hydrophobic moiety. Modified cyclodextrins named “bouquets”, bearing multiple poly(oxyethylene) or polymethylene chains on both the primary and secondary faces, have been shown to be incorporated into lipid vesicles where they act as transmembrane ion channels.2 Recently, we have demonstrated that this membrane incorporation ability could also be observed using amphiphilic monosubstituted CD derivatives. The “cup and ball” molecules, obtained by grafting a linear chain of variable length with a bulky tertbutyloxycarbonyl end group on the primary face of β-cyclodextrin, can indeed be inserted into phospholipid vesicles and still retain their inclusion properties towards external guests.3 On the other hand, the substitution of the linear chain by a cholesterol nucleus led to CD derivatives (cholesteryl-cyclodextrins or chol-CDs) which exhibit very efficient incorporation properties into phospholipid membranes.4 Molecular self-assemblies made of amphiphilic cyclodextrins alone have also been evidenced. Per-(6-dodecylamino-6-deoxy)-β-cyclodextrin5 and per-(6-alkylsulfonyl6-deoxy)-β-cyclodextrins6 have been shown to form stable Langmuir-Blodgett layers, while the corresponding per* Corresponding author. E-mail: [email protected]. (1) (a) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803. (b) Khan, A. R.; Forgo, P.; Stine, K. J.; D’Souza, V. T. Chem. Rev. 1998, 98, 1977. (2) Jullien, L.; Lazrak, T.; Canceill, J.; Lacombe, L.; Lehn, J. M. J. Chem. Soc., Perkin Trans. 2 1993, 1011. (3) Lin, J.; Creminon, C.; Perly, B.; Djedaı¨ni-Pilard, F. J. Chem. Soc., Perkin Trans. 2 1998, 2638. (4) Auze´ly-Velty, R.; Perly, B.; Tache´, O.; Zemb, Th.; Je´han, P.; Guenot, P.; Dalbiez, J. P.; Djedaı¨ni-Pilard, F. Carbohydr. Res. 1999, 318, 82. (5) (a) Yabe, A.; Kawabata, Y.; Niino, H.; Tanaka, M.; Ouchi, A.; Takahashi, H.; Tamura, S.; Tagaki, W.; Nakahara, H.; Fukuda, K. Chem. Lett. 1988, 1. (b) Yabe, A.; Kawabata, Y.; Niino, H.; Matsumoto, M.; Ouchi, A.; Takahashi, H.; Tamura, S.; Tagaki, W.; Nakahara, H.; Fukuda, K. Thin Solid Films 1988, 160, 33. (6) Kawabata, Y.; Matsumoto, M.; Tanaka, M.; Takahashi, H.; Irinatsu, Y.; Tamura, S.; Tagaki, W.; Nakahara, H.; Fukuda, K. Chem. Lett. 1986, 1933.

(6-S-alkyl-6-thio) derivatives7 behave as thermotropic liquid crystals. Moreover, it has been shown that “skirtshaped cyclodextrins” bearing fatty acyl chains on their secondary hydroxyl groups can form stable nanospheres.8 These supramolecular aggregates show promising properties for drug encapsulation and delivery owing to the cumulative effects of size specificity and transport properties of cyclodextrins and organized materials, respectively. To further investigate the properties of supramolecular assemblies of modified CDs, we have undertaken the synthesis of a selectively O-methylated analogue of a cholCD derivative, namely 6I-(cholest-5-en-3R-ylamido)succinylamido-6I-deoxy-per(2,6-di-O-methyl)cyclomaltoheptaose (chol-DIMEB) 6 (Scheme 1). It is indeed well described in the literature that methylation of the parent β-cyclodextrin results in a dramatic increase in water solubility.9 Per(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) has thus an aqueous solubility 30 times higher than that of the parent β-cyclodextrin and still retains efficiency for complexation. Therefore, the modified cyclodextrin 6 is expected to exhibit uncommon properties in water compared to the nonmethylated derivatives. We describe here the synthesis, structural characterization, and aqueous solution behavior of chol-DIMEB 6. Since structurally it is the conjunction of an extremely rigid and hydrophobic part and of a large hydrophilic cyclodextrin headgroup through a spacer, strong cooperativity in self-aggregation in water is expected. Surface tension measurements are used to evidence the selfassembly property of this CD derivative in water. The structure of the chol-DIMEB self-aggregates is further studied using light scattering, small-angle neutron scattering (SANS), and X-ray scattering (SAXS) methods. Experimental Section 6I-Azido-6I-deoxycyclomaltoheptaose

Materials. 1 (Scheme 1) was obtained in two steps from the parent β-cyclodextrin (7) Ling, C. C.; Darcy, R.; Risse, W. J. Chem. Soc., Chem. Commun. 1993, 438. (8) (a) Lemos-Senna, E.; Wouessidjewe, D.; Lesieur, S.; Puisieux, F.; Couarraze, G.; Ducheˆne, D. Pharm. Dev. Technol. 1998, 3, 85. (b) LemosSenna, E.; Wouessidjewe, D.; Ducheˆne, D.; Lesieur, S. Colloids Surf. B 1998, 10, 291. (9) Uekama, K.; Irie, T. In Cyclodextrin and their industrial uses; Ducheˆne, D., Ed.; Edition de Sante´: Paris, 1987; p 395.

