Langmuir 2002, 18, 1945-1948
Aggregation in Water of Nonionic Amphiphilic Cyclodextrins with Short Hydrophobic Substituents Antonino Mazzaglia,† Bart Jan Ravoo,*,‡ Raphael Darcy,‡ Patrizia Gambadauro,§ and Francesco Mallamace§,| Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica dell’ Universita` di Messina and ISMN-CNR, Sezione di Messina, Salita Sperone 31, 98166 Messina, Italy, Department of Chemistry, National University of Ireland, University College Dublin, Belfield, Dublin 4, Ireland, Dipartimento di Fisica, Universita` di Messina, Vill. S. Agata, 98166 Messina, Italy, and INFM, Unita` di Messina, Messina, Italy Received October 11, 2001. In Final Form: December 11, 2001
Introduction A range of chemically modified amphiphilic cyclodextrins has been synthesized with the aim of providing versatile carrier and delivery systems for drug molecules. Amphiphilic cyclodextrins combine the properties of macrocyclic hosts and self-organizing amphiphiles. Initially, the self-organization of various ionic and nonionic amphiphilic cyclodextrins into stable monolayers and multilayers at the air-water interface and also in Langmuir-Blodgett films was investigated.1 Amphiphilic cyclodextrins can be admixed to phospholipid monolayers2 as well as liposomes,3 and they can be dispersed into nanospheres showing promising properties for drug encapsulation.4 However most of these amphiphilic cyclodextrins are not soluble in water, and the (mixed) aggregates in water are systems out of thermodynamic equilibrium. An adequate balance between hydrophobic tails and hydrophilic heads is required for the preparation of equilibrium systems of amphiphilic cyclodextrins in * To whom correspondence may be addressed. Tel.: +353 1 7162449. Fax: +353 1 7162127. E-mail:
[email protected]. † Dipartimento di Chimica Inorganica, Chimica Analitica e Chimica Fisica dell’ Universita` di Messina and ISMN-CNR. ‡ Department of Chemistry, University College Dublin. § Dipartimento di Fisica, Universita ` di Messina. | INFM, Unita ` di Messina. (1) (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) Parazak, D. P.; Khan, A. R.; D’Souza, V. T.; Stine, K. J. Langmuir 1996, 12, 4046. (f) Hamelin, B.; Jullien, L.; Laschewsky, A.; Herve´ du Penhoat, C. Chem. Eur. J. 1999, 5, 546. (2) (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. (3) (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. (4) (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) Skiba, M.; Coleman, A. W.; Fessi, H.; Devissaguet, J. P.; Ducheˆne, D.; Puisieux, F. EP 0646003B1 1996. (d) Gulik, A.; Delacroix, H.; Wouessidjewe, D.; Skiba, M. Langmuir 1998, 14, 1050.
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aqueous solution. Some of us showed that heptakis(6alkylthio-6-deoxy)-β-cyclodextrin derivatives behave as thermotropic liquid crystals but are insoluble in water.5 Efforts have been made to obtain amphiphilic cyclodextrins with increased water solubility. As an example, the grafting of cholesterol onto permethylated β-cyclodextrin produces a highly soluble amphiphilic compound forming small spherical micelles in water.6 The modest water solubility of the parent β-cyclodextrin as well as most of its derivatives results from the efficient intramolecular hydrogen bonding network between OH2 and OH3 of adjacent glucose residues. A low degree of substitution with, for example, methyl, hydroxyethyl, or hydroxypropyl groups disrupts this intramolecular network and results in much greater water solubility. A much increased water solubility was observed upon substitution of β-cyclodextrin with hydroxyethyl and oligo(ethylene glycol) by reaction with ethylene carbonate7 or with ethylene oxide.8 In our case the introduction of hydrophilic oligo(ethylene glycol) onto the secondary side of heptakis(6-deoxy-6-alkylthio)β-cyclodextrins results in the formation of amphiphilic macrocycles with dramatically increased water solubility. Heptakis[6-alkylthio-6-deoxy-2-oligo(ethylene glycol)]-βcyclodextrins, where alkyl is dodecyl or hexadecyl, form bilayer vesicles entirely composed of cyclodextrin molecules upon sonication in water.9 These bilayer vesicles are thermodynamically metastable, but the presence of ethylene glycol oligomers at the vesicle surface increases the colloidal stability of these vesicles while potentially decreasing their adverse immune response in drug delivery systems, much like “stealth liposomes”10 and “niosomes”.11 In this paper we describe the