Self-Assembly of β-Cyclodextrin in Water. 2 ... - ACS Publications

Langmuir 2007, 23, 10959-10967

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Self-Assembly of β-Cyclodextrin in Water. 2. Electron Spin Resonance Simona Rossi, Massimo Bonini, Pierandrea Lo Nostro,* and Piero Baglioni Department of Chemistry and CSGI, UniVersity of Florence, 50019 Sesto Fiorentino, Italy ReceiVed April 20, 2007. In Final Form: July 19, 2007 The interaction of amphipilic spin labels with β-cyclodextrin was investigated using conventional electron spin resonance (ESR) spectroscopy to explore the aggregation of cyclodextrins in water. Methyl 5-doxylstearate (5-DMS) and stearic acid spin probes (n-DSA), which contain a cyclic nitroxide moiety with unpaired electrons covalently linked to the aliphatic chain carbon in positions 5, 7, 12, and 16, show that different dynamic domains coexist in β-CD water solutions above 3 mM. The results are consistent with the formation of β-CD self-assembled structures in water above a critical aggregation concentration and confirm the previous findings that were reported in the part 1 article of this series.

Introduction In this paper we report the second part of a work on the selfassembly of β-cyclodextrin (β-CD) in water. In the first part we showed the presence of β-CD self-assembled structures in water by means of quasi-elastic light scattering (QELS) and cryogenic transmission electron microscopy (cryo-TEM) experiments.1 The results indicated that nanostructures were formed above a critical aggregation concentration of about 3 mM at 298 K, with the main population of particles centered at about 90 nm in terms of hydrodynamic diameter. This conclusion is supported by several other pieces of experimental evidence. The association of R-, β-, and γ-cyclodextrin in water solution has been proposed on the basis of activity coefficient measurements2 and from scanning tunneling microscopy (STM) experiments,3 light scattering,4-6 and adsorption at an electrode surface.7 Moreover, Coleman reported the formation of β-CD rodlike aggregates with an average size of 200 nm,8 and Polarz detected the formation of wormlike assemblies in aqueous solutions of cyclodextrin that act as template for the synthesis of mesoporous silica.9 More recently, Loftsson discussed the formation of cyclodextrin aggregates in water and how this affects the calculation of the complexes’ binding constants.10 Large supramolecular assemblies (pseudopolyrotaxanes) from cyclodextrins and PEG-based polymers in water precipitate only when the concentration of CDs is larger than a critical value.11 Finally, in a previous study we showed that the existence of a cyclodextrin preassembled * To whom correspondence should be addressed. Fax: +39 055 4573036. E-mail: [email protected]. Web: http://www.csgi.unifi.it/. (1) Bonini, M.; Rossi, S.; Karlsson, G.; Almgren, M.; Lo Nostro, P.; Baglioni, P. Langmuir 2006, 22, 1478-1484. (2) Miyajima, K.; Sawada, M.; Nakagaki, M. Bull. Chem. Soc. Jpn. 1983, 56, 3556-3560. (3) Shigekawa, H.; Morozumi, T.; Komiyama, M.; Yoshimura, M.; Kawazu, A.; Saito, Y. J. Vac. Sci. Technol., B 1991, 9, 1189-1192. (4) Georgalis, Y.; Schuler, J.; Umbach, P.; Saenger, W. J. Am. Chem. Soc. 1995, 117, 9314-9322. (5) Gonza´lez-Gaitano, G.; Brown, W.; Tardajos, G. J. Phys. Chem. B 1997, 101, 710-719. (6) Gonza´lez-Gaitano, G.; Rodrı´guez, P.; Isasi, J. R.; Fuentes, M.; Tardajos, G.; Sa´nchez, M. J. Inclusion Phenom. Macrocyclic Chem. 2002, 44, 101-105. (7) Pospisil, L.; Svestka, M. J. Electroanal. Chem. 1997, 426, 47-53. (8) Coleman, A. W.; Nicolis, I.; Keller, N.; Dalbiez, J. P. J. Inclusion Phenom. Mol. 1992, 13, 139-143. (9) Polarz, S.; Smarsly, B.; Bronstein, L.; Antonietti, M. Angew. Chem., Int. Ed. 2001, 40, 4417-4421. (10) Loftsson, T.; Ma´sson, M.; Brewster, M. E. J. Pharm. Sci. 2004, 93, 10911099. (11) Horsky, J.; Porsch, B. J. Inclusion Phenom. 2005, 53, 97-102.

wormlike structure reasonably accounts for the kinetics and mechanism involved in the formation of the pseudopolyrotaxane structures.12 In this second contribution, we performed electron spin resonance (ESR) experiments to study the effect of β-CD selfaggregation in water on the formation of inclusion compounds with two ESR probes, namely methyl 5-doxylstearate (5-DMS) and doxylstearic acid (n-DSA). n identifies the stearic chain carbon atom at which the doxyl, ESR active moiety, is attached (n ) 5, 7, 12, 16). The results indicate that the spectral line shape is strongly affected by the structural and physical properties of the probes and of the surrounding environment. β-cyclodextrin is a macrocyclic oligosaccharide formed by seven glucopyranose units. Its shape resembles a truncated cone with two hydrophilic rims, where the hydroxyl groups are located, and a hydrophobic cavity (see Figure 1).13-15 The formation of inclusion complexes between a guest and cyclodextrins (CDs) primarily results from the hydrophobic interactions that involve the guest and the relatively hydrophobic cavity of the host, and therefore it is driven by the enthalpic contribution. The lipophilic internal cavity of cyclodextrin provides a suitable microenvironment for an apolar guest, if this has the proper cross section to fit within the cavity, as the hydrophobic (van der Waals) interactions strictly depend on the intermolecular distance.16 If the guest carries polar substituents, these may interact with the hydroxyl rims of cyclodextrin and establish stabilizing hydrogen bonds.17,18 Moreover, the release of water from the cyclodextrin cavity (about 6.5 water molecules/β-CD ring, distributed over eight sites) and from the guest molecule into the bulk phase plays a favorable entropic role in the inclusion.19 Cyclodextrins form stable host-guest inclusion complexes also with long polymeric chains, named pseudopolyrotaxanes.20 The reaction is commonly associated with the onset of a strong turbidity in the sample and eventually with the precipitation of (12) Becheri, A.; Lo Nostro, P.; Ninham, B. W.; Baglioni, P. J. Phys. Chem. B 2003, 107, 3979-3987. (13) Szejtli, J. Chem. ReV. 1998, 98, 1743-1754. (14) Del Valle, E. M. M. Process Biochem. 2004, 39, 1033-1046. (15) Ohira, A.; Sakata, M.; Taniguchi, I.; Hirayama, C.; Kunitake, M. J. Am. Chem. Soc. 2003, 125, 5057-5065. (16) Wenz, G.; Han, B.-H.; Mu¨ller, A. Chem. ReV. 2006, 106, 782-817. (17) Rekharsky, M. V.; Inoue, Y. Chem. ReV. 1998, 98, 1875-1918. (18) Liu, Y.; Han, B.-H.; Zhang, H.-Y. Curr. Org. Chem. 2004, 8, 35-46. (19) Lindner, K.; Saenger, W. Angew. Chem., Int. Ed. Engl. 1978, 17, 694695. (20) Rusa, C. C.; Fox, J.; Tonelli, A. E. Macromolecules 2003, 36, 27422747.