10.1021/la991361z CCC: $19.00 © 2000 American Chemical Society Published on Web 03/14/2000

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Auze´ ly-Velty et al. Scheme 1a

a Reagents and conditions: (i) BaO, Ba(OH) ‚8H O, Me SO , DMSO/DMF (1:1 v/v), 8-9 °C, then concentrated NH OH solution, 2 2 2 4 4 RT, 45%; (ii) PPh3, concentrated NH4OH solution, DMF, then purification on Lewatit SP1080 (Merck) resin, 60%; (iii) succinic anhydride, DMF, then DIC, HOBT, 3-R-aminocholesterol 5, THF, 70% over the two steps.

(purchased from Roquette Fre`res, Lestrem, France) as described elsewhere.10 3-R-Aminocholesterol was prepared in two steps from 3-β-cholesterol as previously described.4 Other chemicals for the synthesis of chol-DIMEB were purchased from Fluka (Buchs, Switzerland). CDCl3 and D2O (99.9% D) were obtained from Euriso-Top (Saclay, France). General Methods. 1H NMR experiments were performed using a Bruker DRX500 spectrometer. 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. Electrospray mass spectrometry analysis was performed in positive mode on a ZabSpec TOF (Micromass, UK) mass spectrometer. Chol-DIMEB 6 was dissolved in a mixture of water-acetonitrile 1:1 (v/v) at a concentration of 0.01 mg‚cm-3 and infused into the electrospray ion source. The capillary voltage was set to 4 kV. Poly(ethylene glycol) was used for external calibration. Thin-layer chromatography was performed on Silica Gel 60 plates (Merck, Darmstadt, Germany) followed by charring with 10% H2SO4. For column silica gel and ion-exchange chromatography, Silica Gel 60 (Fluka) and Lewatit SP1080 (H+ form, Merck) were used, respectively. Surface tension of aqueous solutions of chol-DIMEB was measured at 25 °C using the Du Nouy ring method with a digital tensiometer K10 (Kru¨ss, Switzerland). An Anton Paar densitometer (Graz, Austria) was used for density measurements to obtain the molecular volume of cholDIMEB with a 10% accuracy. Static and dynamic light scattering measurements were both performed on an AMTEC SM200 goniometer fitted with an (10) (a) Djedaı¨ni-Pilard, F.; De´salos, J.; Perly, B. Tetrahedron Lett. 1993, 34, 2457. (b) Djedaı¨ni-Pilard, F.; Azaroual-Bellanger, N.; Gosnat, M.; Vernet, D.; Perly, B. J. Chem. Soc., Perkin Trans. 2 1995, 723.

ionized argon laser source (Spectra Physics 2016) at a scattering angle θ ) 90° and a wavelength λ ) 514.5 nm. The correlation function and light intensity measurements were obtained and processed using a BI30 correlator and Brookhaven Instruments software. Refractive indices of aqueous solutions of chol-DIMEB were measured at 25 °C with a 2 × 10-4 uncertainty using an universal refractometer (Abbe type). SANS measurements were performed on the PACE setup at LLB Orphe´e (Saclay, France). 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 0.005 Å-1 < q < 0.55 Å-1. The sample was prepared in D2O and thermostated to 25 °C. Scattering spectra were normalized using water as a standard.11 SAXS measurements were performed on a pinhole collimation Huxley-Holmes type SAXS camera12 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 sample as used for SANS measurements. Data were normalized vs water.13 Synthesis of 6I-(Cholest-5-en-3r-ylamido)succinylamido6I-deoxy-per(2,6-di-O-methyl)cyclomaltoheptaose (cholDIMEB) 6. The synthetic pathway used for the preparation of chol-DIMEB 6 is shown in Scheme 1. It involves the preparation of the methylated mono-6-amino key compound 3 which is further (11) Cotton, J. P. In Neutron, X-ray and light scattering: introduction to an investigative tool for colloidal and polymeric systems; Lindner, P., Zemb, Th., Eds.; Elsevier Science Publishers B. V.: Amsterdam, The Netherlands, 1990; p 19. (12) Le Flanchec, V.; Gazeau, D.; Tabony, J.; Zemb, Th. J. Appl. Crystallogr. 1996, 29, 1. (13) Zemb, Th.; Tache´, O.; Ne´, F. Submitted for publication.

