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Preparation of Vesicles and Nanoparticles of Amphiphilic Cyclodextrins Containing Labile Disulfide Bonds Darren Nolan, Raphael Darcy, and Bart Jan Ravoo* Centre for Synthesis and Chemical Biology, Department of Chemistry, National University of Ireland, University College Dublin, Belfield, Dublin 4, Ireland Received February 25, 2003. In Final Form: March 6, 2003
Amphiphilic cyclodextrin derivatives were prepared in which a disulfide bond connects the hydrophobic substituents to the macrocycle. These compounds were obtained by 1,3-dicyclohexylcarbodiimide-mediated coupling reactions of heptakis(6-amino-6-deoxy)-β-cyclodextrins and disulfide-containing carboxylic acids of increasing hydrophobicity. To improve the water solubility of the cyclodextrins, oligo(ethylene glycol) substituents were grafted to the secondary side of the cyclodextrin molecules. The amphiphilic cyclodextrins form vesicles or nanoparticles in water, which disintegrate in the presence of the disulfide reducing agent dithiothreitol. Hydrophobic guest molecules are released from the nanoparticles upon cleavage of the disulfides.
Introduction Cyclodextrins (CDs) have a rich history as carriers of hydrophobic molecules in water by molecular encapsulation. In pharmaceutical applications, the potential of CDs as drug delivery agents is hampered by the modest strength of drug binding and the unfavorable pharmacokinetics of isolated CD molecules.1 The synthesis of amphiphilic CDs by selective substitution of either the primary or the secondary side, respectively, enables the preparation of lyotropic mesophases and colloids composed of or containing CDs, such as micelles,2 nanoparticles,3 and vesicles,4 in water. These colloids can have improved pharmaceutical properties. Unfortunately, the introduction of hydrophobic substituents onto CDs leads to mostly water-insoluble materials. Recently, we found that counterbalancing hydrophobic substitution of the primary side of β-CD by the introduction of oligo(ethylene glycol) on the secondary side in a graft synthesis5 leads to amphiphilic molecules with high solubility in water. We reported the first examples of bilayer vesicles composed entirely of amphiphilic CDs.4a These vesicles consist of * Present address (to be used for correspondence): Laboratory of Supramolecular Chemistry and Technology, MESA+ Research Institute, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands. Tel: +31 53 4892987. Fax: +31 53 4894645. E-mail:
[email protected]. (1) (a) Frijlink, H. W.; Visser, J.; Hefting, N. R.; Oosting, R.; Meijer, D. K. F.; Lerk, C. F. Pharm. Res. 1990, 7, 1248-1252. (b) Frijlink, H. W.; Franssen, E. F. J.; Eissens, A. C.; Oosting, R.; Lerk, C. F.; Meijer, D. K. F. Pharm. Res. 1991, 8, 380-384. (2) (a) Petter, R. C.; Salek, J. S.; Sikorski, C. T.; Kumaravel, G.; Lin, F.-T. J. Am. Chem. Soc. 1990, 112, 3860-3868. (b) Auze´ly-Velty, R.; Djedaı¨ni-Pilard, F.; De´sert, S.; Perly, B.; Zemb, Th. Langmuir 2000, 16, 3727-3734. (c) Auze´ly-Velty, R.; Pe´an, C.; Djedaı¨ni-Pilard, F.; Zemb, Th.; Perly, B. Langmuir 2001, 17, 504-510. (d) Mazzaglia, A.; Ravoo, B. J.; Darcy, R.; Gambadauro, P.; Mallamace, F. Langmuir 2002, 18, 1945-1948. (3) (a) Skiba, M.; Ducheˆne, D.; Puisieux, F.; Wouessidjewe, D. Int. J. Pharm. 1996, 129, 113-121. (b) Skiba, M.; Coleman, A. W.; Fessi, H.; Devissaguet, J. P.; Ducheˆne, D.; Puisieux, F. European Patent 0646003B1, 1996. (c) Gulik, A.; Delacroix, H.; Wouessidjewe, D.; Skiba, M. Langmuir 1998, 14, 1050-1057. (4) (a) Ravoo, B. J.; Darcy, R. Angew. Chem., Int. Ed. 2000, 39, 43244326. (b) Sukegawa, T.; Furuike, T.; Niikura, K.; Yamagishi, A.; Monde, K.; Nishimura, S.-I. Chem. Commun. 2002, 430-431. (c) Donohue, R.; Mazzaglia, A.; Ravoo, B. J.; Darcy, R. Chem. Commun. 2002, 28642865. (5) Mazzaglia, A.; Donohue, R.; Ravoo, B. J.; Darcy, R. Eur. J. Org. Chem. 2001, 1715-1721.
