Structural Properties of Nonionic Cyclodextrin Colloids in Water

Jan 23, 2004 - Unita` di Messina, Messina, Italy. Received July 28, 2003. In Final Form: November 11, 2003. The amphiphilic character in water of a no...
0 downloads 0 Views 164KB Size
Download PDF
Langmuir 2004, 20, 1057-1064

1057

Articles Structural Properties of Nonionic Cyclodextrin Colloids in Water Domenico Lombardo,*,† Alessandro Longo,‡ Raphael Darcy,§ and Antonino Mazzaglia*,|,⊥ Istituto per i Processi Chimico Fisici (IPCF-CNR), Sezione di Messina, Via La Farina 237, 98123 Messina, Italy, Istituto per lo studio dei Materiali Nanostrutturati (ISMN-CNR), Sezione di Palermo, Via Ugo La Malfa 153, 90146, Palermo, Italy, Centre for Synthesis and Chemical Biology of the Conway Institute, Department of Chemistry, National University of Ireland, University College Dublin, Belfield, Dublin 4, Ireland, ISMN-CNR, Unita` di Messina, c/o Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica Analitica dell’Universita` di Messina, Salita Sperone 31, 98166 Messina Italy, and INFM, Unita` di Messina, Messina, Italy Received July 28, 2003. In Final Form: November 11, 2003 The amphiphilic character in water of a novel class of chemically modified cyclodextrins has been investigated by means of small-angle X-ray scattering and light scattering. The introduction of hydrophilic oligo(ethylene glycol) onto the secondary side of heptakis[6-alkylthio-6-deoxy-2-oligo(ethylene glycol)]-βcyclodextrins produces an enhanced water solubility of these molecules. Shape and dimensions of the generated micellar aggregates, analyzed in terms of a suitable core-shell model, remain stable in the wide concentration range explored. The highly associative behavior of these macromolecules is evidenced by the very low value of the critical micelle concentration (cmc), which is about 2 orders of magnitude smaller than the cmc usually obtained for traditional surfactant. Despite the complex geometry of this novel macromolecule, shape and dimensions of generated micellar aggregates can be properly described according to the thermodynamic approaches generally used for amphiphilic molecules and block copolymers. Results show how the modulation of hydrophobic and hydrophilic components sensitively influence the structural features of the generated aggregates thus offering the possibility to control molecular organization in a manner similar to that for traditional colloids. For all the classes of the investigated systems, the small micelles have been found in equilibrium with polydisperse large aggregates of entangled micelles. These novel nonionic colloidal systems combine inclusion and transport properties of host macrocycles, such as cyclodextrin, together with the increased stability of colloidal aggregates, and may be of interest for their potential application as innovative drug delivery systems.

Introduction Recently there has been increasing interest in the synthesis and characterization of surfactant molecules with novel structural and topological features, which have the tendency to self-aggregate into well-defined supramolecular structures. Synthetic strategies allow a suitable balance between the hydrophobic and hydrophilic components by regulating the thermodynamic equilibrium of generated supramolecular aggregates.1 Particularly interesting in this respect are cyclodextrin (CD) colloids which possess lyotropic and thermotropic properties. It * To whom correspondence may be addressed: Domenico Lombardo, tel. +39 0902939693, fax +39 0902939902, e-mail lombardo@ me.cnr.it; Antonino Mazzaglia, tel + 39 0906765427, fax +39 090393756, e-mail [email protected]. † D.L. Istituto per i Processi Chimico Fisici (IPCF-CNR), Sezione di Messina. ‡ Istituto per lo studio dei Materiali Nanostrutturati (ISMNCNR), Sezione di Palermo. § Centre for Synthesis and Chemical Biology of the Conway Institute, Department of Chemistry, National University of Ireland, University College Dublin. | A.M. ISMN-CNR, Unita ` di Messina, c/o Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica Analitica dell’Universita` di Messina. ⊥ INFM, Unita ` di Messina.

has been shown that macrocyclic amphiphiles of hydrophobically modified CDs can form a variety of supramolecular assemblies, including monolayers, multilayers, and Langmuir-Blodgett films at the air-water interface.2 Moreover amphiphilic CDs can be admixed to phospholipid monolayers3 as well as liposomes,4 and they can be (1) (a) Kaczmarski, J. P.; Glass, J. E. Macromolecules 1993, 26, 5149. (b) Hydrophobic Polymers; Alami, E., Rawiso, M., Isel, F., Beinert, G., Binana-Limbele, W., Franc¸ ois, J., Eds.; Advances in Chemistry Series 248; American Chemical Society: Washington, DC, 1995; Chapter 18. (c) Chassenieux, C.; Nicolai, T.; Durand, D. Macromolecules 1997, 30, 4952. (d) He, L.; Garamus, V. M.; Funari, S. S.; Malfois, M.; Willumeit, R.; Niemeyer, B. J. Phys. Chem B 2002, 106, 7596. (e) Lafle`che, F.; Durand, D.; Nicolai, T. Macromolecules 2003, 36, 1331. (2) (a) Kawabata, Y.; Matsumoto, M.; Tanaka, M.; Takahashi, H.; Irinatsu, Y.; Tamura, S.; Tagaki, W.; Nakahara, N.; Fukuda, K. Chem. Lett. 1986, 1933. (b) Parrot-Lopez, H.; Ling, C. C.; Zhang, P.; Baszkin, A.; Albrecht, G.; de Rango, C.; Coleman, A. W. J. Am. Chem. Soc. 1992, 114, 5479. (c) Tschoreloff, P. C.; Boissonnade, M. M.; Coleman, A. W.; Baszkin, A. Langmuir 1995, 11, 191. (d) Greenhall, M. H.; Lukes, P.; Kataky, R.; Agbor, N. E.; Badyal, J. P. S.; Yarwood, J.; Parker, D.; Petty, M. C. Langmuir 1995, 11, 3997. (e) Matsumoto, M.; Tanaka, M.; Azumi, R.; Tachibana, H.; Nakamura, T.; Kawabata, Y.; Miyasaka, T.; Tagaki, W.; Nakahara H.; Fukuda, K. Thin Solid Films 1992, 210211, 803. (f) Nakahara, H.; Tanaka, H.; Fukuda, K.; Matsumoto, M.; Tagaki, W. Thin Solid Films 1996, 284-285, 687. (g) Parazak, D. P.; Khan, A. R.; D’Souza, V. T.; Stine, K. J. Langmuir 1996, 12, 4046. (h) Hamelin, B.; Jullien, L.; Laschewsky, A.; Herve´ du Penhoat, C. Chem. Eur. J. 1999, 5, 546.