properties of aqueous solutions of heptakis[6-alkylthio-6-deoxy-2-oligo(ethylene glycol)]-β-cyclodextrins where alkyl is ethyl or hexyl. Our study compares three compounds with a different balance of hydrophobic alkyl substituents at C6 and hydrophilic oligo(ethylene glycol) substituents at C2. The preparation of cyclodextrins 3 and 4 was reported recently.12 Cyclodextrin 5 was obtained from cyclodextrin 4. Surface tensiometry, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to study the aggregation of 3-5 in water. We demonstrate that whereas cyclodextrin 4 forms thermodynamically metastable nanoparticles, cyclodextrins 3 and 5 form stable micellar solutions in water. Experimental Methods Synthesis of Cyclodextrins. Cyclodextrins 1 and 2 were obtained in two steps from β-cyclodextrin.5 Cyclodextrins 3 and 4 were synthesized from 1 and 2, respectively, as described.12 Synthesis of Heptakis[6-deoxy-6-ethylthio-2-poly(ethylene glycol)]-β-cyclodextrin (5). Cyclodextrin 4 (460 mg, 0.19 (5) Ling, C.-C.; Darcy, R.; Risse, W. J. Chem. Soc., Chem. Commun. 1993, 438. (6) Auze´ly-Velty, R.; Djedaı¨ni-Pilard, F.; De´sert, S.; Perly, B.; Zemb, T. Langmuir 2000, 16, 3727. (7) 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. (8) 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. (9) Ravoo, B. J.; Darcy, R. Angew. Chem., Int. Ed. 2000, 39, 4324. (10) Lasic, D. D. Angew. Chem., Int. Ed. Engl. 1994, 33, 1685. (11) Uchegbu, I. F.; Vyas, S. P. Int. J. Pharm. 1998, 172, 33. (12) Mazzaglia, A.; Donohue, R.; Ravoo, B. J.; Darcy, R. Eur. J. Org. Chem. 2001, 1715.
10.1021/la015626x CCC: $22.00 © 2002 American Chemical Society Published on Web 01/24/2002
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Langmuir, Vol. 18, No. 5, 2002
mmol), NaH (50 mg), and ethylene carbonate (1.0 g, i.e., 50 molar equiv.) were dissolved in 5 mL of tetra-N-methylurea. The mixture was stirred at 150 °C under N2 for 4 h. No change occurred in thin layer chromatographic analysis (CHCl3/CH3OH/H2O ) 50/ 10/1, Rf ) 0.55), but CO2 emission was observed and ceased at the end of this period. The solvent was removed at 100 °C under reduced pressure. The crude product was dissolved in the minimum quantity of methanol and was purified by size-exclusion chromatography on lipophilic Sephadex LH-20-100 using methanol as eluent. This intermediate product was stirred in NaOMe (0.1 M) in methanol at room temperature overnight.12 The solution was concentrated, and 5 was isolated in 80% yield as an oil by size-exclusion chromatography using methanol as eluent. 1H NMR (CDCl3): δ ) 0.89 (t, 21H, CH3), 1.30 (br s, 42H, CH2), 1.57 (m, 14H, CH2), 2.60 (m, 14H, SCH2), 2.95 (m, 14H, H6), 3.273.50 (m, 14H, H2, H4), 3.5-4.1 (m, ca. 154H, H3, H5, OCH2CH2O), 5.05 (br, 7H, H1) ppm. 13C NMR (CDCl3): δ ) 14.1 (CH3), 22.6 (CH2), 28.7 (CH2), 29.8 (CH2), 31.6 (CH2), 33.7 (CH2S), 33.7 (C6), 61.6 (CH2OH), 71.0-72.5 (C3, C5, CH2O), 80.8 (C2, C4), 101.0 (C1) ppm. ESI-MS: m/z (%) 3006 (86) [M+26EO - Na], 3050 (90) [M+27EO - Na], 3094 (100) [M+28EO - Na], 3138 (82) [M+29EO - Na], 3182 (68) [M+30EO - Na]. Preparation of Aqueous Solutions. Cyclodextrins 3 and 5 readily dissolve in water at concentrations up to 10 wt %. At high concentrations, stirring and gentle heating accelerate dissolution. Cyclodextrin 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. Tensiometry. The surface tension of aqueous solutions of cyclodextrins 3-5 was measured at room temperature with a Lauda TD1 ring-type tensiometer. Doubly distilled water was used to prepare cyclodextrin solutions. The determination of critical micelle concentration (cmc) values covered about 20 surface tension determinations in the concentration range of 0.1 × cmc to 100 × cmc. The reported surface tension is the mean value of at least three measurements for each cyclodextrin concentration and the cmc values were calculated from a plot of the surface tension vs the negative logarithm of the cyclodextrin concentration. Transmission Electron Microscopy. Samples for TEM were prepared on 200 mesh Formvar/carbon-coated copper grids. A drop of cyclodextrin solution (0.5 mg mL-1) was incubated on the grid for 2 min and then gently blotted with filter paper. The specimen was negatively stained with a drop of 2% (w/w) UAc solution, incubated for 5 min, and then gently blotted. The samples were examined in a JEOL 2000 electron microscope operating at 80 kV. Dynamic Light Scattering. DLS measurements were performed using