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Langmuir 2000, 16, 10566-10568
Synthesis of Triarmed Poly(ethylene oxide)-Deoxycholic Acid Conjugate and Its Micellar Characteristics Kang Moo Huh,† Kuen Yong Lee,†,§ Ick Chan Kwon,† Yong-Hee Kim,† Chulhee Kim,‡ and Seo Young Jeong*,† Biomedical Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Korea, and Department of Polymer Science and Engineering, Inha University, Inchon 402-751, Korea Received July 11, 2000. In Final Form: September 27, 2000
Introduction Over the past years, many researchers have focused on polymeric amphiphiles composed of hydrophobic and hydrophilic segments because of their properties of micelle formation in aqueous media.1 In the field of biotechnology and pharmaceutics polymeric micelles have become attractive as a potential delivery system of bioactive agents,2 and there have been many efforts to synthesize new amphiphilic block copolymers3 or hydrophobically modified water-soluble polymers4 that can self-associate to form micelles in aqueous media. Poly(ethylene oxide) (PEO), in particular, has been used as a hydrophilic segment for a number of polymeric amphiphiles because of its low toxicity in biomedical applications.5 We already reported on the synthesis of monosubstituted PEO-deoxycholic acid conjugate and its micellar characteristics in water.6 Deoxycholic acid (DC) is a main component of bile acid, which is the most abundant and biologically detergent-like molecule in the body.7 Bile acids can self-associate in aqueous media and form micelles, * To whom correspondence should be addressed. Tel.: +82-2958-5911. Fax: +82-2-958-5909. E-mail:
[email protected]. † Korea Institute of Science and Technology. ‡ Inha University. § Present address: Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109. (1) (a) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600. (b) Xu, R.; Winnik, M. A.; Hallett, F. R.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 87. (c) Wilhelm, M.; Zhao, C.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 1033. (d) Hurter, P. N.; Hatton, T. A. Langmuir 1992, 8, 1291. (e) Yokoyama, M.; Kwon, G. S.; Okano, T.; Sakurai, Y.; Seto, T.; Kataoka, K. Bioconjugate Chem. 1992, 3, 295. (2) (a) Kwon, G. S.; Okano, T. Adv. Drug Deliv. Rev. 1996, 21, 107. (b) Jones, M. C.; Leroux, J. C. Eur. J. Pharm. Biopharm. 1999, 48, 101. (c) Chung, J. E.; Yokoyama, M.; Okano, T. J. Control. Release 2000, 65, 93. (d) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf. B-Biointerfaces 1999, 16, 3. (3) (a) Yokoyama, M. Crit. Rev. Therap. Drug Carrier Syst. 1992, 9, 213. (b) Lee, S. C.; Chang, Y.; Yoon, J.-S.; Kim, C.; Kwon, I. C.; Kim, Y.-H.; Jeong, S. Y. Macromolecules 1999, 32, 1847. (c) Yasugi, K.; Nakamura, T.; Nagasaki, Y.; Kato, M.; Kataoka, K. Macromolecules 1999, 32, 8024. (d) Akiyoshi, K.; Kohara, M.; Ito, K.; Kitamura, S.; Sunamoto, J. Macromol. Rapid Commun. 1999, 20, 112. (4) (a) Lee, K. Y.; Jo, W. H.; Kwon, I. C.; Kim, Y.; Jeong, S. Y. Langmuir 1998, 14, 2329. (b) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecules 1994, 27, 7654. (c) Miwa, A.; Ishibe, A.; Nakano, M.; Yamahira, T.; Itai, S.; Jinno, S.; Kawahara, H. Pharm. Res. 1998, 15, 1844. (d) Wang, Y. L.; Lu, D. H.; Long, C. F.; Han, B. X.; Yan, H. K.; Kwak, J. C. T. Langmuir 1998, 14, 2050. (5) (a) Kwon, G. S.; Kataoka, K. Adv. Drug Deliv. Rev. 1995, 16, 295. (b) Allen, C.; Han, J. N.; Yu, Y. S.; Maysinger, D.; Eisenberg, A. J Control. Release 2000, 63, 275. (c) Poppe, A.; Willner, L.; Allgaier, J.; Stellbrink, J.; Richter, D. Macromolecules 1997, 30, 7462. (d) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Alakhov, V. Y.; Yaroslavov, A. A.; Kabanov, V. A. Macromolecules 1995, 28, 2303. (6) Lee, S. C.; Kang, S. W.; Kim, C.; Kwon, I. C.; Kim, Y.-H.; Jeong, S. Y. Langmuir 2000, 16, 4792.