10.1021/la7011638 CCC: $37.00 © 2007 American Chemical Society Published on Web 09/19/2007

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Figure 1. Top and side views of the β-CD molecule.

pseudopolyrotaxanes that can be isolated and purified.21 The mechanism of this process involves the threading of several CD units over a single polymer chain and is followed by the aggregation and growth of such assemblies that give rise to the observed relevant turbidity (Rayleigh scattering).22,23 Reactants concentration, temperature, composition of the solvent, and addition of neutral cosolutes or electrolytes largely affect the kinetic of the threading step, as we have already reported in previous papers.12,24-26 Further chemical treatments on the isolated pseudopolyrotaxanes lead to the formation of topologically defined products, such as polyrotaxanes,27 molecular necklaces,28 molecular trains,29,30 and sliding gels,31-34 that are of vast interest for their nanostructured architecture and for their peculiar physicochemical properties, e.g., swelling capacity,35-37 drug delivery,38 and so forth. To further enhance the formation of inclusion compounds from cyclodextrins, their -OH residues may be also functionalized with hydrophobic (e.g., methyl or propyl) or hydrophilic groups (sulfate, phosphate, quaternary amine).39-41 The structure of CDs inclusion complexes and the mechanism of the inclusion process can be studied through different spectroscopic techniques.18,42 Electron spin resonance43-45 (ESR) (21) Harada, A. Coord. Chem. ReV. 1996, 148, 115-133. (22) Ceccato, M.; Lo Nostro, P.; Baglioni, P. Langmuir 1997, 13, 2436-2439. (23) Lo Nostro, P.; Lopes, J. R.; Cardelli, C. Langmuir 2001, 17, 4610-4615. (24) Lo Nostro, P.; Ninham, B. W.; Baglioni, P. In Self Assembly; Robinson, B. H., Ed.; IOS Press: Amsterdam, 2003. (25) Lo Nostro, P.; Lopes, J. R.; Ninham, B. W.; Baglioni, P. J. Phys. Chem. B 2002, 106, 2166-2174. (26) Lo Nostro, P.; Ceccato, M.; Baglioni, P. Preparation of Polyrotaxanes and Molecular Tubes for Host-Guest Systems. In Polysaccharide Applications: Cosmetics and Pharmaceuticals; ACS Symposium Series 737; El-Nokaly, M. A., Soini, H. A., Eds.; American Chemical Society: Washington, DC, 1999. (27) Harada, A.; Okada, M.; Kawaguchi, Y.; Kamachi, M. Polym. AdV. Technol. 1999, 10, 3-12. (28) Miyake, K.; Yasuda, S.; Harada, A.; Sumaoka, J.; Komiyama, M.; Shigekawa, H. J. Am. Chem. Soc. 2003, 125, 5080-5085. (29) Harada, A.; Li, J.; Kamachi, M. Nature 1993, 364, 516-518. (30) Harada, A.; Li, J.; Kamachi, M. Nature 1992, 356, 325-327. (31) Okumura, Y.; Ito, K. AdV. Mater. 2001, 13, 485-487. (32) Guo, X.; Abdala, A. A.; May, B. L.; Lincoln, S. F.; Khan, S. A.; Prud’homme, R. K. Macromolecules 2005, 38, 3037-3040. (33) Fleury, P. G.; Schlatter, G.; Brochon, C.; Hadziioannou, G. Polymer 2005, 46, 8494-8501. (34) Karino, T.; Okumura, Y.; Ito, K.; Shibayama, M. Macromolecules 2004, 37, 6177-6182. (35) Tamura, M.; Gao, D.; Ueno, A. ChemsEur. J. 2001, 7, 1390-1397. (36) Okada, M.; Harada, A. Macromolecules 2003, 36, 9701-9703. (37) Goretzki, C.; Ritter, H. Macromol. Chem. Phys. 1997, 198, 59-69. (38) Ooya, T.; Arizono, K.; Yui, N. Polym. AdV. Technol. 2000, 11, 642-651. (39) Mazzaglia, A.; Monsu` Scolaro, L.; Darcy, R.; Donohue, R.; Ravoo, B. J. J. Inclusion Phenom. Macrocyclic Chem. 2002, 44, 127-132. (40) Mazzaglia, A.; Angelini, N.; Lombardo, D.; Micali, N.; Patane´, S.; Villari, V.; Monsu` Scolaro, L. J. Phys. Chem. B 2005, 109, 7258-7265. (41) Szejtli, J.; Osa, T. ComprehensiVe Supramolecular Chemistry of Cyclodextrins; Elsevier: Oxford, U.K., 1996; Vol. 3. (42) Connors, K. A. Chem. ReV. 1997, 97, 1325-1358. (43) Knyazev, A. A.; Karpov, I. N.; Mikhalev, O. I.; Alfimov, M. V. J. Inclusion Phenom. Macrocyclic Chem. 2001, 40, 77-82. (44) Flohr, K.; Paton, R. M.; Kaiser, E. T. J. Am. Chem. Soc. 1975, 97, 12091218. (45) Eastman, M. P.; Freiha, B.; Hsu, C. C.; Chang, C. A. J. Phys. Chem. 1988, 92, 1682-1685.