an automated homemade goniometer in the angular range 45° e ϑ e 120°, corresponding to scattering wavevectors 12.1 e k e 27.2 µm-1, a sample holder connected to a thermostatic bath ((0.2 °C), a rotating detector Hamamatsu R942 photomultiplier cooled at -30 °C, and a Brookhaven correlator BI9000AT with a logarithmic sampling time scale. Measurements were performed in a delay time range of 5-108 µs with an acquisition time of 20 min. The light source was a Verdi diode pumped NdYVO4 laser (532.4 nm) operating at a power of 100 mW. Refer to the Supporting Information for details of our DLS analysis. Cyclodextrin samples were prepared at room temperature in water (1 mL, Angelini), centrifuged, and filtered using a Millipore Nalgene 0.45 µm filter to remove dust. The samples were stored for a few days before measurements in order to obtain complete aggregation equilibrium and then placed in a Burchard cylindrical quartz cell (Hellman) inside a homemade thermostatic system. Cyclodextrin 3 was studied at 3.0 × 10-4 M and at 1.2 × 10-3 M and cyclodextrin 5 was analyzed at 5.0 × 10-5 M and at 5.0 × 10-4 M. Both were studied in a range of temperatures from 18 to 40 °C and at different values of scattering angle ϑ. Cyclodextrin 4 was studied at 4.0 × 10-5 M at 25 °C and 90° scattering angle.
Notes Chart 1
one ethyl substituent on C6 and about two ethylene oxide units on C2 of each glucose residue (Chart 1). Cyclodextrin 4 has one hexyl substituent on C6 and about three ethylene oxide units on C2 of each glucose residue (Chart 1). The novel amphiphilic cyclodextrin 5 was obtained directly from 4. Further hydroxyethylation of the secondary side of the amphiphilic compound 4 was carried out using ethylene carbonate in the presence of NaH. Attempts at an anionic graft polymerization by increasing the reaction time or directly by using NaH as strong base in the hydroxyethylation of 2 were not successful. As previously observed in the preparation of 4,12 some oligo(ethylene glycol-co-ethylene carbonate) instead of oligo(ethylene glycol) was grafted onto the cyclodextrin. Therefore, compound 5 was treated with sodium methoxide in methanol in order to remove the grafted carbonate and purified by size exclusion chromatography. Cyclodextrin 5 was characterized by positive-ion-mode electrospray ionization mass spectrometry (ESI-MS) and NMR spectrometry. The ESI-MS of 5 shows that the most abundant species has a m/z ratio of 3094, which is 2 with a degree of substitution of 28 ethylene oxide units plus one sodium ion. Neighboring peaks are separated by 44 mass units, which is the molecular mass of one ethylene oxide unit. Correspondingly, the peaks for the doubly charged compounds are separated by 22 mass units and the most abundant species has a m/z of 1559, which is 2 with a degree of substitution of 28 plus two sodium ions. The NMR spectra were assigned by comparison with the corresponding spectra of 3 and 4.5,12 Relative to the spectra of 4, the proton and carbon resonances for 5 are unchanged but integration of the 1H NMR spectrum confirms a higher degree of ethylene oxide substitution for 5. As for 3 and 4, substitution of 5 occurs at C2, not C3. On the basis of ESI-MS and NMR, we suggest the molecular formula of Chart 1 for 5, which is a polydisperse material with a hexyl substituent on C6 and an average of about four ethylene oxide units on C2 of each glucose residue. Thus, the ratio of hydrophobic diethylene substituents at C6 to hydrophilic ethylene oxide substituents at C2 is 0.5 for 3, 1.0 for 4, and 0.75 for 5. Solubility and Surface Tension. Cyclodextrins 1 and 2 are insoluble in water, although it is possible to prepare monolayers of 2 (and long-chain analogues) on the airwater interface.1e, 13 In contrast, cyclodextrin 4 readily dissolves in water upon sonication, and cyclodextrins 3 and 5 spontaneously dissolve in water at all concentrations examined in the course of this worksthat is, up to at least 10% w/w. It was found that both 3 and 5 are highly surface active compounds. From the break in the plot of the surface tension vs the concentration, it was established that the cmc of 3 is 7.1 mg L-1 or 3.4 µM and the cmc of 5 is 4.0 mg L-1 or 1.3 µM (Figure 1). The surface tension at the cmc is 53.2 mN m-1 for 3 and 50.9 mN m-1 for 5. Thus, cyclodextrin 5, with more hydrophobic hexyl substituents, has a lower cmc and lower surface tension at the cmc than cyclodextrin 3, with less hydrophobic ethyl substituents.