Figure 1. Cyclodextrin vesicles consist of bilayers of cyclodextrins (in which the hydrophobic tails are directed inward and the hydrophilic macrocycle headgroups are facing water) enclosing an aqueous interior.
bilayers of CDs, in which the hydrophobic “tails” are directed inward and the hydrophilic macrocycle “headgroups” are facing water, enclosing an aqueous interior (Figure 1). These vesicles combine the properties of liposomes and macrocyclic host molecules and create new possibilities for the development of advanced carrier systems based on CD molecules. We now aim to extend the use of vesicles and other colloids of amphiphilic CDs to the development of carrier and delivery systems by introducing a labile bond linking the hydrophobic substituents to the macrocycle. The labile bond should be sensitive to a biological trigger such as intracellular uptake in endosomes, which present a more reductive and more acidic environment than the extracellular fluids. For example, disulfide bonds are stable in the bloodstream but readily cleaved upon endocytotic uptake and have been previously explored as polymerdrug linkers,6 in cationic lipids for transfection,7 and in stabilization of block-copolymer micelles for drug delivery.8 In our case, we wish to develop CD colloids that are suitable carriers for both hydrophobic and hydrophilic guests but release these guests when the colloid disintegrates upon disulfide cleavage following intracellular uptake. (6) Feener, E. P.; Shen, W. C.; Ryser, H. J. P. J. Biol. Chem. 1990, 265, 18780-18785. (7) (a) Tang, F.; Hughes, J. A. Biochem. Biophys. Res. Commun. 1998, 242, 141-145. (b) Tang, F.; Wang, W.; Hughes, J. A. J. Liposome Res. 1999, 9, 331-347. (c) Tang, F.; Hughes, J. A. S.T.P. Pharma Sci. 2001, 11, 83-90. (8) Kakizawa, Y.; Harada, A.; Kataoka, K. J. Am. Chem. Soc. 1999, 121, 11247-11248.
10.1021/la034330j CCC: $25.00 © 2003 American Chemical Society Published on Web 04/02/2003
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Nolan et al. Scheme 1a
a Reaction conditions: (i) Ph3P, NBS, DMF, 80 °C, 4 h. (ii) NaN3, DMF, 80 °C, 2 days. (iii) Ethylene carbonate, K2CO3, tetraN-methylurea, 150 °C, 4 h. (iv) (a) Ph3P, methanol, room temperature, 2 h; (b) NH3 (33% aq), room temperature, 12 h. (v) 6-8, DCC, HOBT (only in the synthesis of 9), N-ethylmorpholine, DMF, 0 °C - room temperature, 4 days. (vi) DCC, DMAP, ROH, DMF, 0 °C - room temperature, 3 h.
In this paper, we describe in detail the synthesis of a series of amphiphilic CDs (9-12 in Scheme 1) which contain a disulfide linking the hydrophobic substituents to the macrocycle. The formation of vesicles and nanoparticles of CDs 10-12 in water was observed using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Both the vesicles and the nanoparticles disintegrate in the presence of the reducing agent dithiothreitol (DTT), which cleaves the disulfide bonds linking the hydrophobic substituents to the CD macrocycle. Using fluorescence spectroscopy, we demonstrate that hydrophobic guest molecules are released from the CD nanoparticles upon cleavage of the disulfide bond. Results and Discussion The synthesis of CDs 9-12 is outlined in Scheme 1. In brief, the CDs were obtained through coupling of heptakis(6-amino-6-deoxy)-β-cyclodextrins 4 and 5 with disulfidecontaining carboxylic acids 6-8. The hydrophobicity of the primary side of the CDs was varied according to the hydrocarbon chain length of the acids 6-8, while the hydrophilicity of the secondary side was increased by the introduction of oligo(ethylene glycol) through graft synthesis of CD 3, leading via 4 to 10-12. CD 9 was obtained by 1,3-dicyclohexylcarbodiimide (DCC)-mediated coupling of acid 6 to heptakis(6-amino-6-deoxy)-β-cyclodextrin 5 (isolated as the hydrochloride salt), which was prepared from β-cyclodextrin 1 via heptakis(6-azido-6-deoxy)-β-
cyclodextrin 2 following literature procedures.9,10 The oligo(ethylene glycol) substituents in 10-12 were introduced onto heptakis(6-azido-6-deoxy)-β-cyclodextrin 2 by a graft synthesis using ethylene carbonate.5 The resulting CD 3 was reduced to CD 4, and the acids 6-8 were introduced in a DCC-mediated coupling reaction. It was found that a graft synthesis starting from heptakis(6-amino-6-deoxy)β-cyclodextrin 5 leads to formation of (6-hydroxyethylamino-6-deoxy)-β-cyclodextrin11 due to the greater nucleophilicity of the amines at C6 relative to the hydroxyls at C2. Also, the graft synthesis starting from 9 leads to decomposition of the disulfide instead of the desired product 10. CD 3 was synthesized in high yield by reaction of 2 with ethylene carbonate