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of spin probes is a suitable technique for structural and dynamic studies of multidomain systems.46,47 In particular, spin probes can be tailored in such a way that they either solubilize in one kind of microenvironment or, alternatively, partition and exchange between different domains. Previous ESR studies on cyclodextrin solutions in the presence of aminoxyl free radicals (i.e., DTBN, di-tert-butyl nitroxide; TEMPO, 2,2,6,6,-tetramethylpiperidine-1-oxyl; TEMPOL, 2,2,6,6,tetramethylpiperidine-N-oxyl-4-ol; R-phenyl-2,4,6-trimethoxybenzyl tert-butyl nitroxide, etc.) were carried out to investigate the conformational and dynamic properties of the inclusion complexes.48-55 Some articles have already reported that the complexed radicals exhibited different nitrogen hyperfine splitting and rotational correlation times with respect to the noncomplexed radicals.44,54-56 More recently, other works have shown a pressure dependence of the inclusion equilibrium in β- and γ-CD complexes with different spin probes.57 To the best of our knowledge, there is only one report on the interaction between doxyl carboxylic acid-based ESR probes and cyclodextrins in water solutions, where Tanaka and coworkers studied the interaction of n-doxylpalmitic acid probes with γ-CD.58 In this work we used a nonlinear least-squares program (NLLS)59,60 for the interpretation of ESR spectra obtained from 5-, 7-, 12-, and 16-DSA and 5-DMS spin labels in water solutions of β-cyclodextrin at 298 K. The line shape analysis provided a quantitative description of the n-DSA/β-CD and 5-DMS/β-CD interaction, revealing new insights into the self-aggregation of β-cyclodextrin in water. Our results indicate that the aliphatic chain of the spin probes is included in the β-CD cavity, even at low concentrations. Moreover, when [β-CD] g 3 mM, ESR results account for different spin relaxation modes. The spin probes experience multiple domains, with different polarity and dynamic properties, which are reflected in the recorded spectra. Materials and Methods The n-doxylstearic acid spin labels (n-DSA) and 5-DMS (see Chart 1) were purchased from Sigma Chemicals (Milan, Italy) and used without further purification. β-cyclodextrin was purchased from Aldrich (Milan, Italy; cat. no. 85,608-8; lot no. 07413-026) and used as received. All cyclodextrin solutions were prepared in bidistilled water (resistivity: 18.2 MΩ·cm), stirred for 3 h at 50 °C, and left overnight at 25 °C. Different CD/n-DSA and CD/5-DMS mole ratios, ranging from 504 to 27, were used to study the inclusion equilibrium: probe + m(β-CD) / [probe:(β-CD)m]. 5-DMS is not soluble in water, while the critical micelle concentrations (cmc) of 5- and 7-DSA were (46) Zana, R. Surfactant solution: New Methods of InVestigation; Marcel Dekker Inc.: New York, 1987. (47) Berliner, L. J. Spin Labelling: Theory and Applications; Academic Press: New York, 1976. (48) Kotake, Y.; Janzen, E. G. J. Am. Chem. Soc. 1988, 110, 3699-3701. (49) Kotake, Y.; Janzen, E. G. Chem. Phys. Lett. 1988, 150, 199-202. (50) Kotake, Y.; Janzen, E. G. J. Am. Chem. Soc. 1989, 111, 5138-5140. (51) Kotake, Y.; Janzen, E. G. J. Am. Chem. Soc. 1989, 111, 2066-2070. (52) Kotake, Y.; Janzen, E. G. J. Am. Chem. Soc. 1989, 111, 7319-7323. (53) Eastman, M. P.; Freiha, B.; Hsu, C. C.; Lum, K. C.; Chang, C. A. J. Phys. Chem. 1987, 91, 1953-1956. (54) Okazaki, M.; Kuwata, K. J. Phys. Chem. 1984, 88, 4181-4184. (55) Okazaki, M.; Kuwata, K. J. Phys. Chem. 1984, 88, 3163-3165. (56) Martinie, J.; Michon, J.; Rassat, A. J. Am. Chem. Soc. 1975, 97, 18181823. (57) Sueishi, Y.; Tobisako, H.; Kotake, Y. J. Phys. Chem. B 2004, 108, 1262312627. (58) Tanaka, H.; Kato, N.; Kawazura, H. Bull. Chem. Soc. Jpn. 1997, 70, 1255-1260. (59) Meirovitch, E.; Nayeem, A.; Freed, J. H. J. Phys. Chem. 1984, 88, 34543465. (60) Budil, D. E.; Lee, S.; Saxena, S.; Freed, J. H. J. Magn. Reson. 1996, 120, 155-189.

Self-Assembly of β-Cyclodextrin in Water

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Chart 1

taken from the literature.61 Their values are about 1.5 × 10-3 and 2.5 × 10-3 M, respectively. 12- and 16-DSA are more soluble. The maximum probe concentration in the investigated aqueous solutions (1 × 10-3 M) was therefore always below its cmc. This is reflected by the narrow lines in the ESR spectra (see Figure 2A, spectra a-c) and the absence of a broad line due to probe aggregation. For labeling, an appropriate amount of each spin label dissolved in ethanol was added to a vial. After evaporation of the ethanol, the proper amount of β-CD solution was added to the dry probe and gently shaken. After 30 min at 25 °C, the mixed solutions were transferred into a glass capillary (1 mm internal diameter), flame sealed, and placed in the ESR cavity. The ESR measurements were performed at 25 °C on a 200D Bruker ESR spectrometer. Data acquisition was performed with the STELAR software. The temperature was controlled with a Bruker VTB 3000 accessory (accuracy: (0.5 °C) and monitored through a thermocouple placed directly in the proximity of the sample cavity. The microwave frequency was about 9.51 GHz, while the central magnetic field was set to 3390 G with scans of 100 G. To prevent inhomogeneous line broadening, the modulation amplitude was usually kept around 1/10 of the peak-to-peak line width of the narrowest line. To simulate the ESR spectra, NLLS analyses based on the stochastic Liouville equation62,63 were performed using the latest version of the fitting program.60,64 The magnetic g and A tensors are defined in a molecule-fixed frame, where the rotational diffusion rates around the x-, y-, and z-axis are included. According to the conventional procedure, the x-axis points along the N-O bond, the z-axis is parallel to the 2pz axis of the nitrogen atom, and the y-axis is perpendicular to x and z.65 The program provides also the rotational diffusion rate of the nitroxide radical around the axis perpendicular to the main symmetry axis for the rotation (R⊥). This symmetry axis is the direction of preferential orientation of the spin-labeled molecule.66 For n-DSA, R⊥ represents the rotational wagging motion of the long axis of the acyl chains.64 The value of τ⊥, the perpendicular component of the reorientation time, was calculated as τ⊥ ) 1/6R⊥.66 The spectral analysis also yields RII, which is related to the motion about the symmetry axis. The software is also capable of performing multidomain fitting and provides quantitative information on the probe fraction that experiences each environment.