10.1021/la035370q CCC: $27.50 © 2004 American Chemical Society Published on Web 01/23/2004

1058

Langmuir, Vol. 20, No. 4, 2004

dispersed into nanospheres thus showing promising properties for drug encapsulation.5 Most of these CDs are not soluble in water, and the (mixed) aggregates in water are systems out of thermodynamic equilibrium. On the other hand highly soluble amphiphilic compounds forming spherical micelles in water have been produced.6 Some of us showed that heptakis(6-alkylthio-6-deoxy)β-cyclodextrin derivatives behave as thermotropic liquid crystals but are insoluble in water.7 A much increased water solubility was observed upon substitution of β-cyclodextrin with hydroxyethyl and oligo(ethylene glycol) by reaction with ethylene carbonate8 or with ethylene oxide.9 To increase the solubility as well as to provide a versatile carrier system, some of us synthesized nonionic amphiphilic CDs (heptakis[6-alkylthio-6-deoxy-2-oligo(ethylene glycol)]-β-CD, where alkyl is ethyl, hexyl, dodecyl, and hexadecyl).10-12 Furthermore, cationic analogs were prepared by modifying OH terminal groups into NH2 functions in ethylene glycol conjugates, and their interaction with anionic porphyrins was studied.13 In particular nonionic and cationic bilayer vesicles entirely composed of the dodecyl and hexadecyl derivative11,13 are still thermodynamically metastable, but the presence of ethylene glycol oligomers at the vesicle surface increases the colloidal stability of these vesicles while potentially decreasing their immunogenicity in drug delivery systems, much like “stealth liposomes”14 and “niosomes”.15 In a previous note12 the synthesis of a novel class of chemically modified amphiphilic cyclodextrins composed of 6-alkylthio 2-oligo(ethylene glycol)]-β-CDs (where alkyl is ethyl and hexyl) has been presented. In water solution these macromolecules showed an enhanced water solubility and the formation of supramolecular aggregates. The main features and properties of the investigated systems are summarized respectively in Chart 1 and Table 1. The aim of the this paper is to outline the structural properties of these macromolecules by probing in detail (3) (a) Coleman, A. W.; Kasselouri, A. Supramol. Chem. 1993, 1, 155. (b) Kasselouri, A.; Coleman, A. W.; Baszkin, A. J. Colloid Interface Sci. 1996, 180, 384. (c) Kasselouri, A.; Coleman, A. W.; Albrecht, G.; Baszkin, A. J. Colloid Interface Sci. 1996, 180, 398. (4) (a) Jullien, L.; Lazrak, T.; Canceill, J.; Lacombe, L.; Lehn, J. M. J. Chem. Soc., Perkin Trans. 2 1993, 1011. (b) Lin, J.; Creminon, C.; Perly, B.; Djedaı¨ni-Pilard, F. J. Chem. Soc., Perkin Trans. 2 1998, 2639. (c) Auze´ly-Velty, R.; Perly, B.; Tache´, O.; Zemb, T.; Je´han, P.; Guenot, P.; Dalbiez, J. P.; Djedaı¨ni-Pilard, F. Carbohydr. Res. 1999, 318, 82. (d) Lesieur, S.; Charon, D.; Lesieur, P.; Ringard-Lefebvre, C.; Muguet, V.; Duchene, D.; Wouessidjewe, D. Chem. Phys. Lipids 2000, 106, 127. (5) (a) Skiba, M.; Morvan, C.; Ducheˆne, D.; Puisieux, F.; Wouessidjewe, D. Int. J. Pharm. 1995, 126, 275. (b) Skiba, M.; Ducheˆne, D.; Puisieux, F.; Wouessidjewe, D. Int. J. Pharm. 1996, 129, 113. (c) Gulik, A.; Delacroix, H.; Wouessidjewe, D.; Skiba, M. Langmuir 1998, 14, 1050. (d) Lemos-Senna, E.; Wouessidjewe, D.; Ducheˆne, D.; Lesieur, S. Colloids Surf., B. (6) (a) Petter, R. C.; Salek, J. S.; Sikorski, C. T.; Kumaravel, G.; Lin, F. T. J. Am. Chem. Soc. 1990, 112, 3860. (b) Auze´ly-Velty, R.; Djedaı¨niPilard, F.; De´sert, S.; Perly, B.; Zemb, T. Langmuir 2000, 16, 3727. (7) Ling, C.-C.; Darcy, R.; Risse, W. J. Chem. Soc., Chem. Commun. 1993, 438. (8) Friedman, R. B. In Proceedings of the 4th International Symposium on Cyclodextrins; Huber, O., Szejtli, J., Eds.; Kluwer Academic Publishers: Dordrecht, 1988; pp 103-111. (9) Topchieva, I. N.; Mischnick, P.; Ku¨hn, G.; Polyakov, V. A.; Elezkaya, S. V.; Bystryzky, G. I.; Karezin, K. I. Bioconjugate Chem. 1998, 9, 676. (10) Mazzaglia, A.; Donohue, R.; Ravoo, B. J.; Darcy R. Eur. J. Org. Chem. 2001, 1715. (11) Ravoo, B. J.; Darcy, R. Angew. Chem., Int. Ed. 2000, 39, 4324. (12) Mazzaglia, A.; Ravoo, B. J.; Darcy, R.; Gambadauro, P.; Mallamace, F. Langmuir 2002, 18, 1945. (13) (a) Donohue, R.; Mazzaglia, A.; Ravoo, B. J.; Darcy, R. Chem. Commun. 2002, 2864. (b) Mazzaglia, A.; Monsu` Scolaro, L.; Darcy, R.; Donohue, R.; Ravoo, B. J. J. Inclusion Phenom. Macrocyclic Chem. 2002, 44, 127. (14) Lasic, D. D. Angew. Chem., Int. Ed. Engl. 1994, 33, 1685. (15) Uchegbu, I. F.; Vyas, S. P. Int. J. Pharm. 1998, 172, 33.