Results and Discussion Synthesis. Cyclodextrins 3 and 4 were obtained from cyclodextrins 1 and 2 as described.12 Cyclodextrin 3 has
(13) (a) O’Keeffe, D. Ph.D. Thesis National University of Ireland, 1998. (b) Kobayashi, K.; Kajikawa, K.; Sasabe, H.; Knoll, W. Thin Solid Films 1999, 349, 244.
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Figure 3. Electron microscopy of nanoparticles of cyclodextrin 4. Table 1. DLS Results for Cyclodextrin 3 (1.2 mM) at 30 °Ca ϑ (deg)
k (µm-1)
D (108 cm2 s-1)
Rh (Å)
45 60 90 105 120
12.1 15.8 22.3 25.0 27.3
3.16 4.08 3.49 3.46 3.33
700 542 633 639 663
a Legend: ϑ, scattering angle; k, wave vector, D, diffusion coefficient; Rh, hydrodynamic radius.
Figure 1. Surface tensiometry of cyclodextrins 3 and 5.
Figure 2. Size distribution according to Contin analysis for DLS of cyclodextrin 5.
These values are similar to those measured for a monocholesteryl derivative of permethylated β-cyclodextrin,6 which has a cmc of 5 ( 2 µM and a surface tension at the cmc of about 43 mN m-1 and for mono-6-(dodecylamino)β-cyclodextrin,14 which has a cmc of 1.4 µM. In contrast, cyclodextrin 4, with less hydrophilic ethylene oxide substituents than 5, is much less surface active (the surface tension is 62.5 mN m-1 at 300 mg L-1) and no cmc could be detected. Dynamic Light Scattering and Transmission Electron Microscopy. DLS measurements were performed above the cmc of cyclodextrin 5 at different angles for each concentration. Figure 2 shows an example of a Contin analysis in terms of hydrodynamic radii Rh and relative weights under the experimental conditions reported. In all cases, two major contributions are detected around (14) Petter, R. C.; Salek, J. S.; Sikorski, C. T.; Kumaravel, G.; Lin, F.-T. J. Am. Chem. Soc. 1990, 112, 3860.
Rh ) 3 nm (0.6 nm and at Rh ) 32 nm (5 nm. The small dimension corresponds to the hydrodynamic radius Rh of a monomer or a dimer of cyclodextrin 5. It is possible that the larger aggregates are elongated micelles. In contrast, it was found that cyclodextrin 4 forms a polydisperse solution of aggregates with an average size of 50-150 nm. These particles were also observed using TEM (Figure 3). It was found by DLS and TEM that it was difficult to reproduce the size distribution of aggregates of 4 in independently prepared samples. We contend that 4 forms polydisperse nanoparticles4 upon sonication in water. These particles are thermodynamically metastable, and their size would depend critically on temperature, concentration, and ultrasound intensity during sample preparation.15 A more detailed DLS study was carried out for cyclodextrin 3 for which we have obtained the correlation decay rate and the decay time from a fitting function taken as the sum of two contributions, a single-exponential decay and a Williams-Watts function. Two characteristic relaxation times of the order of 6.8 × 10-4 and 0.4 s were observed, and from the cumulant analysis we obtained an estimate of Rh from the short relaxation time τ1. The deviation of the correlation function from a single exponential is detected as a second contribution on a longer time scale τ2. Autocorrelation functions at different angles (ϑ ) 45-120°) measured in the temperature range from 18 to 40 °C provided estimates for τ1 and τ2. Table 1 shows results obtained at 30 °C. The diffusion coefficient D is independent of the scattering wave vector k at each temperature. Results obtained at 25 and 36 °C are reported in the Supporting Information. Figure 4 shows the autocorrelation functions at different temperatures for [3] ) 1.2 × 10-3 M. At this concentration (15) In collaboration with Dr. G. N. Misevic (Universite´ de Lille, France), we have carried out some preliminary atomic force microscopy (AFM) work on nanoparticles of cyclodextrin 4. AFM shows that these particles are mostly thicker than 25 nm, indicating that they are not multilamellar vesicles. The larger particles are considerably flattened, i.e., their height is always less than their diameter, indicating the fluid or soft nature of these particles.