Results and Discussion Representative ESR spectra of 5- and 12-DSA in water and in aqueous β-cyclodextrin solutions are shown in Figure 2A,B, (61) Ge, M.; Rananavare, S. B.; Freed, J. H. Biochim. Biophys. Acta 1990, 1036, 228-236. (62) Polimeno, A.; Freed, J. H. J. Phys. Chem. 1995, 99, 10995-11006. (63) Meirovitch, E.; Igner, D.; Igner, E.; Moro, G.; Freed, J. H. J. Chem. Phys. 1982, 77, 3915-3938. (64) Ge, M.; Budil, D. E.; Freed, J. H. Biophys. J. 1994, 67, 2326-2344. (65) Schneider, D. J.; Freed, J. H. Biological Magnetic Resonance. Spin Labeling. Theory and Applications; Plenum Press: New York, 1989; Vol. 8. (66) Freed, J. H. In Spin Labeling: Theory and Applications; Berliner, L. J., Ed.; Academic Press: New York, 1976; pp 53-132.

Figure 2. (a) ESR spectra at 298 K from spin label 5-DSA (A) and 12-DSA (B) in water and (b-d) in β-cyclodextrin solutions at different concentrations: (b) 1 mM; (c) 3 mM; (d) 15.4 mM. The β-CD/n-DSA mole ratio is 136:1 in all spectra. The two magnifications in (A) at low and high fields are related to the spectrum d. Upward arrows point to the broad lines indicating a strong spinspin interaction. The inset in (B) shows the enlargement of the third line of spectrum d, and the arrow indicates the sharp peak of the fast component (component 1).

respectively. All ESR spectra presented in stack plots were normalized to the same integrated intensity and, eventually, expanded or reduced vertically by the factor indicated on the left of the spectrum. In water, n-DSA spin probes produce ESR profiles with three narrow hyperfine lines of nearly equal intensity that result from fast tumbling motions of the spin labels67 (see spectra “a” in Figure 2A,B). The uncomplexed free spin probes in solution can

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Table 1. Parameters Used for the Computation of the ESR Spectrum of n-DSA in Different Environments, in Water Solution with and without β-CD environment

gxx

gyy

gzz

〈g〉

Axx

Ayy

Azz

〈A〉

10-10τ⊥

10-10τ|

n

5.9 5.9 5.8

34.6 35.8 35.4

15.3 15.6 15.8

5.6 2.8 0.8

1.0 1.2 0.8

5.4 2.3 1

component 2 component 1 in water

2.008 2.008 2.008

2.007 2.006 2.006

2.003 2.003 2.003

2.006 2.006 2.006

5-DSA 5.3 5.2 6.2

component 2 component 1 in water

2.008 2.008 2.008

2.007 2.006 2.006

2.003 2.003 2.003

2.006 2.006 2.006

7-DSA 5.0 5.2 6.2

5.5 5.9 5.8

34.8 35.8 35.4

15.1 15.6 15.8

7.2 3.3 0.9

1.3 1.4 0.9

5.5 2.4 1

component 2 component 1 in water

2.009 2.008 2.008

2.007 2.006 2.006

2.003 2.003 2.003

2.006 2.006 2.006

12-DSA 5.0 5.0 6.2

5.3 5.8 5.8

34.5 35.7 35.4

14.9 15.5 15.8

7.8 3.4 1.3

1.0 1.5 1.3

8 2.3 1

component 2 component 1 in water

2.009 2.008 2.008

2.007 2.006 2.006

2.003 2.003 2.003

2.006 2.006 2.006

16-DSA 5.3 5.2 6.2

5.6 5.8 5.8

35.2 35.9 35.4

15.4 15.6 15.8

3.0 1.8 0.5

0.8 1.2 0.5

3.5 1.5 1

freely rotate with no restriction, and the position of the doxyl group along the aliphatic chain has a little influence on the spectrum shape. In the presence of β-CD, even at 1 mM concentration, the high field lines are slightly broadened as a result of the reduction of the spin probe mobility in the water matrix (Figure 2A,B, spectra “b”). This broadening effect becomes larger when [β-CD] ) 3 mM (Figure 2A,B, spectra “c”). The ESR spectra obtained for [β-CD] ) 15.4 mM show a significant asymmetry of the line around 3375 G, clearly indicating the presence of n-DSA in more than one microenvironment (Figure 2A,B, spectra “d”). The characteristic two-component feature of the ESR spectra is shown in the inset of Figure 2B that reports a magnification of the 3375 G line of ESR signal obtained from 12-DSA in a 15.4 mM β-CD solution. The arrow in the inset indicates the effect of the faster component, hereinafter referred to as component 1. This consideration stands also for 7- and 16-DSA. Radicals in two different microenvironments and in a slow-exchange regime in the ESR time scale often generate different ESR signals that overlap and produce the whole spectrum.55,68 In a large range of β-CD/n-DSA mole ratios (from 136 to 504) the ESR signals of all n-DSA solutions at a fixed β-CD concentration remain unchanged. This is an unambiguous evidence that the inclusion equilibrium n-DSA + m(β-CD) / [n-DSA:(β-CD)m] is completely shifted toward the formation of the inclusion compound, and no free spin probe is present in solution in this β-CD/n-DSA ratio range. As reported in the literature, CDs include long-chain hydrophobic chains (12 carbon atoms or longer) with a stoichiometric ratio “m” of 2:1.16,69 In PEG/CDs pseudopolyrotaxanes, depending on the molecular weight of the guest species, the ethylene oxide:cyclodextrin stoichiometric ratio is at most 2:1.16 Since the recorded ESR spectra, also at a β-CD concentration as low as 1 mM, indicate different motional parameters that cannot be ascribed to the free uncomplexed probe in water, we suggest that component 1, identified by the arrow in the inset in Figure 2B, refers to n-DSA molecules that experience a relatively fast motion microenvironment. This is also confirmed by the 〈A〉 value found through the fitting and discussed in more details later on (see Table 1). (67) Carrington, A.; McLachlan, A. D. Introduction to Magnetic Resonance with Application to Chemistry and Chemical Physics; Harper and Row: New York, 1967. (68) Gelamo, E. L.; Itri, R.; Alonso, A.; Vasques da Silva, J.; Tabak, M. J. Colloid Interface Sci. 2004, 277, 471-482. (69) Funasaki, N.; Ishikawa, S.; Neya, S. J. Phys. Chem. B 2004, 108, 95939598.