Lombardo et al. Chart 1. Compositional and Topological Features of the CD 3, CD 4, and CD 5 Surfactant Molecules

Table 1. Properties of Amphiphilic Cyclodextrin Monomers system

molar mass Ma (g/mol)

molecule volb (Å3)

carbon chain volc (Å3)

headgroup vol (Å3)

3 4 5

2061 2763 3071

2926 3921 4359

568.4 1321.6 1321.6

2357.6 2599.4 3037.4

a Main peaks found in Maldi spectra. b Calculated from density measurements. c Calculated by using the Tanford relation16 (i.e., 7vC ) 7(27.4 + 26.9nc)).

their amphiphilic behavior. We describe, in particular, the morphological features of the aggregates that form spontaneously when these molecules are dissolved in water. More specifically we study the possibility to control molecular organization and self-assembly processes as a function of the geometrical properties of individual macromolecules (such as its surface area or headgroup hindrance) and relate it with the recent thermodynamic approaches relative to the traditional colloids. The use of scattering techniques, such as small-angle X-ray scattering (SAXS) and light scattering, is proven to be a powerful method for the investigations of the structural details of the generated aggregates. Experimental Methods Materials. CDs 1 and 2 were obtained in two steps from a β-cyclodextrin parent. CDs 3 and 4 were synthesized from 1 and 2, respectively, as described.10 CD 5 was obtained from 4 as reported.12 Preparation of Aqueous Solutions. CDs 3 and 5 readily dissolve in water at concentrations up to 10 wt %. At high concentrations, dissolution can be accelerated by stirring and gentle heating. CD 4 does not dissolve spontaneously in water, but a homogeneous dispersion can be obtained by sonication in an ultrasound bath (Bransonic 1510) for 30-60 min at room temperature. Density Measurements and Volumetric Properties (Partial Molar Volume). The apparent molar volume Vm of 3, 4, and 5 has been evaluated according to the following relation

Cyclodextrin Colloids Vm )

Langmuir, Vol. 20, No. 4, 2004 1059 Mw 1000(F - F0) F mFF0

(1)

where Mw is the solute molar mass, m is the solution molality, and F and F0 are the mass density of the solution and of the solvent, respectively. These last quantities were measured as a function of concentration at the constant temperature of T ) 25 °C using a vibrating method with a DMA 5000 density meter (Anton Paar, Graz, Austria). Combining this information with an empirical formula relating the volume of a hydrocarbon chain V to the number of carbon molecules nC in the chain (i.e., V ) 27.4 + 26.9nc Å3),16 the volumes of the hydrophilic and hydrophobic parts of the molecule have been determined. Small-Angle X-ray Scattering. The SAXS patterns have been recorded by a laboratory instrumentation consisting of a Philips PW X-ray generator (providing Cu KR, Ni-filtered X-ray radiation of wavelength 1.5418 Å) with a Kratky-type smallangle camera in the “finite slit height geometry” equipped with step scanning motor and scintillator counter as detector. The range of scattering vector covered is 0.005 Å-1 < q < 0.6 Å-1. All measurements were carried out at the temperature of T ) 25 °C. The scattering data were normalized with respect to transmission and were corrected by the empty cell and solvent contribution. Best-fit analysis was performed using homemade programs based on the CERN minimization procedure MINUITS. The smearing effect, due to instrumental setup, was taken into account during the minimization to the data according to the standard procedure.17,18 Light Scattering. Both elastic light scattering (ELS) and quasi-elastic light scattering (QELS) measurements were carried out using a computerized homemade goniometer and the 532 nm line of a duplicated Nd:YAG laser as exciting source. A Malvern 4700 correlator was used to collect the intensity autocorrelation function g(2)(t). The temperature in the light scattering experiment was fixed at T ) 298 ( 0.1 K. The investigated scattering angle range was 20° e θ e 150° corresponding to exchanged wavevector values in the range 5.5 e k e 30.5 µm-1 (being k ) [(4πn)/λ] sin(θ/2), where n is the refractive index of the sample and λ the vacuum incident wavelength). To convert the photomultiplier output into an absolute scattering intensity, a calibration procedure was performed using toluene as reference (with Rayleigh scattering cross section per unit volume Rtoluene ) 1.406 × 10-5 cm-1). This allows the determination of the calibration constant (the socalled Rayleigh ratio). The refractive index increments dn/dc were measured using a differential refractometer.