in the presence of K2CO3 at elevated temperature. This graft synthesis of oligo(ethylene glycol) by stepwise addition of ethylene carbonate and elimination of carbon dioxide leads to substitution exclusively at C2, not C3.5 In the 1H NMR spectrum of 3, the methylene protons of the oligo(ethylene glycol) substituents are observed at 3.5-3.7 ppm. The signal for H1 at 4.97 ppm shows line broadening that is characteristic for this polymerization reaction. In the 13C NMR spectrum of 3, the ether carbons and the terminal alcohol carbon of the (9) (a) Gadelle, A.; Defaye, J. Angew. Chem., Int. Ed. Engl. 1991, 30, 78-79. (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, 54795480. (10) Guillo, F.; Hamelin, B.; Jullien, L.; Canceill, J.; Lehn, J. M.; de Robertis, L.; Driguez, H. Bull. Soc. Chim. Fr. 1995, 132, 857-866. (11) Ahern, C.; Darcy, R.; O’Keeffe, F.; Schwinte´, P. J. Inclusion Phenom. Mol. Recognit. Chem. 1996, 25, 43-46.
Vesicles and Nanoparticles of Cyclodextrins
oligo(ethylene glycol) substituents are observed around 72 ppm and at 61.5 ppm, respectively. C2 has shifted from 70.69 to 80.9 ppm, which indicates substitution at C2, whereas C3 and C6 are observed (unchanged) near 72 ppm and at 51.5 ppm, respectively. According to IR, the azide at C6 is unaffected (2107 cm-1). Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) confirmed the formation of a polydisperse product 3 with a degree of substitution between 15 and 25 units CH2CH2O per CD. The m/z ratios of the products differ by increments of 44, corresponding to the weight of one unit CH2CH2O. The most intense peaks occurred for sodium adducts of the products with a degree of substitution of 19, 20, 21, and 22, which means that n ) 1-2 for all compounds in Scheme 1. Finally, CD 4 was prepared from 3 in good yield following the conventional procedure for azide reduction.10 In the 13C NMR spectrum of 4, C6 has shifted from 51.5 to 43.8 ppm, and in the IR spectrum the absorbance of the azide has disappeared. A series of disulfide-containing carboxylic acids was obtained in moderate yield by DCC-mediated coupling of 3,3′-dithiodipropionic acid and 1 equiv of n-alcohols of increasing hydrophobicity. The monoesters 6-8 were isolated from the undesired diesters, which formed in significant amounts, and residual diacid by silica gel chromatography. CD 9 was obtained in 34% yield by DCC-mediated coupling of 5 and the disulfide-containing carboxylic acid 6 according to a modified literature procedure.12 As a result of amidation, H6A,B shift from ca. 3.15 ppm in 5 to 2.59 ppm in 6 and C6 shifts from 40.4 to 35.0 ppm. CDs 10-12 were obtained in 35-40% yield by DCC-mediated coupling of 4 and the disulfide-containing carboxylic acids 6-8, respectively. To achieve amidation of each of the seven amino residues, additional acid and DCC were added after 2 days, and the reaction was continued for a further 5 days. Under these conditions, no hydroxybenzotriazole (HOBT), which is very hard to remove from the final product, was required. For CDs 11 and 12, incomplete amidation was the inevitable result when fewer equivalents of acid were used or when the acid and DCC were added all at once or when shorter reaction times were used. As a result of amidation, H6A,B shift from 3.37 ppm in 4 to 2.66 ppm in 10-12 and C6 shifts from 43.8 to 40.7 ppm. When CD 9 is compared to its oligo(ethylene glycol) conjugate 10, the characteristic shifts of H2 (from 3.38 to ca. 3.65 ppm) and of C2 (from ca. 72.5 to 80.4 ppm) are clearly observed. Aqueous solutions of CDs 11 and 12 were prepared by sonication of a hydrated film of CD cast by slow evaporation from chloroform. The solutions have the characteristic opalescence of liposome and vesicle solutions. When investigated by TEM using negative staining, flattened vesicles with a diameter of 50-300 nm were observed (Figure 2). These observations are reminiscent of the behavior of other nonionic amphiphilic CDs.4a According to DLS, the vesicles have an average hydrodynamic diameter of ca. 80 nm for 11 as well as for 12. The size distribution is monomodal but relatively broad (polydispersity index, 0.20-0.30). The solutions are colloidally stable at room temperature, low ionic strength, and neutral pH for at least 2 weeks. A solution of CD 10 was prepared as described for 11 and 12. Using TEM, we observed particles (50-400 nm) (12) Beulen, M. W. J.; Bu¨gler, J.; de Jong, M. R.; Lammerink, B.; Huskens, J.; Scho¨nherr, H.; Vancso, G. J.; Boukamp, B. A.; Wieder, H.; Offenha¨user, A.; Knoll, W.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Chem.sEur. J. 2000, 6, 1176-1183.