Micellization of Hydrophobically Modified CDs

Langmuir, Vol. 16, No. 8, 2000 3729 Table 1. Solubility in Water of the Native β-Cyclodextrin and Some Hydrophobically Modified Derivatives

Figure 1. 1H NMR spectrum (500 MHz, 25 °C, 7 × 10-3 M in CDCl3) of compound 6. coupled to 3-R-aminocholesterol 5 through a succinyl spacer. For the synthesis of 3, barium oxide (3.6 g, 24 mmol) and barium hydroxide octahydrate (3.8 g, 12 mmol) were successively added to a solution of 6I-azido-6I-deoxycyclomaltoheptaose 1 (2 g, 1.7 mmol) in dry dimethyl sulfoxide/dimethylformamide (DMSO/ DMF) 1:1 (v/v, 30 cm3). The mixture was stirred under nitrogen at 8-9 °C for 30 h. It is noteworthy that selective alkylation of the hydroxyl groups at C-2 and C-6 positions implies carefully maintaining the reaction temperature at 8-9 °C to avoid C-3O-methylation. Concentrated ammonium hydroxide (5 cm3) was then added and the mixture was stirred for a further 3 h at room temperature (RT). After evaporation of most of the solvents, the resulting solid cake was extracted with dichloromethane (3 × 70 cm3). The combined organic phases were washed with saturated aqueous sodium chloride (3 × 20 cm3), and water (2 × 20 cm3), dried over sodium sulfate, and concentrated under reduced pressure. The crude product was precipitated by addition of n-hexane (100 cm3), filtered, washed with n-hexane, and dried. 6I-Azido-6I-deoxy-per(2,6-di-O-methyl)cyclomaltoheptaose 2 was isolated (1 g, 45%) as a white amorphous powder. A solution of 2 (0.75 g, 0.56 mmol) in DMF (35 cm3) was treated by triphenylphosphine (0.75 g, 2.86 mmol). The mixture was stirred for 2 h at RT. After cooling to 0 °C, concentrated ammonium hydroxide (14 cm3) was added and stirring was continued for 18 h at RT. The solvent was evaporated and water (30 cm3) was added. After elimination of the precipitates of triphenylphosphine and of the corresponding oxide by filtration, the aqueous solution was concentrated under reduced pressure. The resulting solid was purified by ion-exchange chromatography (elution with 6% ammonium hydroxide) affording 6I-amino-6I-deoxy-per(2,6-diO-methyl)cyclomaltoheptaose 3 (0.44 g, 60%) as a white powder. The subsequent coupling of the cholesterol moiety through a succinyl spacer was performed in a one-pot reaction. Succinic anhydride (0.019 g, 0.19 mmol) dissolved in dry DMF (2 cm3) was added to a solution of freeze-dried 3 (0.25 g, 0.19 mmol) in dry DMF (6 cm3). The reaction mixture was stirred for 5 h and treated with diisopropylcarbodiimide (DIC) (0.11 cm3, 0.76 mmol) and hydroxybenzotriazole (HOBT) (0.014 g, 0.09 mmol) dissolved in DMF (2 cm3). The mixture was stirred under nitrogen for 30 min at RT and 3-R-aminocholesterol (0.089 g, 0.23 mmol) dissolved in dry THF (5 cm3) was added. The mixture was stirred at RT for 48 h. The reaction was quenched by addition of water (0.5 cm3), and the solvents were evaporated under reduced pressure. The resulting solid was purified by silica gel chromatography (elution with CH2Cl2-MeOH 95:5 then 90:10 (v/v)) affording the target 6I-(cholest-5-en-3R-ylamido)succinylamido-6I-deoxy-per(2,6di-O-methyl)cyclomaltoheptaose 6 as a white amorphous powder (0.24 g, 70% yield). Rf 0.50 (CH2Cl2-MeOH 9:1 (v/v)). The 1H NMR analysis performed in CDCl3 demonstrated that the purified final compound 6 was free of any included byproduct or reagent as shown by Figure 1. The identification of most proton signals was derived from two-dimensional COSY and successive RELAY experiments.14 Being located in a very specific spectral domain, anomeric protons were used as a starting point for stepwise assignments. The cholesterol and cyclodextrin moieties were then (14) Berthault, P.; Djedaı¨ni, F.; Perly, B. In New trends in cyclodextrins and derivatives; Ducheˆne, D., Ed.; Edition de Sante´: Paris, 1991; p 215.