Results and Discussion Formation of Micellar Aggregates: Morphological Features and Thermodynamic Considerations. Selfassociation of amphiphilic cyclodextrins can be described by close analogy with low molecular weight surfactants.16,19 Micellar formation is driven by the competition between interfacial energy of the micelle core with solvent and the conformational distortion energy of the soluble chains emanating from the core. According to the closed association model the detected critical micellar concentration (cmc) can be used to obtain information on the thermodynamic parameters of the micellization process. The standard Gibbs free energy change ∆G for the transfer of 1 mol of amphiphile from solution to the micellar phase (free energy of micellization) can be approximated by the relation19 (16) Israelachvili, J. N.; Mitchel, D. J.; Ninham B. W. J. Chem. Soc., Faraday Trans. 1976, 72, 1525. (17) (a) Feign, L. A.; Svergun, D. I. Structure Analysis by SmallAngle X-ray and Neutron Scattering; Plenum Press: New York, 1987. (b) Glatter, O.; Kratky, O. Small-Angle X-ray Scattering; Accademic Press: London, 1982. (18) Strobl, G. Acta Crystallogr. 1970, A26, 367. (19) Israelachvili, J. N. Physics of Amphiphiles: Micelles, Vesicles and Microemulsions; Degiorgio, V., Corti, M., Eds.; North-Holland: Amsterdam, 1985.

Figure 1. Plot of the scaled inverse excess scattering intensity Kc/R as a function of concentration c for the CD 3 sample (measured at θ ) 90° and T ) 25 °C). The upturn in the intensity indicates the onset of the micellization and allows the determination of the cmc for the system.

∆G ≈ RT ln(Xcmc)

(2)

where R is the ideal gas constant, T is the absolute temperature, and Xcmc is the cmc (expressed in molar fraction) at temperature T. In Figure 1 we report the concentration dependence of the scaled inverse excess scattering intensity Kc/R for the CD 3 system, where K is the usual optical constant, c is the concentration, and R is the so-called Rayleigh ratio. The highly associative behavior is evidenced by the very low value detected for the cmc as determined by light scattering measurements. In fact while no cmc could be detected for CD 4 in water solution, the cmc’s for 3 and 5 were found to be 10 mg/L (Xcmc ) 8.7 × 10-8) and 5 mg/L (Xcmc ) 2.9 × 10-8), respectively. These values confirm the determination of cmc for 3 and 5 by surface tension measurements12 and are about 2 orders of magnitude smaller than cmc’s usually obtained for traditional surfactant with large hydrophilic headgroups.20 The corresponding micellization free energy values are ∆G ) -40.28 kJ/mol for 3 and ∆G ) -43.00 kJ/mol for 5, and provide evidence that thermodynamically stable micelles are formed spontaneously in water solution. The resolution of the stability problem in water solution of parent β-CD has been a nontrivial problem, as documented by the large amount of literature that appeared in recent years.21 This problem is mainly related to the poor water solubility of these cyclodextrins, which is probably caused by an intramolecular hydrogen bonding network between OH2 and OH3 of adjacent glucose residues. In this respect the substitution of hydroxyl group by alkyl or ester groups produces a disruption of the unfavorable intramolecular interaction, thus realizing a sensitive enhancement of water solubility. In our specific case the introduction of hydrophilic oligo(ethylene glycol) onto the secondary side of heptakis[6-alkylthio-6-deoxy2-oligo(ethylene glycol)]-β-CDs produces a sensitive increase of water solubility of these molecules. The presence of complex compositional and topological features makes these systems rather different with respect to most of the investigated amphiphiles. The corresponding supramo(20) Rosen, M. J. Surfactant and interfacial phenomena, 2nd ed.; John Wiley & Sons: New York, 1989; Chapter 3. (21) (a) Coleman, A. W.; Nicolis, I.; Keller, N.; Dalbiez, J. P. J. Inclusion Phenom. Macrocyclic Chem. 1992, 13, 139. (b) Ha¨usler, O.; Mu¨ller-Goymann, C. C. Starch 1993, 45, 183. (c) Azaroual-Bellanger, N.; Perly, B. Magn. Reson. Chem. 1994, 32, 8. (d) Valleton, J. M.; Alexandre, S.; Coleman, A. W.; Kasselouri, A. Thin Solid Films 1996, 284-285, 765. (e) Gaitano, G. G.; Brown, W.; Tardajos, G. J. Phys. Chem. B 1997, 101, 710. (f) Gonzales Gaitano, G.; Rodriguez, P.; Isasi, J. R.; Fuentes, M.; Tadayos, G.; Sanchez, M. J. Inclusion Phenom. Macrocyclic Chem. 2002, 44, 101.

1060

Langmuir, Vol. 20, No. 4, 2004

Lombardo et al.

an uniform sphere of radius R, the corresponding form factor P(q) can be written as17

P(q) ) [3J1(qR)/(qR)]2 ) [sin(qR) - (qR) cos(qR)] 3 (qR)3

{

Figure 2. (a) Characteristic SAXS spectra for different concentrations of the CD 4 system at T ) 25 °C. (b) Scaling of the SAXS curves with respect to concentration.

lecular aggregates, in fact, are very sensitive to the geometrical properties of the individual molecule, such as its surface area or headgroup hindrance.22 SAXS Measurements: Structural Features of Micellar Aggregates. The characteristic SAXS intensity for different concentrations of CD 4 is shown in Figure 2a. The different curves scaled with respect to the concentration (Figure 2b) show that the main structural features of the aggregates are not influenced by the change in the concentration. Small deviations in the low q region of more concentrated spectra, as evidenced in the square region of the figure, are mainly connected with interparticles correlations. Moreover the presence of an upturn in the low q region is probably due to the presence of big aggregates (R > 500 Å). A similar trend has been obtained for samples of CD 3 and CD 5. If we assume the micelle solution as a monodisperse system, the SAXS scattering intensity I(q) can be expressed as a product of the form factor P(q), which contains information on the shape and dimension of the scattering particles and the structure factor S(q) describing the interparticle interaction17 2