which play an important role in the emulsification, solubilization, and absorption of cholesterol, fats, and lipidsoluble vitamins in the body.8 Therefore, it was expected that the introduction of deoxycholic acid as an end group of the PEO chain would induce self-association of the PEO-DC conjugate and form polymeric micelles in water.6 However, monosubstituted PEO-DC conjugates formed micelles at a fairly high critical micelle concentration (cmc) compared to those of other amphiphilic block copolymers.3b,6,9 In addition, the length of the PEO chain did not significantly affect the size and cmc of the monosubstituted PEO-DC conjugates. In this context, we synthesized a new type of a triarmed PEO-DC conjugate, consisting of one hydrophilic chain and three hydrophobic groups to impart more hydrophobic character to the PEO-DC conjugate. We also investigated its micellar characteristics using a dynamic light scattering method, atomic force microscopy, and fluorescence spectroscopy. Experimental Section Materials. Poly(ethylene oxide) methyl ether with the numberaverage molecular weight of 2000 (g/mol) was purchased from Aldrich (St. Louis, MO) and used after drying in a vacuum at 80-90 °C for over 12 h. Acetonitrile was dried over CaCl2 and distilled. Dimethyl sulfoxide (DMSO) was purified by vacuum distillation. Tetrahydrofuran (THF) was dried over sodium metal and distilled. DC, N-(dimethylamino)pyridine (DMAP), and dicyclohexylcarbodiimide (DCC) were purchased from Aldrich (St. Louis, MO) and used as received. All other chemicals were used with reagent grade and double-distilled water was used in all procedures. Polymer Synthesis. p-Nitrophenylchloroformate (25 mmol) was dissolved in acetonitrile. PEO (2 mmol) and triethylamine (12.5 mmol) were added to the solution and the reaction mixture was stirred for 24 h. The precipitated triethylammonium chloride was removed by filtering, and the solution was precipitated by diethyl ether. The precipitate was washed with diethyl ether and dried in a vacuum (1, yield: 95%). Tris(hydroxymethyl) aminomethane (trisamine, 10 mmol) was dissolved in DMSO by heating and p-nitrophenyl ester of PEO (1, 2 mmol), which was also dissolved in DMSO, was added to it. After 24 h, the polymer solution was diluted with distilled water, dialyzed with dialysis membranes (molecular weight cutoff: 1000) against water, and freeze-dried (2, yield: 65%). The trisamine derivative of PEO (2, 1.5 mmol) and deoxycholic acid (5 mmol) were dissolved in 40 mL of THF, followed by the addition of DCC (5 mmol) and DMAP (0.5 mmol). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 24 h. After the precipitated dicyclohexylurea was filtered out, the filtrate was concentrated under reduced pressure and then poured into the excess amount of cold diethyl ether. The precipitate was extracted twice with the mixture of diethyl ether and ethyl alcohol (50/50, v/v) to remove unreacted deoxycholic acid residue and dried in a vacuum (3, yield: 30%). 1H NMR (CDCl ) δ: 0.65, 0.90, 0.95, 1.20-2.00, 3.60, 3.85, 3 3.98, 4.20 ppm. IR (KBr pellet): 1655 (NHCdO) cm-1. Measurements. The chemical composition of the polymeric amphiphile was characterized by 1H NMR (300 MHz). Each sample was dissolved in CDCl3 or D2O at the concentration of 0.5%. GPC measurements were performed using a Waters LC (7) Coello, A.; Meijide, F.; Nu´n˜ez, E. R.; Tato, J. V. J. Pharm. Sci. 1996, 85, 9. (8) Cai, X.; Grant, D. W.; Wiedmann, T. S. J. Pharm. Sci. 1997, 86, 372. (9) (a) Nagasaki, Y.; Okada, T.; Scholz, C.; Iijima, M.; Kato, M.; Kataoka, K. Macromolecules 1998, 31, 1473. (b) Kwon, G. S.; Naito, M.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1993, 9, 945.