compound

solubility at 20 °C (g‚dm-3)

β-CD DIMEB 7 8a 8b chol-β-CD 9 chol-β-CD 10 chol-DIMEB 6

16.4 570 493 28 insoluble insoluble 800

sequenced using a phase-sensitive T-ROESY experiment.15 1H NMR (500 MHz, CDCl3, 25 °C): δ 6.49 (NH CD), 5.70 (NH chol), 5.38 (H-6 chol), 5.28-4.88 (H-1, OH-3 CD), 4.11 (H-3 chol), 3.993.16 (H-2, H-3, H-4, H-5, H-6, H-6′, OCH3 CD), 2.60-2.48 (CH2 succ, H-4 chol), 2.04-0.68 (H chol). The final compound 6 was further characterized by electrospray ionization mass spectrometry (ESI-MS, low and high resolution). The accurate mass data obtained by ESI-HRMS confirmed the molecular formula of the target chol-DIMEB 6 since ESI-HRMS showed a major peak at m/z 1805.9534, corresponding to the [M + Na]+ ion (exact mass 1805.9553) for 6. An additional peak at m/z 1819.9703, corresponding to the [M + CH2 + Na]+ ion (exact mass 1819.9710) was also observed, indicating the presence of the overmethylated homologue of the expected chol-DIMEB 6 as a minor component, as already described in the case of the synthesis of the per(2,6-di-O-methyl)β-cyclodextrin derivative.16 Since the separation of the minor component by recrystallization and/or HPLC is very tedious and since the physicochemical properties of both compounds are expected to be very similar, the final product 6 was further investigated without removing the minor overmethylated derivative.

Results and Discussion Properties of chol-DIMEB. The parent β-cyclodextrin displays a surprisingly low solubility in water.17 This cyclic oligosaccharide is indeed much less soluble in water at room temperature than R- and γ-cyclodextrin (cyclohexaose and cyclooctaose, respectively). It has been observed however that almost any chemical or enzymatic modification, substitution of any hydroxyl group by any alkyl or ester group, results in a dramatic increase in water solubility. It is supposed that breaking the symmetry of β-CD leads to strong perturbations of its interactions with the network of water, inducing increased solubility even if the grafted substituent is hydrophobic. This is illustrated in Table 1. Hydrophobically modified compound 8a (structure shown in Scheme 2) exhibits a higher solubility in water than the parent β-CD although it is still in the monomeric form.3 However, as the size of the aliphatic chain and/or hydrophobic group increases, the effect of the latter drives the properties toward more crystalline materials and therefore to poorly soluble derivatives as shown by compounds 8b, 9, and 10 (Table 1). These compounds show the classical behavior of all amphiphilic derivatives of β-cyclodextrin reported in the literature. For the same reasons discussed above, methylation of OH-2 and OH-6 groups of β-CD leads to higher solubility of the resulting compound (DIMEB 7) in water, although this derivative exhibits inverse solubility behavior; i.e., its solubility decreases at high temperature. Therefore, the combination of methylation and monosubstitution by a hydrophobic unit is expected to afford highly water soluble derivatives. Chol-DIMEB 6 indeed shows (15) Hwang, T. L.; Shaka, A. J. J. Magn. Reson. Ser. B 1993, 102, 155. (16) Spencer, C. M.; Stoddart, J. F.; Zarzycki, R. J. Chem. Soc., Perkin Trans. 2 1987, 1323. (17) Fro¨mming, K. H.; Szejtli, J. In Cyclodextrins in pharmacy; Kluwer: Dordrecht, The Netherlands, 1994; p 1.

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Auze´ ly-Velty et al. Scheme 2

a dramatically high solubility in water (see Table 1), but this seems to be connected to the existence of an aggregation process as indicated by the 1H NMR spectrum of 6 performed in D2O which displays very broad signals (data not shown). Characterization of the Structure of chol-DIMEB Aggregates in Aqueous Medium. Surface Tension Measurements. Surface tension was measured for a series of chol-DIMEB solutions of increasing concentrations in pure water at 25 °C. As shown in Figure 2, surface tension levels off at 40 mN‚m-1 for an extremely low concentration of solute. The critical micellar concentration (cmc) was indeed found as (5 ( 2) × 10-6 M. This indicates a highly associative behavior for cyclodextrin derivative 6, which is very surprising in view of the large volume of the hydrophilic head. It is indeed known that for nonionic surfactants having a fixed hydrophobic group, the larger the hydrophilic headgroup, the higher the cmc.18 This is the first time, to our knowledge, that a clear-cut cmc behavior in water is reported for pure and fully characterized CD derivatives. Further thermodynamic data were therefore determined to provide a better