I(q) ) N(∆F) P(q)S(q)

(3)

where N is the number density of the micelles, and ∆F ) (F - F0) is the so-called “contrast” (i.e., the difference between the scattering length density of the particle F and that of the solvent F0). In the dilute region the interparticle interaction can be neglected (i.e., S(q) ≈ 1), so that the analysis of scattering intensity I(q) can furnish direct information of morphological features of the scattering particles. Assuming our aggregates as (22) (a) Cantu, L.; Corti, M.; Sonnino, S.; Tettamanti, G. Chem. Phys. Lipids 1986, 41, 315. (b) Cantu, L.; Corti, M.; Del Favero, E.; Digirolamo, E.; Raudino, A. J. Phys. II 1996, 6, 1067.

}

2

(4)

where J1(x) ) [sin(x) - x cos(x)]/x2 is the first-order spherical Bessel function. This expression has been used to fit our data in the dilute regime (c < 2% w/w), where interparticles interference effects are assumed to be negligible. Results of the fitting, for CD 3 are presented in the inset of Figure 3a. It is clear that while scattering data are well reproduced in the small q region, a sensitive discrepancy is detectable for higher q values. These scattering features are typical of internal interference caused by intraparticle contrast effects and are indicative of a layered structure of the scattering particles. If we assume that the generated aggregates are composed of hydrocarbon chains in the core and hydrophilic headgroups in a surrounding shell, the corresponding form factor can thus be described by a core-shell model expressed as a function of core and shell radius, R1 and R2, respectively, and core and shell scattering-length densities, Fc and Fs (Figure 3b). The scattering-length density (SLD) is expressed as F ) Σibi/V, where V is the volume of the head or tail groups while bi is the scattering length of the component atoms.17 Furthermore if we consider that water molecules penetrate into the hydrophilic region, the scattering-length density of the hydrophilic shell can be expressed as Fs ) ∑ibi/(VHG + hVW), where VHG and VW are the headgroup and water volume respectively, and h is the hydration number. Actually a priori determination of headgroup SLD is quite difficult due to the complex hydration process involved in that region. The high hydration in the corona region produces, in fact, the shift of the corresponding effective SLD toward that of the solvent. Two micellar geometries, namely, the ellipsoidal and cylindrical (core-shell) model, were used to fit the scattering data. While the results obtained using the cylindrical models were poor (data not shown) and were rejected for further analysis, both spherical and ellipsoidal core-shell models provide good fitting quality. On the other hand the very low ellipticity ( ) 1.04) obtained from the fitting, together with an ellipsoid major radius a very close to the sphere radius, suggests that a spherical morphology is the most appropriate for the description of CD 3, 4, and 5 aggregates. The corresponding form factor (spherical core-shell model) can be expressed as17

P(q)(∆F)2 )

[

3J1(qR1) 4π 3 R1 (FC - FS) + 3 qR1 3J1(qR2) 4π 3 R (F - F0) 3 2 S qR2

]

2

(5)

Results obtained for CD 3 are presented in Figure 3a and clearly indicate how the main features of SAXS data are well reproduced by the adopted model in the whole q range explored. The core-shell approach has been successfully employed to describe the SAXS experiments in water solution of hydrophobically modified cyclodextrin.6b By fitting the SAXS curves for the CD 3, CD 4, and CD 5 systems, the average values of the corresponding core radius R1 and the external radius R2 of the aggregates can be obtained, as reported in Table 2.

Cyclodextrin Colloids

Langmuir, Vol. 20, No. 4, 2004 1061

Figure 3. (a) Fitting of the experimental SAXS form factor P(q) for CD 3 (c ) 0.028 M) with an uniform sphere (inset) and a core-shell model. (b) Sketch of the adopted core-shell model for the micellar aggregates of chemically modified β-cyclodextrins. Table 2. Structural Features of the CD 3, CD 4, and CD 5 Aggregates system R1 (Å)a R2 (Å)a 3 4 5

8.0 12.0 9.0

23.0 40.0 32.5

Nagg (11.2)c

3.8 5.5 (7.7)c 2.3 (3.2)c

RHF (Å)b

RHS (Å)b

Rg (Å)

26 44 31

750 570 820 (280)d

950 840 910

a Obtained from SAXS measurements by means of the coreshell model. b Average values obtained from DLS by means of the double exponential fit (eq 11). c Calculation accounting for the possible inclusion of hydrophobic alkyl chains in the cyclodextrin cavity. d Average value obtained from DLS by means of a triple exponential fit.

The obtained SAXS results clearly indicate that in water solution the investigated samples form stable micellar aggregates with a very small dimension. More specifically a detected internal radius of R1 of the order of 10 Å is incompatible with the dimension of the single β-cyclodextrin core (which measures RβCD ) 3.9 Å)23 and excludes the possibility that the adopted core-shell approach can be attributed to the scattering of a single amphiphilic monomer. From the values of R1 and R2 useful information can be derived about the generated micellar structures. Assuming that no water is present in the core region, from the corresponding volume Vcore an estimate of the micelles aggregation number Nagg can be obtained by means of the relation

Vcore )

4 πR13 ) Nagg(7vC) 3

(6)

where 7vC )7(27.4 + 26.9nc)16 accounts for the total volume (23) (a) Li, S.; Purdy, W. C. Chem. Rev. 1992, 92, 1457.