It is worthwhile to note that an increase in the viscosity of the solution should result in a decreased mobility of the probes. To check this effect, we prepared a 0.33 M solution of methyl glucoside in water containing n-DSA probes, but no changes in the ESR spectrum were detected. Moreover, as already reported in the literature,44 the viscosity of β-CD solutions does not significantly differ from that of pure water, even at concentrations close to the solubility limit. An additional broad spectral component with large outer peak separation (greater than 60 G) occurred for [β-CD] ) 15.4 mM and low β-CD/n-DSA mole ratios. In the case of 5-DSA, even at a mole ratio as large as 136, the presence of this broad line is evident (Figure 2A, spectrum “d” and its magnification). Presumably, this is due to the presence of large cyclodextrin aggregates, as the β-CD/n-DSA mole ratio is lowered and more lipophilic probe molecules are bound to the aggregate. Since the ESR signal height is inversely proportional to the square of the line width, the broad component makes only a small contribution to the composite line shape. Computer simulation of this spectral component was achieved and will be discussed later. A further indication of the presence of large aggregates in the cyclodextrin solution was given from the formation of a precipitate after 1 day at 25 °C in all the samples with [β-CD] ) 15.4 mM and higher n-DSA content. This behavior is typical of cyclodextrin solutions in the presence of hydrophobic compounds.12 In Figure 3 we show the broadening of the baseline for 5-DSA spectra obtained from the precipitate (full line). This is due to the inclusion of the probe within a highly hindering environment, consistently with the segregation of 5-DSA inside a β-CD aggregate. It is worthy to note that the broad signal is small, corresponding to a minor amount of probe. A similar behavior was observed for 7-DSA spin probes, when the β-CD/n-DSA mole ratio was set to 27 and [β-CD] was set at 15.4 mM. Under these conditions, both 5-DSA and 7-DSA solutions became turbid a few minutes after mixing the cyclodextrin solution to the spin probe, indicating that inclusion occurred. The intensity of the broader line for 5-DSA in 15.4 mM β-CD at a 27 mole ratio (Figure 3, dotted line) is more pronounced than that given by 7-DSA in the same system (data not shown). Moreover, the same signal appeared at a lower β-CD concentration (12 mM), confirming that 5-DSA shows the highest tendency within the investigated n-DSA spin probes to produce the broad line signal. Figure 4 shows a typical composite spectrum a obtained as the sum of an ESR signal with relatively fast motion features

Self-Assembly of β-Cyclodextrin in Water

Figure 3. Vertical expansion of 5-DSA spectrum in β-CD water solution (15.4 mM) at β-CD:5-DSA ) 136 registered after 24 h positioning of the precipitate into the ESR cavity (full line) and in β-CD water solution at β-CD:5-DSA ) 27 (dotted line).

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Figure 5. Experimental (full lines) and simulated (dotted lines) ESR spectra at 298 K of 7-DSA in presence of 3, 6, 9, 12, and 15.4 mM β-CD.

Figure 6. Component 1 fractions in overall ESR absorptions for 7-DSA (b), 5-DSA (9), 16-DSA (]), and 12-DSA (4) as a function of β-CD concentration. Figure 4. Experimental spectrum of 12-DSA in β-CD 15.4 mM (a, full line), simulation (a, dotted line), and the calculated two components (b, dotted line, and c) by NLLS analysis.

(component 1; Figure 4b, dotted line) and a slower spectrum (Figure 4c), henceforth called component 2. The two components were obtained as the result of multidomain fitting with the NLLS program (see Materials and Methods). Interestingly, the component 1 signal resulting from the NLLS fitting is the same for all β-CD concentrations and almost identical to the spectrum of 12-DSA in a 1 mM β-CD solution (Figure 4b, full line). On the other hand, the component 2 signal changes as a function of [β-CD]. The same behavior was found for all the experimental results at [β-CD] g 3 mM. In all the cases the agreement between the experimental and fitted spectra is very good. As an example, the ESR spectra from 7-DSA in β-CD solutions at 25 °C are reported in Figure 5. The solid lines are the experimental spectra, while the dotted lines are the results of the fittings obtained from the sum of the two weighted components. We recall here that any attempt at including a contribution from the free probe in water in the ESR simulation in the presence of β-CD above 1 mM results in a dramatic worsening of the fitting spectrum. In Figure 6 the contributions of component 1 are reported as a function of β-CD concentration. At [β-CD] ) 1 mM, the ESR signal is symmetric, indicating that the probes experience a single

domain and confirming that component 1 represents the totality of the ESR signal. As the β-cyclodextrin concentration is increased, the contribution of component 1 decreases. In particular, in the case of 16-DSA, this decrement is more pronounced and appears at lower β-CD concentrations. On the other hand, in the case of 7-DSA the contribution of component 1 shows the highest value at all β-CD concentrations, while 5- and 12-DSA have an intermediate behavior. Since the component 1 and 2 contributions quantitatively account for the partition of the spin probes between two different microenvironments, the results indicate that 7-DSA has a higher affinity for the molecular arrangement characterized by a faster motion (reflected in component 1), while 16-DSA is preferentially associated to the more constrained domain (corresponding to component 2). Besides the component 1 and 2 relative percentages in the composite spectrum, the NLLS program provides also the isotropic nitrogen hyperfine coupling constant 〈A〉 and the correlation times parallel and perpendicular to the molecular symmetry axis (τII and τ⊥, respectively) for the two components. The values are reported in Table 1, together with the parameters used for the spectral computation of the spin probes in pure water solutions. In passing, we note here that the values of the rotational parameters do not allow us to distinguish between a 1:1 and a 2:1 β-CD:probe complex. In fact the difference between the internal available area of β-CD and the cross section of the