Figure 2. Plot of surface tension of chol-DIMEB 6 solutions in pure water at 25 °C as a function of concentration.

understanding of the original behavior of this new class of surfactants. For nonionic surfactants, the standard free energy of micellization is given by18

∆G°mic ) RT ln xcmc where xcmc is the mole fraction of the surfactant in the bulk phase at the cmc. A value of -40 kJ/mol, i.e., -16 kT/molecule, was found for the free energy of micellization of chol-DIMEB. Comparing this value with those reported in standard tables of free energy of micellization,18 we notice that ∆G°mic values of compound 6 and of efficient nonionic surfactants such as polyoxyethylenated derivatives are in the same range. Chol-DIMEB has a cmc of (5 ( 2) × 10-6 M. Double chain lipids have a cmc 3-10 times smaller than the cmc of chol-DIMEB, whereas single chain surfactants with large polar headgroups have a cmc 100 times higher. Since chol-DIMEB seems to behave closer to lipid molecules than to single chain surfactants, it is of interest to focus on the study of the mass and shape of the aggregates formed above the cmc considering the molecular volumes of the cyclodextrin and hydrophobic moiety. The question is therefore to know if chol-DIMEB derivative self-assembles into single-walled closed vesicles19 or more classical globular micelles.20 Light Scattering Experiments. Light scattering provides information about the size of colloidal particles. Neglecting interparticle interactions and hydrodynamic corrections, the hydrodynamic radius of chol-DIMEB aggregates measured in pure water at 25 °C by dynamic light scattering (DLS) for various concentrations (c ) 15, 7.5, and 5 mM) is (30 ( 2) Å. This indicates that the aggregates are likely micelles. Moreover, the method of cumulants21,22 (18) Rosen, M. J. Surfactants and interfacial phenomena, 2nd ed.; John Wiley & Sons: New York, 1989; Chapter 3. (19) Gershfeld, N. L.; Stevens, W. F., Jr.; Nossal, R. J. Faraday Discuss. Chem. Soc. 1986, 81, 19. (20) Chevalier, Y.; Zemb, Th. Rep. Prog. Phys. 1990, 53, 279. (21) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814. (22) Thomas, J. C. J. Colloid Interface Sci. 1997, 117, 187.

Micellization of Hydrophobically Modified CDs

Figure 3. Determination of the average molecular weight of chol-DIMEB aggregates using static light scattering: plot of the quantity Kc/R as a function of chol-DIMEB 6 concentration c in pure water at 25 °C.

shows that the aggregates are relatively monodisperse with a polydispersity degree Q of 10-2 given by the following equation:

Q ) µ2/(〈D〉q2)2 Where µ2 is the second moment (cumulant), 〈D〉 the average diffusion coefficient, and q the scattering vector. This polydispersity degree has no unit, and its square root gives the relative standard deviation of the distribution. This polydispersity degree in decay time obtained from dynamic measurements corresponds to a mass polydispersity index Mw/Mn of 1.1, where Mw is the weightaverage molecular weight and Mn is the number-average molecular weight. We further characterized the chol-DIMEB micellar structure using static light scattering (SLS), which allows the determination of the average molecular mass M of the aggregates. Owing to the small size of the particles (,λ/ 20), we determined M using the classical equation:23

Kc/R ) 1/M + 2A2c where

R ) (I - Is)/Ib(ns/nb)2Rb

Langmuir, Vol. 16, No. 8, 2000 3731

Kc/R is (43 000 ( 8600) g‚mol-1, corresponding to an average aggregation number N of (24 ( 5). SANS and SAXS Measurements. The combined use of small-angle neutron and X-ray scattering provides an extremely sensitive approach to investigate the structure of colloidal solutions, since contrasts in the two types of radiation are very different.24,25 These two techniques offer the possibility to determine aggregation number, aggregate shape, solvent included in the excluded volumes of micelles, i.e., hydration number, and effective charge of micellar aggregates with precision. SANS is more sensitive to the total volume of the micelle while SAXS is more prone to evidence the outside shell, exhibiting a higher electron density compared to the solvent. In the general case of an isotropic solution of interacting spherically symmetric monodisperse particles, the observed scattered intensity I(q) as a function of the scattering vector q is given by