of the 7 hydrophobic chains of the amphiphilic molecules (hydrocarbon unit are respectively nc ) 2 for 3 and nc ) 6 for 4 and 5 molecules). As shown in Table 2, these calculations furnish a very small aggregation number for the investigated micellar aggregates. More specifically our results show that the aggregation number Nagg increases with the increase of the length of hydrophobic alkyl moieties (i.e., when passing from CD 3 to CD 4) while decrease with increasing length of hydrophilic ethylene oxide substituents (i.e., when passing from CD 4 to CD 5). This approach may suffer some uncertainty caused by possible inclusion of hydrocarbon chain into the hydrophobic cavity of the cyclodextrin. In this respect we also performed calculations accounting for the possible inclusion of hydrophobic alkyl chains in the β-CD cavity. Following this approach, the total volume of the hydrocarbon chains present in the core region is reduced by the cavity volume (Vcav ) 377 Å3),23 as reported in Table 2. From the thermodynamic point of view the morphology of the generated micellar aggregates can be explained according to the theory of Israelachvili.19 According to this approach the dimensionless packing parameters v/(a0lc) determines in fact the preferred shape of the generated aggregates (where a0 is the area per hydrophilic headgroup and v and lc are volume and critical chain length of the hydrophobic chains, respectively). For hydrocarbon chains these last two quantities can be approximated by16 v ≈ (27.4 + 26.9n) Å3 and lc ≈ (1.5 + 1.265n) Å. Following this approach, the generated structures, such as spherical micelles (v/a0lc < 1/3), nonspherical micelles (1/3 < v/a0lc < 1/2), or bilayers (1/2 < v/a0lc < 1), correspond to minimum-sized aggregates satisfying the requisite of minimum in free energy.19 The calculation of this quantity is nontrivial for our investigated systems, due to the presence an hydrophilic headgroup with a complex geometry. Assuming a constant value for a0, given by larger side radius β-cyclodextrin ring,23 we obtain the values of v/a0lc ) 0.110 for CD 3, v/a0lc ) 0.112 for CD 4, and v/a0lc ) 0.113 for CD 5, respectively, thus confirming that spherical micelles are the preferred morphology for all types of investigated systems. Derived quantities for the packing parameters should be considered as a rough estimation, while for a more precise calculation hydration of the CDs should be taken into account, but their effects are difficult to rule out.24 The small size of the generated micellar aggregates as well as the variation in the aggregation number can be explained, at least in a qualitative way, at the light of recent models of micellization of amphiphilic block copolymers in selective solvents.24 According to these theoretical approaches, it has been found that micelles aggregation number Nagg increases with increasing hydrophobic block length NA and with decreasing length of the hydrophilic one NB, according to the scaling relation Nagg ∼ NAP/NBQ (where the exponents P and Q strongly depend on the nature of specific the interaction between solvent and soluble block).25 Thus a low ratio between (24) The big hydrophilic headgroup is composed of the β-cyclodextrin ring, (larger side radius is r1 ) 7.65 Å)23 with seven grafted PEG hydrophilic chains. Fully stretched PEG chains may contribute with a maximum additional radius of r2 ≈ 9 Å for CD 3, r2 ≈ 13 Å for CD 4, and r2 ≈ 17 Å for CD 5. A more realistic picture is obtained assuming that only a fraction of r2/2 of the fully stretched PEG chains provides additional area to the headgroup. The corresponding area per hydrophilic headgroup will be a0 ) [π(r1 + r2/2)2] ) 464 Å2 for CD 3, a0 ) 629 Å2 for CD 4, and a0 ) 819 Å2 for CD5. This provides the even lower values for the packing parameters of v/a0lc ) 0.043 for CD 3, v/a0lc ) 0.033 for CD 4, and v/a0lc ) 0.025 for CD 5, respectively. (25) (a) Whitemore, M.; Noolandi, J. Macromolecules 1985, 18, 657. (b) Nagarajan, R.; Ganesh, K. J. Chem. Phys. 1989, 90, 5843. (c) Zang, L.; Barlow, R. J.; Eisenberg, A. Macromolecules 1995, 28, 6055.

1062

Langmuir, Vol. 20, No. 4, 2004

Lombardo et al.

hydrophobic/hydrophilic components of the amphiphile, as in our case, will favor formation of micellar aggregates with small aggregation number Nagg. Another interesting feature of the associating properties regard the hydration process of the β-cyclodextrin and the ethylene glycol chains of the corona region of the micelles. If we assume, in fact, that the micellar aggregates are composed of Nagg amphiphilic molecules each bringing an average number h of bound water molecules (hydration number), the following relation can be established

Vmic )

4 πR23 ) Nagg(VSC + hVW) 3

(7)