10964 Langmuir, Vol. 23, No. 22, 2007

alkyl chain allows the rotation of the included probe without the simultaneous rotation of the host. The analysis of the 〈A〉 values clearly indicates that no free spin probe molecules are present; in fact 〈A〉 for the faster component is significantly lower than in the case of the free, randomly moving n-DSA probe dispersed in pure water. This conclusion accounts for the absence of any effect on the ESR spectra when the β-CD:n-DSA mole ratio is changed between 136 and 504. As a matter of fact, the values obtained for component 1 (15.5 G for 12-DSA and 15.6 G for 5-, 7-, and 16-DSA) significantly differ from the results obtained in pure water, where all the n-DSA’s show an 〈A〉 value of 15.8 G. A previous study on composite ESR spectra has been of great help in the interpretation of our results.55 In that paper, the inclusion of small hydrophilic spin probes (TEMPO and TEMPOL) in β-CD was investigated through ESR experiments. The separation of the two components in the high field peak was explained by the authors as an effect due to the molecular arrangement of the spin probes within the cyclodextrin cavity. In the case of TEMPOL, where the shift of the high field peak is the largest (2.2 G), the authors concluded that the N-O group was deeply included in the cavity, a much less polar environment as compared to bulk water. On the other hand, the separation of the two ESR signals in TEMPO/β-CD solutions was only 0.2 G. In this latter case the probe was considered to be included in the β-CD cavity, but the N-O group was supposed to be exposed to water. Similarly, we relate component 1 to the inclusion of the aliphatic chain inside the apolar cavity of cyclodextrin, while the N-O ring remains confined on the top of the hydrophilic rim. As a matter of fact, in our case the faster component is shifted by 0.2-0.3 G with respect to the probe in water, in very good agreement with the value expected for an N-O group in a similar situation.55 The good matching between the cross section of the probe chain and the internal diameter of the β-CD cavity (see Figure 7A, top view) further supports that the hydrophobic tail of n-DSA stretches across the cavity, with the doxyl ring anchored to the polar rim, and the carboxylic group fully exposed to bulk water (side view). In principle, for an n-DSA probe, the threading of the β-CD rings can take place from both sides of the chain, e.g., from the polar headgroup and from the terminal -CH3 moiety. But in the case of 5-DSA, the inclusion can occur only from the methyl group, as depicted in Figure 7A, due to steric restrictions. In the case of 7-DSA (see Figure 7B) there are two possibilities: “out” and “in” that differ for the position of the paramagnetic ring and of the carboxylic group. In the “out” structure the two CD rings thread the alkyl chain and the headgroup is exposed to water. In the “in” arrangement the nitroxide ring is sandwiched between the two hosts. The latter structure seems to be unfavored, as the strongly polar -COOH moiety would reside in the hydrophobic cavity of β-CD and the alkyl tail would be partially exposed to the aqueous medium. Moreover, the “in” configuration implies a larger characteristic rotational time, as the probe would drag one β-CD macrocycle in its rotation around the aliphatic chain’s main axis. Therefore, in the case of 7-DSA we expect the faster component in the spectra (component 1) to be related to the inclusion of 1 or 2 β-CD rings in the “out” arrangement and the slower component to derive from another kind of probe-cyclodextrin complex. For 12-DSA, Figure 7C shows the location of the two β-CD rings in the host-guest complex. This configuration is similar to the “in” arrangement depicted in Figure 7B for 7-DSA, but here the hydrophilic group is facing the water phase and the aliphatic chain is totally included. Therefore, in this case we

Rossi et al.

Figure 7. Three-dimensional views of the inclusion compounds formed by n-DSA and β-CD.

conclude that the faster component 1 is due to the complexation of one single β-CD. The slower component 2 can be ascribed to two different adducts: one requires the inclusion of two β-CD rings (one over the C-1 group and the other over the C-18 terminal end), and the second involves the interaction of the probe with a large aggregate of β-CD. The inclusion compound produced by 16-DSA with two β-CD is illustrated in Figure 7D. It has the same structure of the adduct formed by 5-DSA. On the basis of a detailed analysis of the recorded data, one may argue that the two spectral components reflect the presence of n-DSA/β-CD complexes with different stoichiometric ratios, 1:1 and 1:2. If this was the case, we should observe component 2 only in the spectra produced by 7-DSA and 12-DSA, where the inclusion of a second CD brings about a much slower rotation, due to the larger size of the rotating unit. Instead, this signal shows up with all the probes, and it appearssregardless of the probe usedsfor [β-CD] g 3 mM. Incidentally, this value is very close to the critical aggregation concentration of β-CD in water at room temperature that we found in our previous study (part 1)1 and to those reported in the literature sources.8-12 Finally, we recall that the 1:1 and 1:2 complexes produce the same spectral

Self-Assembly of β-Cyclodextrin in Water

profile. Therefore, the consistent decrement in component 1 for 5-DSA and 16-DSA (see Figure 6) has to be related to another mechanism, and our interpretation is that it reflects the different interaction of the probe with β-CD aggregates. For the component 2, the 〈A〉 values are shifted by 0.4-0.9 G, indicating that the nitroxide groups are located in a quite apolar environment for all n-DSA probes. To explain these results, we have examined different options. We first considered the deep inclusion of the doxyl ring into the β-CD cavity, with two possible orientations: the doxyl ring plane oriented either perpendicularly or parallel to the plane that contains the hydrophilic β-CD rims. Due to its size, the deep inclusion of the doxyl ring in the parallel direction is not possible, as previously suggested by Lajzerowicz-Bonnetau.70 Moreover, according to the results reported by Okazaki,55 we should expect a much higher decrease in the 〈A〉 value. Similarly, also the perpendicular inclusion of the probe can be excluded from the analysis of the relaxation times. In fact, the rotation of the spin probe around the axis of the aliphatic chain would result in the slower motion, and we should expect τ| . τ⊥. This is not the case for our results, where τ| e τ⊥. Instead, we suggest that the presence of two components in the experimental ESR spectrum results from the simultaneous presence of two forms of β-CD in water: monomers and aggregates. In this framework, component 1 accounts for the spin probes interacting with monomeric cyclodextrins, while component 2 is related to the interaction between the spin probes and the preformed β-CD aggregates. The geometry of inclusion is the same in both cases (see Figure 7), but in the latter case the spin probe interacts with the cyclodextrins located at the surface of the aggregate. This conclusion finds a further independent support in the results that we previously obtained on β-CD/water samples.1 Useful information can be extracted from the dependence of 〈A〉 on the position of the doxyl group along the acyl chain (see Table 1). In both component 1 and 2, 〈A〉 decreases continuously from the C-5 to the C-7 and to the C-12 position, while it increases when the probe is located at the terminal methyl group, in the C-16 position. This indicates that both components reflect the same mechanism of inclusion. The difference between the two components is therefore related only to the different polarity of the microenvironments experienced by the spin probes. The presence of monomers and aggregates fairly accounts for this evidence for two reasons: (1) The geometrical parameters of probes and β-CD molecules that regulate the inclusion process are the same regardless of the β-CD state (either monomers or aggregate). (2) However, the hydrophobicity offered by an aggregate of several β-CD is significantly greater than that of a monomeric cyclodextrin. Furthermore, this interpretation is consistent with the observed difference in the dynamics of the probes: in fact, the parallel relaxation times of the two components do not significantly differ, while the values of τ⊥ of component 2 in all the spin probes are about twice those of the fast motion component; i.e., the microenvironment surrounding the n-DSA molecule is much slower, as expected for a colloidal assembly, and cannot be related to the presence of free, uncomplexed probe moieties in solution. Finally, it is interesting to note that the trend of 〈A〉 for both components, and particularly for component 2, is similar to those obtained for the solubilization of n-DSA in micellar aggregates.71 If the doxylstearic acid spin probe has a predominantly all-anti conformation, as n increases the nitroxide group should be probing (70) Lajzerowicz-Bonnetau, J. Spin Labeling, Theory and Applications; Academic Press: New York, 1976. (71) Baglioni, P.; Bongiovanni, R.; Rivara-Minten, E.; Kevan, L. J. Phys. Chem. 1989, 93, 5574-5578.