I(q) ) npP(q) S(q) where np is the concentration (cm-3) of particles. The function P(q) is the particle form factor, which depends on the size and shape of the micelle, while S(q) is the structure factor accounting for intermicelle interactions. When the shape of the aggregate is unknown and the dilution is such that interparticle interactions can be neglected, the scattering of the sample can be compared to the scattering produced by spheres, cylinders, or bilayers. If the q-range extends to low enough values, the shape of the aggregate can be detected unambiguously, since the scattered intensities produced by aggregates for different shapes differ by orders of magnitude at low q.26 For homogeneous noninteracting (S(q) ) 1) spheres of radius R, the intensity I(q) can be written as

I(q) ) φV(∆F)2{f(q,R)}2 where

f(q,R) ) 3[sin(qR) - qR cos(qR)]/(qR)3 φ is the volume fraction, V (cm3) is the dry volume of the spheres, and ∆F (cm-2) the difference between the scattering length density (SLD) of the aggregates (〈F〉) and the solvent (Fs). For homogeneous noninteracting and infinite cylinders of radius R, the intensity I(q) can be written as27

I(q) ) 4π(VL/q)(∆F)2φ{J1(q,R)/(qR)}2

and

K ) 4π2ns2/(λ4Na)(dn/dc)2 A2 is the second virial coefficient, c the concentration, nb the refractive index of benzene, ns the refractive index of the solvent, and Rb the benzene Rayleigh factor (3.2 × 10-5 cm-1). I, Is, and Ib are the scattering intensities of the solution, solvent, and benzene, respectively. dn/dc is the refractive index increment (0.139 cm3‚g-1) and Na the Avogadro number. The Rayleigh ratio R was measured at a scattering angle θ of 90° for various concentrations c of chol-DIMEB 6 in pure water. As shown in Figure 3, the value of Kc/R is insensitive to c, indicating that the interparticle interactions can be neglected considering the accuracy of the measurements. The average molecular mass M which could be deduced from the quantities of (23) Chu, B. In Laser light-scattering: basic principles and practice, 2nd ed.; Academic Press: San Diego, 1991; Chapter 1.

where J1(x) is the first-order Bessel function and VL (cm3) the volume of the cylinders per unit length. For bilayer type aggregates (vesicles) of thickness δ, the intensity I(q) can be written as28

I(q) ) (4π/q2)Σ{(δ/2)∆F sin(qδ/2)/(qδ/2)}2 where Σ is the specific area of the bilayer. (24) Zemb, Th.; Charpin, P. J. Phys. 1985, 46, 249. (25) Cabane, B. In Surfactant solutions: new methods of investigation; Zana, R., Ed.; Marcel Dekker Publishers: Dordrecht, The Netherlands, 1987; p 57. (26) Glatter, O. In Neutron, X-ray and light scattering: introduction to an investigative tool for colloidal and polymeric systems; Lindner, P.; Zemb, Th., Eds.; Elsevier Science Publishers B. V.: Amsterdam, The Netherlands, 1990; p 33. (27) Bardez, E.; Nguyen, Cao Vy; Zemb, Th. Langmuir 1995, 11, 3374. (28) Cantu`, L.; Corti, M.; Del Favero, E.; Dubois, M.; Zemb, Th. J. Phys. Chem. B 1998, 102, 5737.

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Table 2. Molecular Parameters for the Amphiphilic Cyclodextrin chol-DIMEB 6 molecular volumea Vmol molar mass M volume of the hydrophobic coreb Vchol volume of the polar headgroup VCD

2329 Å3 1783 g‚mol-1 602 Å3 1727 Å3

a The molecular volume of chol-DIMEB was determined from densitometry with a 10% accuracy. b The volume of the hydrophobic part was assumed to be the volume of the cholesterol nucleus.

Figure 5. SANS spectrum obtained for a solution of cholDIMEB in D2O at 25 °C (10-2 M). The dotted lines represent the calculated model curves for independent homogeneous spherical micelles with N ) 20 and 28.

Figure 4. SANS spectrum obtained for a solution of cholDIMEB in D2O at 25 °C (10-2 M). The solid line represents the calculated model curve for independent homogeneous spherical micelles. The dotted lines represent the calculated model curves for noninteracting infinite cylinders and bilayer type aggregates.