where Vmic is the overall micelle volume, VSC is the volume of the single amphiphilic molecule determined by density measurements, and VW ) 30 Å3 is volume of a water molecule. Calculation furnishes an average hydration parameter of h ) 349 for CD 3. A similar result for the h parameter has been recently obtained by Liu et al. in a micellar system of PEO-based amphiphilic block copolymers.26 The obtained high hydration number is the consequence of the complex geometry of the hydrophilic headgroups as well as by the high hydration process of the ethylene glycol chains. In this respect the even higher value of the h parameters for the CD 4 (h ) 1494) and CD 5 (h ) 1939) systems can be explained considering that the h parameter determined by the fitting of the scattering data includes, together with the hydrating water, the number of free water molecules present in the hydrophilic shell region. Light Scattering Analysis: Evidence of Large Aggregates. As previously stated, the presence of an upturn in the SAXS spectra of the investigated systems is probably due to the presence of aggregates whose dimensions (R > 500 Å) go beyond the limit of resolution of the SAXS technique. With the aim to clarify this point as well as to gain further insight into the main structural features of the investigated systems, we extended our analysis with the use of the elastic light scattering technique. Note that the size of the micelles is so small compared with the wavelength of light that they do not contribute to the angular dependence of the scattering intensity. For larger aggregates, on the contrary, the scattering intensity in the so-called Guinier region (i.e., for qRg , 1) can be approximated as I(q) ) I0 exp(-q2Rg2/ 3). In such a way, from the linear portion of the plot of ln I(q) as a function of q2, an average gyration radius of Rg ) 950 ( 30 Å for 3, Rg ) 840 ( 30 Å for 4, and Rg ) 910 ( 30 Å for 5 is obtained. Those values remain constant in the wide range of concentrations investigated (i.e., for 0.01 < c < 2% w/w), thus pointing out the equilibrium nature of those generated large supramolecular structures. The main structural features obtained by (X-ray and light) static scattering experiments are confirmed by dynamic light scattering measurements. In a quasi-elastic light scattering (QELS) experiment the measured intensity-intensity time correlation function g(2)(t) is related to the electric field correlation function g(1)(t) by the Siegert relation27

g(2)(t) ) B(1 + f|g(1)(t)|2)

(8)

Figure 4. Double exponential fit of the scattered intensity correlation function for CD 4 (T ) 25 °C and c ) 1% w/w). Two main relaxation rates (arrows) are indicative of the presence of two populations of particles with different dimensions. The corresponding relaxation time distribution, obtained by the inverse Laplace transformation algorithm REPES is also reported (inset).

coefficient. From the diffusion coefficient D, the mean hydrodynamic radius RH is calculated using the StokesEinstein relation

D)

kBT 6πηRH

(9)

where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. The correlation function g(1)(t) may also be expressed as the Laplace transform of a continuous distribution G(Γ) of decay times (relaxation rates Γ). This allows the calculation of relaxation time distributions τA(τ) ) ΓG(Γ) by means of a regularized inverse Laplace transformation.

g1(t) )

∫0∞ G(Γ) exp(-Γt) dΓ ) ∫0∞ τA(τ) exp(- τt) ln τ

(10)

As shown in Figure 4 the scattered intensity correlation function for CD 4 presents two relaxation rates, which indicates the presence of two main populations of particles characterized by different dimensions. These features are confirmed by the analysis of the relaxation time distribution τA(τ) obtained by the inverse Laplace transformation algorithm REPES,28 as reported in the inset of Figure 4. The observed relaxation rates ΓF and ΓS (fast and slow contribution, respectively) connected with the two peaks of the distribution are linearly dependent on the square of the scattering wavevector k, thus indicating the diffusive nature of the detected mode.27,29 To obtain useful information from the fast and slow contribution, a double exponential fit of the experimental correlation function has been performed, according to the relation

g(1)(t) ) AF exp(-k2DFt) + AS exp(-k2DSt) (11)

where B is the baseline and f is a spatial coherence factor. In the case of a dilute solution of monodisperse particles, g(1)(t) ) exp(-k2Dt), where D is the translational diffusion

The corresponding diffusion coefficients DF and DS have been used to calculate the average hydrodynamic radius from the Stokes-Einstein relation. The fast mode furnishes the value of RhF ) 44 ( 2 Å, which is certainly

(26) Liu, Y. C.; Chen, S. H.; Huang, J. S. Phys. Rev. E 1996, 54, 1698. (27) Berne, B. J.; Pecora, R. Dynamic Light Scattering; WileyInterscience: New York, 1976.

(28) Jakes, J. Czech. J. Phys. 1988, B38, 1305. (29) (a) Corti, M.; Degiorgio, V. Phys. Rev. Lett. 1980, 45, 1945. (b) Corti, M.; Degiorgio, V. J. Phys. Chem. 1981, 85, 711.

Cyclodextrin Colloids

Figure 5. Relaxation time distribution curves obtained from REPES approach as a function of the hydrodynamic radius Rh of the aggregates for the samples CD 3 (a), CD 4 (b), and CD 5 (c). All systems are at T ) 25 °C and c ) 1% w/w.

connected with the micelles previously detected with the SAXS technique. The slow contribution (with average radius RhS ) 570 Å) corresponds to the large aggregates previously detected by the static light scattering technique. Those last structures, which present a higher polydispersity, are probably the result of the entanglement process between smaller micelles. Similar results are obtained for CD 3 and CD 5 systems (see Table 2). To gain a deeper insight into the main structural features of the investigated systems, the distribution curves obtained from REPES approach have been reported as a function of the hydrodynamic radius of the aggregates Rh (Figure 5). These distributions are indicative of the relative populations of the aggregates with different dimensions. Is it clear that large polydisperse aggregates are present in the systems in equilibrium with small micelles. Large structures are probably spherical ag-