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Figure 2. Transmission electron microscopy of vesicles of cyclodextrins 11 (left) and 12 (right). Negative staining with uranyl acetate was used. The scale bars represent 400 nm.
that tend to cluster together and merge on the surface of the (hydrophobic) surface sample grids. Using DLS, we observed nanoparticles with a diameter of ca. 80 nm and a relatively narrow size distribution (polydispersity index, 0.1) which are colloidally stable for several days. These observations are reminiscent of the behavior of other nonionic amphiphilic CDs with short alkyl substituents2d,4c as well as CD nanoparticles described in the literature.3 In contrast to 10-12, CD 9 is hardly soluble in water, even after prolonged sonication at elevated temperature. We attribute the insolubility of this CD to the absence of ethylene glycol substituents on the secondary side of the macrocycle. The different behavior of CDs 9-12 demonstrates once more how the mode of aggregation of these amphiphilic macrocycles is determined by the balance between hydrophobic alkyl chains and hydrophilic oligo(ethylene glycol) headgroups, as previously observed for nonionic and cationic CDs.2d,4a,4c To evaluate the potential of vesicles and nanoparticles of 10-12 as carrier and release systems, the effect of reductive cleavage of the disulfide bond linking the hydrophobic substituents to the macrocycle was investigated. DTT is a potent reductor that efficiently reduces disulfides to the corresponding thiols under mild conditions in aqueous solution.13 As anticipated, the addition of DTT to solutions of 10-12 had a dramatic effect on the CD vesicles and nanoparticles. Invariably, the addition of excess of DTT resulted in a slow transition of the bluish, not fully transparent vesicle or nanoparticle solution to a clear, transparent solution. We contend that DTT reduces the disulfides linking the hydrophobic substituents to the macrocycle, resulting in disintegration of the vesicles and nanoparticles. No aggregates larger than 5 nm were detected by TEM or DLS in solutions of 10-12 after exposure to excess DTT for 3 h or more. To quantify the disintegration of vesicles and nanoparticles of 10-12 in the presence of DTT, the intensity of light scattered by the colloidal solution was measured as a function of time. The disintegration of nanoparticles of CD 10 is presented in Figure 3. The half-life of disintegration of the nanoparticles is ca. 1 h at the DTT concentrations and temperatures studied. If no DTT is added, the intensity of light scattered by the solution remains constant over the entire time scale of the experiment, reflecting the colloidal stability of the nanoparticles at ambient conditions. The disintegration of vesicles of CD 12 is also presented in Figure 3. The rate of disintegration of the vesicles is clearly dependent on the DTT concentration. If no DTT is added, the intensity of light scattered by the solution remains constant over the entire time scale of (13) (a) Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P. J. Org. Chem. 1977, 42, 332-338. (b) Lees, W. J.; Whitesides, G. M. J. Org. Chem. 1993, 58, 642-647.