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Notes
Figure 4. Temperature dependence of the correlation function obtained for a solution of cyclodextrin 3 (1.2 mM): (a) 20 °C; (b) 25 °C; (c) 30 °C; (d) 36 °C; (e) 40 °C. Table 2. Temperature Dependence of the Correlation Times τ1 and τ2 from DLS of Cyclodextrin 3 (1.2 mM, T ) 90°)a T (°C)
τ1 (µs)
Rh (Å)
τ2 (s)
β
18 20 22 25 28 30 32 34 36 38 40
931 1084 827 965 681 656 599 548 534 497 478
981 1150 883 1041 742 720 662 609 598 560 542
0.49 0.69 0.47 0.58 0.39 0.40 0.37 0.30 0.35 0.32 0.32
0.83 0.77 0.83 0.78 0.87 0.84 0.87 0.87 0.88 0.88 0.89
a Legend: ϑ, scattering angle; R , hydrodynamic radius; β, h exponential coefficient for τ2.
the behavior of the correlation functions at different wavevectors and at different temperatures does not change significantly. In all cases an estimate of Rh of 50-80 nm is obtained from the first characteristic time (of the order of 6.8 10-4 s). τ1 and τ2 are reported in Table 2. An Arrhenius dependence characteristic of diffusive behavior is observed in a graph of relaxation times τ1 and τ2 versus the inverse of temperature 1/T. The correlation function is independent of the concentration of 3 above the cmc. Interestingly, the second contribution in the long time scale suggests the presence of aggregates much larger than 50-80 nm. We assume that elongated micellar aggregates with an Rh of about 60 nm for cyclodextin 3 are present in solution. It is possible that the oligo(ethylene glycol) chains drive attractive interactions which yield dynamic larger networks of smaller aggregates, for example by inclusion of oligo(ethylene glycol) in the cyclodextrin cavity.16 Alternatively, the polydisperse cyclodextrin 3 could form elongated micellar aggregates
characterized by fast relaxation and Rh ) 60 nm from molecules with longer oligo(ethylene glycol) chains, which are more strongly hydrated and tend to form aggregates with a more positive curvature.17 Extended and entangled micellar aggregates characterized by slow relaxation would form from molecules with shorter oligo(ethylene glycol) chains, which are less hydrated and tend to form aggregates with less curvature.17 We note that the correlation function for the more hydrophobic cyclodextrin 5 does not show the long correlation time contribution found for 3, which indicates that no aggregates larger than Rh ) 32 ( 5 nm are present. These results stimulate further investigation of the formation and dynamics of these aggregates using elastic light scattering and other techniques. Acknowledgment. Tensiometry was carried out in the Department of Pharmaceutics and Pharmaceutical Technology, Trinity College, Dublin. We are indebted to Mrs. G. Fitzpatrick for NMR experiments and to Dr. D. Cottell for providing TEM facilities. We thank Professor K. Dawson and Dr. A. Gorelov for providing DLS facilities and assistance with DLS measurements in UCD. B.J.R. is a Schering-Plough (Avondale) Co. Newman Scholar in Organic Chemistry at UCD. A.M. is grateful to MURST, CNR, and COST P1 for financial support. Supporting Information Available: General description of the analysis of the DLS data and DLS results for cyclodextrin 3 at 25 and 36 °C. This material is available free of charge via the Internet at http://pubs.acs.org. LA015626X (16) (a) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803. (b) Nepogodiev, S. A.; Stoddart, J. F. Chem. Rev. 1998, 98, 1959. (17) Israelachvili, J. Intermolecular and Surface Forces; Academic Press: London, 1991; pp 366-394.