In the present case, we suppose first that the cholDIMEB molecules self-assemble into homogeneous noninteracting dry aggregates (no hydration of the cholDIMEB molecules in the micelles) of averaged scattering length density 〈F〉. We use a monodisperse model to fit the scattering data, since the polydispersity index Mw/Mn should be of the order of 1.1. For a fixed area per molecule σ at the solvent-micelle interface, I(q) can be calculated since the quantities R, δ, V, Φ, and Σ can be expressed as a function of σ using simple relations. As an example, in the case of spherical structures, using the following relations between N and R 2

3

4πR ) Nσ and 4/3πR ) NVmol where Vmol is the known molecular volume measured by densitometry (see Table 2 for chol-DIMEB), it becomes

R ) 3Vmol/σ and N ) 4πR2/σ In Figure 4, the small-angle neutron scattering curve obtained from a diluted aqueous solution of chol-DIMEB is compared to calculated models for independent homogeneous spheres, infinite cylinders, and bilayer type aggregates, taking an area per molecule at the watermicelle interface σ of 295 Å2, i.e., an aggregation number N of 24. Due to the large q-range measured on an absolute scale, it is clear that an overall spherical shape is the only model in agreement with the experimental data. Fitting the scattering results with a sphere model requires only one parameter which may be the aggregation number N or the area per headgroup σ, which are linked via a packing constraint. Choosing a value of N (or σ) produces both a shape and a scale for the simulated I(q). This is the fully

self-consistent fitting procedure introduced by Hayter29 which avoids separate fitting of mass and volume. Moreover, the scattering curve proves that interactions between micelles are negligible, i.e., S(q) ) 1 within experimental uncertainties. Assuming an area per molecule σ of 295 Å2, which affords the best result in this first simplified model, the radius of the micelle is 24 Å. The value of N is obtained independently of the light scattering results. In Figure 5 is plotted in dotted lines the calculated I(q) obtained for N ) 20 and 28 corresponding to σ ) 313 and 279 Å2, respectively. From visual inspection and known accuracy of neutron scattering scale calibration,11 we estimate the uncertainty to be 20%. The values found for N and R are in full agreement with the light scattering data. No oscillation is observed before the first side maximum of P(q). This may be a combination of detector resolution and micellar shape fluctuation in time. If we suppose that the chol-DIMEB molecules aggregate into spherical droplets with the cholesterol nucleus in the inner hydrophobic core and the cyclodextrin moiety in the surrounding shell, the micelles can be described as a twoshell object and the general intensity I(q) can be written as

I(q) ) np{V1(F1 - F2)f(q,R1) + V2(F2 - F3)f(q,R2)}2 where f(q,R) is defined above. The inner hydrophobic core of radius R1 has a scattering length density F1 and a volume V1 (V1 is assumed here to be the volume of the cholesterol core Vchol; see Table 2). V2 is the volume of the whole micelle of radius R2, F2 is the scattering length density of the hydrophilic corona, and F3 is the scattering length density of the solvent, i.e., D2O. It should be noted here that, when the area per molecule at the water-micelle interface σ2 and thus the aggregation number N are fixed, then the radius of the hydrophobic core R1 is fixed according to

4/3πR13 ) NVchol Figure 6 shows the neutron scattering produced by the diluted aqueous chol-DIMEB solution compared to calculated models for spherical homogeneous and two-shell micelles, taking an area per molecule at the hydrophobic core-corona interface σ1 of 120 Å2 and at the water(29) Hayter, J. B. In Proceedings of the XC Corso International School of Physics; Degiorgio, V., Corti, M., Eds.; North-Holland: Amsterdam, 1985.

Micellization of Hydrophobically Modified CDs

Figure 6. SANS spectrum obtained for a solution of cholDIMEB in D2O at 25 °C (10-2 M). The solid line represents the calculated model curve for independent two-shell spherical micelles. The dotted line represents the calculated model curve for independent homogeneous spherical micelles.

Figure 7. Illustration of the scattering length density profiles for neutrons (A) and X-rays (B) of dry chol-DIMEB micelles.

micelle interface σ2 of 295 Å2, as in the previous case. The difference between the two models proposed for the cholDIMEB micelles remains very small. This is not surprising if we consider the “bulk-like” SLD profile for neutrons of the dry chol-DIMEB micelles (see Figure 7A). In contrast to the SLD profile for neutrons, the SLD one for X-rays is “shell-like”. The core has an electron density which is similar to that of the solvent, but the corona has a higher electron density as shown in Figure 7B. This means that SAXS is very sensitive to the high electron density of the polar layer and thus to the water inclusion in the hydrophilic corona of the micelle, as shown below. The chol-DIMEB micelles are now assumed to be twoshell objects as above, but with hN water molecules inside the corona which contains N cyclodextrin headgroups. The number of water molecules per cyclodextrin headgroup h is a second free parameter in addition to the area per molecule σ2 in the fit. Since crystalline cyclodextrins bind

Langmuir, Vol. 16, No. 8, 2000 3733

Figure 8. SAXS spectrum obtained for a solution of cholDIMEB in D2O at 25 °C (10-2 M). The solid line represents the calculated model curve for independent spherical micelles with h ) 18 and σ2 ) 340 Å2. The dotted line represents the calculated model curve for independent spherical micelles with h ) 0 and σ2 ) 295 Å2.