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Figure 3. Disintegration of nanoparticles of cyclodextrin 10 and vesicles of cyclodextrin 12 in the presence of DTT, shown as normalized scattering intensity versus time. Legend: (O) CD 10, [DTT] ) 44 mM, T ) 25 °C; (0) CD 10, [DTT] ) 125 mM, T ) 25 °C; (9) CD 10, [DTT] ) 125 mM, T ) 37 °C; (4) CD 12, [DTT] ) 25 mM, T ) 37 °C; (2) CD 12, [DTT] ) 50 mM, T ) 37 °C; (b) CD 12, [DTT] ) 125 mM, T ) 37 °C.
the experiment, reflecting the colloidal stability of the vesicles at ambient conditions. The rate of disintegration of the vesicles is not increased by the shorter alkyl chains of 11 relative to 12 (data not shown). When comparing the data for 10 and 12, it is striking that at identical conditions the rate of disintegration of the vesicles (τ1/2 ) 15 min) is much larger than the rate of disintegration of the nanoparticles (τ1/2 ) 1 h). Clearly, the bilayer vesicles are more fragile than the nanoparticles, despite the longer alkyl chains of 12. Thus, it is the structure of the aggregate (vesicle versus nanoparticle) rather than the hydrophobicity of the CD (hexadecyl > dodecyl > octyl) that determines the lability toward cleavage of the disulfide linking the substituents to the macrocycle. We propose that the hydrophilic DTT diffuses slower into the large hydrophobic interior of the nanoparticle than into the thin hydrophobic interior of the bilayer membrane vesicles. Finally, Nile Red was chosen as a fluorescent model compound to investigate the fate of hydrophobic guests absorbed in nanoparticles of 10 upon disintegration of the nanoparticles in the presence of DTT. Nile Red is a hydrophobic fluorophore that is highly fluorescent in apolar media (λem ) 615 nm) but much less fluorescent in water.14 The fluorescence intensity of Nile Red in a solution of nanoparticles of 10 is approximately 70 times higher than in pure water, indicating that Nile Red is efficiently absorbed in the hydrophobic interior of the nanoparticles. Upon addition of DTT to a solution of nanoparticles of 10 loaded with Nile Red (with a 10:1 ratio of CD to probe), a gradual decrease of the intensity of Nile Red fluorescence was observed (Figure 4). After ca. 3 h, the fluorescence had almost entirely disappeared. These experiments demonstrate that upon disintegration of the nanoparticles Nile Red is expelled from the hydrophobic interior of the particles and released into the aqueous environment, resulting in fluorescence quenching. If the rate of release of Nile Red is taken as the rate of fluorescence decrease, it is characterized by a half-life of ca. 1 h, similar to the rate of disintegration of the nanoparticles. If no DTT is (14) (a) Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781789. (b) Sackett, D. L.; Wolff, J. Anal. Biochem. 1987, 167, 228-234.
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Figure 4. Release of Nile Red from nanoparticles of cyclodextrin 10 in the presence of DTT, shown as normalized fluorescence at 460 nm versus time. Legend: (0) [DTT] ) 125 mM, T ) 25 °C; (9) [DTT] ) 125 mM, T ) 37 °C.
added, the fluorescence intensity of Nile Red in the solution remains constant over the entire time scale of the experiment, reflecting the colloidal stability of the nanoparticles loaded with Nile Red at ambient conditions. Also, when DTT was added to a solution of Nile Red in a mixture of Hepes buffer and DMF, the fluorescence intensity of Nile Red in the solution remained constant over the entire time scale of the experiment, reflecting the stability of the probe to the reducing agent. We take these results as a proof of concept that nanoparticles of 10 can be used a carrier and delivery system for hydrophobic guests. Conclusion A family of amphiphilic cyclodextrin derivatives was prepared in which a disulfide connects the hydrophobic substituents to the macrocycle. The lipophilic/hydrophilic balance of the macrocycles was varied by the introduction of disulfide esters of increasing hydrophobicity on the primary side and/or oligo(ethylene glycol) moieties on the secondary side. In water, the amphiphilic macrocycles form vesicles or nanoparticles. Both the vesicles and the nanoparticles disintegrate upon cleavage of the disulfide by the reducing agent DTT. Disintegration of the nanoparticles leads to release of a model hydrophobic guest from the nanoparticle. Acknowledgment. We sincerely thank Dr. A. Mazzaglia, Ms. P. Malvagna, and Dr. D. Garozzo for the MALDI-MS experiments carried out at the University of Catania, Italy. We are indebted to Mrs. G. Fitzpatrick (Chemical Services Unit, University College Dublin (UCD)) who measured the 13C and 2D NMR spectra and to Dr. D. Cottell (Electron Microscopy Unit, UCD). We thank Professor K. Dawson and Dr. A. Gorelov for providing DLS facilities. D.N. is supported by an Enterprise Ireland postgraduate grant. Schering-Plough (Avondale) Co. is acknowledged for the Newman Scholarship in Organic Chemistry awarded to B.J.R. Supporting Information Available: Synthesis of amphiphilic cyclodextrins 9-12 and protocols for preparation and disintegration of vesicles and nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org. LA034330J