Langmuir, Vol. 23, No. 22, 2007 10965

more deeply into the inner region of β-CD aggregates and farther from their surface. For n-DSA/β-CD systems, 〈A〉 shows a minimum for 12-DSA, and it significantly increases for 16DSA. This effect can be explained by simply assuming that the aliphatic chain is twisted through a trans-to-gauche transition, allowing both the carboxylic and the nitroxide groups to approach the aggregate surface, resulting in an increase in the hyperfine coupling constant. The energy required for such transition is only about 2.5 kJ/mol,72 which can be easily accessed at room temperature by the lowering of the chemical potential ∆µ due to the stabilizing interactions of the COOH and N-O groups with the hydrophilic rims of cyclodextrin. The entire picture is also consistent with the higher tendency of 5-DSA to interact with the β-CD aggregate. In fact, the shorter distance between the carboxylic group and the doxyl ring in 5-DSA allows the location of the doxyl ring between two β-CD molecules arranged in a head-to-head fashion. This would imply the segregation of the probe inside the β-CD aggregate and justify the slow motion parameters describing the broad component as shown in Figure 3. To better understand the behavior of stearic acid spin probes interaction with cyxlodextrin aggregates, and to further test our hypothesis, we repeated the ESR experiments with a more lipophilic spin label, i.e., methyl 5-doxylstearate (5-DMS; see Chart 1). The spectrum for a water solution of 5-DSA in the concentration range between 10-5 M and 6 × 10-4 M showed a sharp three-line profile with τ ) 8 × 10-11 s, whereas the intensity of the ESR absorption of 5-DMS is significantly lower than the value expected for a 1 mM nitroxide solution. This is due to the very poor solubility of the probe in water. This means that the solubilization of the probe in water in the presence of β-CD is a clear indication of the formation of the inclusion compound and that the concentration of the free uncomplexed probe is certainly negligible. In Figure 8A the comparison between 5-DMS and 5-DSA in water solution is shown. Although the concentration is 5 times higher, it is clear that 5-DMS signal is much smaller than that of 5-DSA (the spectra were recorded in the same experimental conditions), indicating that most of 5-DMS is not solubilized in water. As shown in the inset, the very weak ESR signal of 5-DMS in water consists of a single broad line reflecting a strong spinspin interaction due to the clustering of the nitroxide probes superimposed to a very weak triplet. This is the reason the 5-DMS probe is not suitable for detecting the cac of β-CD in water. In fact, even at extremely low concentrations of 5-DMS, the probe does necessarily form inclusion complexes with β-CD. On the other hand, the position of the doxyl ring between two hydrophobic moieties (the aliphatic chain and the ester group) promotes the inclusion of two β-CD molecules. Figure 8B shows the ESR spectra for 5-DMS in 1 mM (dotted line) and 3 mM (full line) β-CD solution at the same CD:5-DMS ratio. The concentration of the spin probes in the two solutions is 3.7 × 10-5 and 1.1 × 10-4 M, respectively. It is clear that, in the case of the hydrophobic 5-DMS, the probe is solubilized by the cyclodextrin, by inclusion in its apolar cavity. Figure 9 reports the ESR spectra for 5-DMS in β-CD solutions at different concentrations and the corresponding simulated spectra. The analysis of the ESR spectra indicates that: (i) The spectra of 5-DMS in the entire range of β-CD concentration show a lower intensity as compared to 5-DSA after the same scanning time. This is shown by the decreased signal-to-noise ratio of 5-DMS spectra with respect to 5-DSA (see Figure 2). Moreover, for a β-CD concentration ranging (72) Lemaire, B.; Bothorel, P. Macromolecules 1980, 13, 311-318.

10966 Langmuir, Vol. 23, No. 22, 2007

Figure 8. (A) 298 K ESR spectrum of 5-DSA (full line) 0.2 mM in water solution and 5-DMS (dotted line) 1 mM in water solution recorded with 5 and 50 scannings, respectively. In the inset, the enlargement of the 5-DMS spectrum is reported. (B) Comparison of 5-DMS in 1 mM (dotted line) and 3 mM (full line) β-CD solution at the same molar ratio (β-CD:5-DMS ) 27).

Figure 9. Experimental (full line) and simulated (dotted line) ESR spectra of 5-DMS at 298 K in the presence of (a) 3 mM, (b) 9 mM, (c) 12 mM, and (d) 15 mM β-CD. The β-CD:probe ratio is 136.

between 3 and 9 mM the high-field line at M ) -1 of 5-DMS is symmetric, as the probe experiences one single environment,

Rossi et al.