10-18 wt % water17 (h ) 9-14 for β-CD) on the one hand, and since the hydration number of classical glycosidic surfactants such as dodecyl β-D-maltoside is ca. 1030 on the other hand, the hydration number of chol-DIMEB is expected in the 15-20 range. For SAXS, we found a best fit of the high q-oscillation for h ) 18. However, since the accuracy of such experiments remains low, correct fits could be obtained in the range h ) 10-30. Figure 8 displays the SAXS signal produced by the diluted aqueous solution of chol-DIMEB compared to fitted scattering curves for independent spherical micelles with h ) 0, σ2 ) 295 Å2 and with h ) 18, σ2 ) 340 Å2. The oscillation at high q is direct evidence of the layered structure of the micellar aggregates. The structural parameters of chol-DIMEB micelle are reported in Table 3. It is interesting, at this stage, to compare the radius of chol-DIMEB micelle obtained from experimental data with the extended length of the molecule. The length of the cholesterol nucleus is ca. 20 Å and the height of the CD ring 8 Å.17 If we assume that the length of the succinyl spacer arm is 4 Å, an average length of 32 Å is found for chol-DIMEB molecule. This value is in correct agreement with the radius of the chol-DIMEB micelle (25 Å) deduced from scattering experiments. If we consider molecular packing as the main term in self-aggregates of surfactant molecules,31 we can derive a theoretical aggregation number for chol-DIMEB micelle from geometrical considerations only. In the case of spherical micelles based on closely packed surfactant molecules, the aggregation number is

Nσ ) 4πl2 where σ is the area of the molecule at the micelle-solvent interface and l the fully extended length. Since the area of the larger side of the β-CD ring is 186 Å2,17 a theoretical aggregation number of 69 is found for chol-DIMEB micelle. This value is more than twice that obtained experimentally, indicating that the hydrophobic core (cholesterol moiety) drives the final diameter of the micelle. The higher area of the polar head observed compared to pure geometrical considerations is likely related to the effects (30) Cecutti, C.; Focher, B.; Perly, B.; Zemb, Th. Langmuir 1991, 7, 2580. (31) Israelachvili, J. N.; Mitchell D. J., Ninham, B. W. J. Chem. Soc., Faraday. Trans. 1 1976, 72, 1525.

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Auze´ ly-Velty et al.

Table 3. Structural Parameters Describing the Pure chol-DIMEB Micelle at 1.8% (w/w) in Water at 25 °Ca R2 R1 N h σ2 σ1 F1′ F2′ 〈F′〉 F1 F2 〈F〉 a

total radius of the micelle short radius of the apolar hydrophobic core aggregation number water molecules per cyclodextrin molecule area per molecule at the water-micelle interface area per molecule at the hydrophobic core-corona interface X-ray scattering length density of the micellar core X-ray scattering length density of the cyclodextrin headgroup region averaged X-ray scattering length density of the micelle neutron scattering length density of the micellar core neutron scattering length density of the cyclodextrin headgroup region averaged neutron scattering length density of the micelle

25 Å 15 Å 24 18 340 Å2 120 Å2 9.7 × 1010 cm-2 11.6 × 1010 cm-2 11.2 × 1010 cm-2 0.1 × 1010 cm-2 2.9 × 1010 cm-2 2.3 × 1010 cm-2

The exchangeable protons are taken into account.

of methylation and hydration of the CD and to the contribution of the relatively hydrophilic spacer. Conclusion Monodisperse spherical micelles with a well-defined aggregation number N ) 24 have been evidenced for aqueous solutions of chol-DIMEB 6 using various scattering techniques. This aggregation number is lower than the value expected from geometrical considerations alone, indicating that the methylation of the polar head and hydration effects play key roles in the final area per polar head in the micelles. A very low free energy of micellization has been found, indicating a highly associative behavior for chol-DIMEB molecule. Moreover, the core-shell structure has been shown to be retained in these original

nonionic spherical aggregates, making the CD moieties available for the inclusion of hydrophobic guests. The next step will be to estimate the solubilization power of these highly water soluble aggregates for hydrophobic compounds and to evidence the inclusion properties of the CD moiety. Acknowledgment. This work was supported by the European Commission (DG XII) under the FAIR Program CT 95-0300. We thank P. Guenot and P. Je´han (CRMPO, Rennes, France) for the mass spectrometry measurements. We gratefully acknowledge the assistance of O. Tache´ (CEA Saclay) with the SAXS measurements. LA991361Z