10.1021/la000978+ CCC: $19.00 © 2000 American Chemical Society Published on Web 11/29/2000
Notes
Langmuir, Vol. 16, No. 26, 2000 10567 Scheme 1. Synthetic Scheme for a Triarmed PEO-DC Conjugate
system equipped with a Waters 410 differential refractometer. The degassed THF was eluted at a flow rate of 1.0 mL/min through four microstyragel columns (pore size; 102, 5 × 102, 103, and 104 Å, Waters). Dynamic light scattering experiments were performed with an argon ion laser system (Lexel Laser Model 95) tuned at a wavelength of 488 nm. The intensity autocorrelation of the sample was measured at a scattering angle (θ) of 90° with a Brookhaven BI-9000AT digital autocorrelator at 25 ( 0.1 °C. Steady-state fluorescence spectra were recorded on an ISS K2 fluorometer (ISS, Champaign, IL). A sample solution containing pyrene (6.0 × 10-7 M) was excited using a 300-W xenon arc lamp (ILC, Sunnyvale, CA). For the measurement of the intensity ratio of the first and the third highest energy bands (I1/I3) in the emission spectra of pyrene, the slit openings for excitation and emission were set at 1 and 0.5 mm, respectively. The excitation wavelength (λex) was 336 nm, and the spectra were accumulated with an integration time of 5 s/nm. Atomic force microscopic (AFM) images were obtained by a commercial Autoprobe CP system (Park Science, Sunnyvale, CA) using contact mode under ambient conditions. A silicon nitride tip on a cantilever with a spring constant of 0.12 N/m was used.
Results and Discussion For the synthesis of a triarmed PEO-DC conjugate, we initially introduced a triol group to the end of the PEO chain using trisamine and then followed by coupling reaction with deoxycholic acid. The schemes for introduction of a triarmed structure to the end of the PEO chain and subsequent coupling reaction are illustrated in Scheme 1. Products from each synthetic step of the conjugation process were characterized by 1H NMR and infrared spectroscopy. The amount of p-nitrophenyl groups in the intermediate 1 was calculated from the 1H NMR spectrum by comparing the integration values of aromatic protons of the p-nitrophenyl group (δ ) 7.4 and 8.3 ppm) with those of methylene protons in PEO repeating units (δ ) 3.6 ppm). After the reaction between the intermediate 1 and trisamine, the peaks of p-nitrophenyl groups disappeared clearly in the spectrum, indicating a complete substitution reaction. An increase in molecular weight of PEO was measured by GPC to confirm successful conjugation. The number-average molecular weight (Mn) and the molecular weight distribution (Mw/Mn) for PEO were 2400 and 1.06, respectively, whereas Mn and Mw/Mn for the PEO-DC conjugate were 3400 and 1.12, respectively. No peaks corresponding to the PEO homopolymer or deoxycholic acid were observed after purification. The chemical composition of the conjugate was characterized by 1H NMR spectroscopy. Figure 1a shows the
Figure 1. 1H NMR spectra of the triarmed PEO-DC conjugate dissolved in (a) CDCl3 and (b) D2O.
characteristic peaks of two components, indicating that conjugation has taken place between PEO and DC. The amount of deoxycholic acid attached to the end of the PEO chain was calculated from the integration values of the methylene peak of PEO at 3.6 ppm and the proton peaks of dexoycholic acid at 2.4-0.6 ppm. The average number of deoxycholic acids attached to one PEO chain was found to be 2.6. The spectrum of the PEO-DC conjugate in CDCl3 was compared to that of the micelle solution in D2O (Figure 1b). The solution for 1H NMR characterization was prepared by dissolution of freeze-dried micelles in each solvent. In D2O, the peaks from deoxycholic acid were not visible, but the ethylene peak of PEO was clearly visible in both solvents. This might indicate that the conjugate associated in water to form a micellar structure with a hydrophobic core of deoxycholic acid residues. Dynamic light scattering measurements confirmed the size and distribution of micelles in the concentration range of 0.02-5 mg/mL. The diameter of micelles prepared from the triarmed PEO-DC conjugate was 95 nm with unimodal size distribution. The size of the micelles was not significantly affected by the change of polymer concentrations. The mean diameter of micelles from monosubstituted PEO-DC conjugates was in the range of 120-180
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Notes
Figure 3. Changes in the intensity ratios (I1/I3) from pyrene emission spectra in the presence of deoxycholic acid sodium salt (b) and triarmed PEO-DC conjugate (O) as a function of concentration. Figure 2. Atomic force microscopic image of micelles of the triarmed PEO-DC conjugate. X-Y scan was at 4 × 4 µm scale.