Langmuir, Vol. 20, No. 4, 2004 1063

gregates of entangled micelles, although the possibility of different morphologies, such as elongated aggregates or vesicles, are not excluded. Dimensions of large aggregates have been found to remain constant with time, over more than 10 days. The presence of large aggregates in equilibrium with smaller micelles has also been observed in a number of recent investigations in water solution of PEObased amphiphilic block copolymers.30 But despite the growing number of those observations, up to now, no clear understanding of the phenomenon has merged. This unexpected colloidal phenomenon possesses substantial different features with respect to those observed in the other investigations, such as the random (fractal) clusters formed during irreversible aggregation in colloidal systems.31 Moreover what limits large structures to welldefined dimensions, as in our case, seems to be inexplicable in terms of conventional thermodynamic theory.19,24 The strong analogy between those observations30 and the main features of our results suggests that the presence of hydrophilic PEO chains at the surface of micelles may play an important role in order to promote, by means of the hydrogen bonds, the connectivity between micelles. In this respect the entanglement process can be driven by the presence of an attractive interaction between micelles caused at the molecular level by the depletion interaction between interpenetrating oligo(ethylene glycol) chains of different micelles32 or by the inclusion of the oligo(ethylene glycol) chains into the cyclodextrin cavity.33 Interestingly, a closer inspection of Figure 5a points out the presence of even smaller particles corresponding to an average hydrodynamic radius of Rh0 ≈ 7.5 Å. The presence of this contribution in CD 3 samples, although sensitively weak, has been found to be present in a systematic way in all the different diluted samples down to c ) 0.05 w/w and can be attributed to the presence of free molecules in solution. On the contrary the presence of residual monomers has not been detected for CD 4 and CD 5 in a wide range of concentrations. This result suggests that the efficient inclusion of the short hydrocarbon chain into the hydrophobic cavity of the β-cyclodextrin may, in part, induce a sensitive reduction of the amphiphilic character to the CD 3 molecule. The extension of our analysis in the intensity correlation function at higher concentrations (c > 2% w/w) reveals the presence of a further relaxation component in long time region (τ > 0.01 s), which points out the presence of even bigger aggregates of the order of several thousands of angstroms in the investigated CD systems. This contribution, which becomes more pronounced with increasing concentration, confirms the highly associative behavior of these novel amphiphiles and indicates that the entanglement process (30) (a) Khan, T. N.; Mobbs, R. H.; Price, C.; Quintana, J. R.; Stuberfield, R. B. Eur. Polym. J. 1987, 23, 191. (b) Brown, W.; Rymden, R.; Stam, J.; Almgren, M.; Svenks, G. J. Phys. Chem. 1989, 93, 2512. (c) Brown, W.; Schillen, K.; Almgren, M.; Hvidt, S.; Bahadur, P. J. Phys. Chem. 1991, 95, 1850. (d) Xu, R.; Winnik, M. A.; Hallet, F. R.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 87. (e) Mortensen, K.; Brown, W.; Amdal, K.; Alami, A.; Jada, A. Langmuir 1997, 13, 3635. (f) Vivare`s, D.; Belloni, L.; Tardieu, A.; Bonnete´, F. Eur. Phys. J. E 2002, 9, 15. (g) Pispas, S.; Hadjichristidis, N. Langmuir 2003, 19, 48. (31) (a) Witten, T. A.; Sander, L. M. Phys. Rev. Lett. 1981, 47, 1400. (b) Chen S. H.; Teixeira, J. Phys. Rev. Lett. 1986, 57, 2583. (c) Mallamace, F.; Micali, N.; Lombardo, D. In Structure and Dynamics of Strongly Interacting Colloids and Supramolecular Aggregates in Solutions; Chen, S. H., Huang, J. S., Tartaglia, P., Eds.; Kluwer Academic Publishers: Dordrecht, 1992; pp 405-417. (d) Gimel, J. C.; Durand, D.; Nicolai, T. Phys. Rev. B 1995, 51, 11348. (32) (a) Chen, S. H.; Mallamace, F.; Faraone, A.; Gambadauro, P.; Lombardo, D.; Chen, W. R. Eur. Phys. J. E 2002, 9, 283. (b) Mallamace, F.; Beneduci, R.; Gambadauro, P.; Lombardo, D.; Chen, S. H. Physica A 2001, 302, 202. (33) (a) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803 (b) Nepogodiev, S. A.; Stoddart, J. F. Chem. Rev. 1998, 98, 1959.

1064

Langmuir, Vol. 20, No. 4, 2004

between supramolecular aggregates involves different length scales in the system. Conclusions Combination of small-angle X-ray scattering (SAXS) and light scattering experiments allows a detailed investigation of supramolecular aggregates formed in water solutions of modified amphiphilic cyclodextrins. The highly associative behavior of these novel macromolecules is evidenced by the very low value of the critical micelles concentration and by the presence of an entaglement process between supramolecular aggregates which is active at different length scales of the system. Thermodynamic approaches generally used for traditional amphiphilic systems can be used to explain, at least qualitatively, the main structural features of the generated micellar aggregates. In particular by means of the modulation of hydrophobic and hydrophilic components the structural features of the generated micellar aggregates can be changed in the desired way thus offering the possibility to control molecular organization in a manner similar to those of traditional colloids. Interestingly small micelles have been found in equilibrium with polydisperse large aggregates of entangled micelles. Connectivity between micelles is probably favored by the

Lombardo et al.

presence of an attractive depletion interaction between interpenetrating oligo(ethylene glycol) chains of different micelles or caused by the penetration of these chains into the cyclodextrin cavity. The complex geometry of those systems constitutes an important difference with respect to most of the investigated amphiphiles and stimulates novel approaches to the control of molecular organization and the self-assembly processes on size scales comparable to those of traditional colloids. These novel nonionic colloidal systems (cuttle-fish amphiphiles) combine, in fact, the inclusion and transport properties of host macrocycles, such as cyclodextrin, together with the increased stability of colloidal aggregates and may be of interest for their potential application as innovative drug delivery systems. Acknowledgment. We gratefully acknowledge Dr. N. Micali (IPCF-CNR, Messina) and Professor L. Monsu` Scolaro (University of Messina) for helpful comments. We are indebted to B. J. Ravoo (University of Twente, The Netherlands) for his collaboration with synthetic procedures. We sincerely thank Dr. Domenico Garozzo and Dr. Luisa Sturiale (ICTP-CNR Catania) for the MALDI-MS experiments. MIUR (PRIN-Cofin 2002-01), CNR, Enterprise Ireland provided financial support. LA035370Q