Figure 10. β-CD concentration dependence of the (A) isotropic hyperfine splitting (a0) and (B) perpendicular component of rotational correlation time of 5-DMS.

whereas for 5-DSA the line shape can be depicted as the overlapping of two different contributes (components 1 and 2). (ii) For β-CD concentration above 12 mM, the spectra of 5-DMS are characterized by an additional broad ESR signal which is assigned to species that slowly move in restricted conditions. The separation of the outer peaks 2A′II is 62 G, and its variation with increased β-CD concentration (up to 15.4 mM) is within the experimental error. Increasing the β-CD/5-DMS ratio from 136 to 271 results in an increment in the slow/fast motion ratio (data not shown). As mentioned above, an additional slow motion component appears in the spectrum at the highest concentration of β-CD (15.4 mM), containing 5-DSA at the same mole ratio (136) [Figure 2A (see the magnifications at the bottom) and Figure 3]. This broad triplet is identical to that obtained for 5-DMS probe at 12 and 15 mM. However, the relative intensity of the slow motion component is significantly lower for 5-DSA. In both systems, the same environment was sensed by a population of 5-DSA and 5-DMS spin probes, giving rise to a slow motion component. The analysis of the spectra results in lower percentage of 5-DSA probes associated with the β-CD aggregate. The occurrence of a higher slow motion component in β-CD solution containing 5-DMS reflects the higher affinity of this lipophilic probe for the hydrophobic region of large aggregates. And since this probe is completely insoluble in water, this finding confirms that component 2 is not produced by the free uncomplexed probe. From a double integration of the ESR spectrum at 15 mM and

Self-Assembly of β-Cyclodextrin in Water

Langmuir, Vol. 23, No. 22, 2007 10967

Table 2. Parameters Used for the Computation of the ESR Spectrum of 5-DMS in Water Solution at Different Concentrations of β-CD component

gxx

gyy

gzz

〈g〉

Azz

〈A〉

2.008

2.006

2.003

2.006

In Water 6.1 5.8

35.3

15.7

0.8

0.8

1

2.008

2.007

2.003

2.006

1 mM 6.0 4.8

35.7

15.5

3.3

2.1

1.6

35.6

15.5

3.6

2.6

1.4

Axx

Ayy

10-10τ⊥

10-10τ|

S20

n

2.008

2.007

2.003

2.006

3 mM 6.0 4.8

2.008

2.007

2.003

2.006

6 mM 6.0 4.4

35.7

15.4

3.9

2.6

1.5

2.006

9 mM 6.1 4.4

35.4

15.3

4.3

2.1

2

35.3 33.9

15.3 15.2

4.8 320.1

2.2 5.3

0.16

2 61

35.3 33.9

15.2 15.2

5.9 320.0

3.0 5.3

0.16

2 61

2.008

2.007

2.003

fast component slow component

2.008 2.008

2.007 2.007

2.003 2.003

2.006 2.006

12 mM 6.1 4.4 5.9 5.7

fast component slow component

2.008 2.008

2.007 2.007

2.003 2.003

2.006 2.006

15 mM 6.1 4.3 5.9 5.7

12 mM β-CD, the contribution of the fast moving radical was estimated as 7 and 10% of the total intensity, respectively. The magnetic and motion parameters obtained from the best fit calculation of 5-DMS ESR spectra reported in Figure 9 are summarized in Table 2. The A0 values of the fast motion component of 5-DMS in β-CD solution, measured directly from the spectra, are plotted in Figure 10A and indicate that the nitroxide group of 5-DMS experiences a rather apolar environment (with respect to pure water), i.e., the internal cavity of β-cyclodextrin. Figure 10B shows the dependence of τ⊥ of the fast motion component on the concentration of β-CD. The rotational motion of the 5-DMS in the presence of 1 mM β-CD was significantly lower than in buffer solution. Interestingly, the mobility of the spin probes does not vary significantly by increasing the β-CD concentration up to 6 mM. Instead, a consistent concentration effect is revealed above 9 mM, with a dramatic lowering in the mobility of 5-DMS. The motional and polarity values are consistent with the interaction of 5-DMS molecules segregated in the β-CD aggregates: in fact, the probe mobility is highly hindered. A mere interaction of the probe with the surface of the aggregate does not account for such effect, as the probe would be free to rotate in the β-CD cavity. Regarding the fast motion component, the τ⊥ values of the slow motion component are 2 orders of magnitude larger and comparable to the values obtained for the same kind of ESR probes when they are confined in a lipid bilayer. Moreover, the environment experienced by the probe is much less polar than water. It is worthy to note that 5-DSA provided similar results, although the amount of probe segregated within the aggregate is much smaller. This difference is due to the hydrophilic nature of the carboxylic moiety and its interactions with the aqueous solvent. In the case of 5-DMS, the polarity of the ester group is much lower and favors the inclusion of a second β-CD molecule.

Conclusions In this work, ESR spectroscopy has been used to study the structure and dynamics of β-CD aqueous solutions at 298 K. The data suggest that the probes n-DSA and 5-DMS intercalate in the cyclodextrin aggregates, but the different probes preferentially interact with different sites depending on the local polarity, hydration, and dynamics. Two main components were found in the ESR produced by n-DSA/β-CD systems. The faster ESR component (component 1), which was detected in all the investigated n-DSA/β-CD systems, has been simulated to extract the dynamic and polarity

properties experienced by the spin probes (τ, 〈A〉). The comparison of the simulation results with the spectrum obtained from the n-DSA in pure water definitely shows that the component 1 signal derives from the inclusion of the stearic chain in the β-cyclodextrin inner cavity. However, the decrease in 〈A〉 is consistent with the location of the doxyl ring close to the β-CD hydrophilic rim. The broader lines and lower 〈A〉 values observed for the slower ESR spectral component (component 2) detected in n-DSA/βCD systems indicate that the rotational diffusion of the probes is slower and that they experience a less polar microenvironment. This result probably suggests that the probes interact with large β-CD aggregates that offer different domains with different polarities. The appearance of two separated signals shows that the frequency of exchange between the two sites is low, if compared to the experimental time scale accessible through ESR, supporting the existence of stable aggregates of β-CD in water in equilibrium with the monomers. Component 1 arises from the interaction of the spin probes with monomeric units, while component 2 reflects both the magnitude of the aggregation phenomenon and the different affinity of the probes for the aggregates. Results obtained from the 5-DMS/β-CD systems are consistent with these conclusions and show that a molecule with appropriate size and polarity properties can be included inside a β-CD aggregate. The formation of self-assembled structures in β-CD aqueous solutions explains the results of the ESR experiments carried out in this work and confirms the cryo-TEM and QELS measurements reported in our previous paper,1 as well as the kinetics of the threading process that takes place during the formation of pseudopolyrotaxanes.12 We expect that temperature and solvent composition have a dramatic effect on the inclusion of spin probes in the hydrophobic CD’s cavity and on the association of cyclodextrins in water and will be the subjects of future works. We wish to thank the reviewers for their criticism and suggestions. Acknowledgment. We gratefully acknowledge the Consorzio per lo Sviluppo dei Sistemi a Grande Interfase (CSGI, Italy) and Ministero per l’Istruzione, l’Universita` e la Ricerca (PRIN-2003, MIUR, Italy), for partial financial support. We wish to thank the reviewers for their criticism and suggestions. LA7011638