nm, depending on the length of the PEO chain.6 Considering the block length of PEO-DC conjugates and the micellar sizes, it could be suggested that the micelles of PEO-DC would be a multicore structure formed by the association of individual micelles rather than a simple core-shell-type structure as previously reported.6 In particular, it was reported that the formation of large aggregates of individual micelles was more pronounced in the case of micelles with a shell from a relatively short PEO block (Mn ) ∼2000).10 The spherical shape of micelle particles was directly observed by AFM (Figure 2), which revealed that the size observed from AFM images was quite consistent with one determined by the dynamic light scattering method. A sample was prepared by air-spraying the micelle solution onto the surface of a glass plate and was followed by freeze-drying. It is generally known that polymeric amphiphiles form a micellar structure at a lower critical micelle concentration (cmc) than low molecular weight surfactants, as polymeric amphiphiles have more interaction sites than low molecular weight surfactants.11 The fluorescence technique proved useful in the study of the micellar structure as we measured and compared cmc values of the conjugate and deoxycholic acid. The cmc was determined by measuring the intensity ratio (I1/I3) of the first and the third highest energy band in the emission spectra of pyrene.12 It should be recalled that I1/I3 values of pyrene depend on the wavelength of excitation and the change in fluorescent intensity could be influenced by the partitioning of pyrene molecules rather than the polymer association at dilute concentrations of polymeric amphiphiles. Therefore, one needs to understand the par(10) Allen, C.; Yu, Y.; Maysinger, D.; Eisenberg, A. Bioconjugate Chem. 1998, 9, 564. (11) Lee, K. Y.; Jo, W. H.; Kwon, I. C.; Kim, Y.; Jeong, S. Y. Macromolecules 1998, 31, 378. (12) Astafieva, I.; Zhong, X. F.; Eisenberg, A. Macromolecules 1993, 26, 7339.
titioning of pyrene in detail to determine the correct cmc.12 Figure 3 presents a plot of the intensity ratio versus logarithmic concentration of the triarmed PEO-DC conjugate. The cmc was determined by the interception of two straight lines at a low concentration region. The obtained cmc values for deoxycholic acid and the PEODC conjugate were 0.7 and 1.5 × 10-3 g/L, respectively. The triarmed PEO-DC conjugate showed a significantly lower cmc than deoxycholic acid or even much lower than the monosubstituted PEO-DC conjugate. This might be one of the most important characteristics of polymeric micelles as a delivery system because they could maintain a stable micellar structure at dilute conditions. The cmc of monosubstituted PEO-DC conjugates was in the range of 3.6-4.3 × 10-3 g/L, depending on the molecular weight of PEO.6 The I1/I3 values of pyrene for deoxycholic acid sodium salt after micelle formation were lower than those of the PEO-DC conjugate, indicating the formation of less polar microdomains inside the micelles of deoxycholic acid sodium salt. In conclusion, the triarmed PEO-DC conjugate was successfully synthesized by the introduction of a triol group to the end of the PEO chain and by a subsequent coupling reaction with deoxycholic acid. These polymeric amphiphiles formed stable and spherical micelles in aqueous media with unimodal size distribution. The triarmed PEO-DC conjugate formed micelles of smaller size at a lower cmc than those of monosubstituted ones. This was considered to result from an increase in hydrophobicity of the triarmed conjugate. Regarded as a potential delivery system due to its very low cmc value, micelle systems containing multiarmed hydrophobic groups may open opportunities in designing more versatile types of delivery systems. Acknowledgment. The authors would like to thank Dr. Youngro Byun for his help in taking photographs of AFM. This work was supported by the KIST-2000 project. LA000978+
