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Physicochemical Characteristics of Self-Assembled Nanoparticles Based on Glycol Chitosan Bearing 5β-Cholanic Acid Seunglee Kwon, Jae Hyung Park, Hesson Chung, Ick Chan Kwon,* and Seo Young Jeong Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Haweolgog-dong, Sungbook-gu, Seoul 136-791, Korea
In-San Kim Department of Biochemistry, School of Medicine, Kyungpook National University, 101 Dongin-dong, Jung-gu, Daegu 700-422, Korea Received June 16, 2003. In Final Form: September 5, 2003 The self-aggregation behavior and microscopic characteristics of hydrophobically modified glycol chitosans (HGCs), prepared by covalent attachment of 5β-cholanic acid to glycol chitosan, were investigated by using 1H NMR, dynamic light scattering, fluorescence spectroscopy, and transmission electron microscopy (TEM). The HGCs formed self-aggregates in an aqueous phase by intra- or intermolecular association between hydrophobic 5β-cholanic acids attached to glycol chitosan. The critical aggregation concentrations (cacs) of the HGCs were dependent on the degree of substitution (DS) of 5β-cholanic acid and were significantly lower than those of low molecular weight surfactants. The mean diameters of the self-aggregates decreased with the increase in the DS of 5β-cholanic acid because of the formation of compact hydrophobic inner cores. The TEM images demonstrated that the shape of the self-aggregates, on the basis of the HGCs, is spherical. The partition equilibrium constants (Kv) of pyrene, measured in the self-aggregate solutions of the HGCs, indicated that the increase in the DS enhances the hydrophobicity of inner core of self-aggregates. The aggregation number of 5β-cholanic acid per one hydrophobic microdomain, estimated by the fluorescence quenching method using cetylpyridinium chloride, increased with increasing the DS, which suggested that several HGC chains were needed to form one hydrophobic domain.
Introduction Polymeric amphiphiles have received increasing attention because of their potential biotechnological and pharmaceutical applications.1-3 Upon contact with an aqueous environment, polymeric amphiphiles spontaneously form micelles or micellelike aggregates via undergoing intra- or intermolecular associations between hydrophobic moieties, primarily to minimize interfacial free energy. These polymeric micelles exhibit the unique characteristics, depending on hydrophilic/hydrophobic constituents, such as unusual rheological feature, small hydrodynamic radius (less than microsize) with coreshell structure, and thermodynamic stability.2-6 In particular, polymeric micelles have been recognized as a promising drug carrier, since their hydrophobic domain, surrounded by a hydrophilic outer shell, can serve as a preservatory for various hydrophobic drugs.3,7 For the past two decades, therefore, many efforts have been made to develop novel polymeric amphiphiles such as amphiphilic block copolymers1,5,7-10 and hydrophobically modified water-soluble polymers11,12 which self-assemble to form * Corresponding author. Tel: +82-2-958-5912; fax: +82-2-9585909; e-mail:
[email protected]. (1) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science 1994, 263, 1600-1603. (2) Tobio, M.; Gref, R.; Sanchez, A.; Langer, R.; Alonso, M. J. Pharm. Res. 1998, 15, 270-275. (3) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113-131. (4) Cudd, A.; Bhogal, M.; O’Mullane, J.; Goddard, P. Proc Natl. Acad. Sci. U.S.A. 1991, 88, 10855-10859. (5) Park, S. Y.; Han, D. K.; Kim, S. C. Macromolecules 2001, 34, 8821-8824. (6) Tae, G.; Kornfield, J. A.; Hubbell, J. A.; Lal, J. Macromolecules 2002, 35, 4448-4457. (7) Nakanishi, T.; Fukushima, S.; Okamoto, K.; Suzuki, M.; Matsumura, Y.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. J. Controlled Release 2001, 74, 295-302.
compact micellar structure in an aqueous media. In recent years, self-assemblies based on naturally occurring polymers have been of particular interest because of their potential for biomedical applications.13-18 Chitosan, the N-deacetylated derivative of chitin, has attracted significant interest in the broad range of scientific areas, including biomedical, agricultural, and environmental fields.19 Recently, much attention has been paid to chitosan as a drug20-22 or gene23-26 carrier because of its biocompatibility and biodegradability. However, the extended applications of chitosan are frequently limited because it is insoluble in biological solution (pH 7.4), which has stimulated studies to prepare water-soluble chitosan (8) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Alakhov, V. Y.; Yaroslavov, A. A.; Kabanov, V. A. Macromolecules 1995, 28, 2303-2314. (9) Poppe, A.; Willner, L.; Allgaier, J.; Stellbrink, J.; Richter, D. Macromolecules 1997, 30, 7462-7471. (10) Lee, S. C.; Chang, Y.; Yoon, J.; Kim, C.; Kwon, I. C.; Kim, Y.; Jeong, S. Y. Macromolecules 1999, 32, 1847-1852. (11) Kim, C.; Lee, S. C.; Kang, S. W.; Kwon, I. C.; Kim, Y.; Jeong, S. Y. Langmuir 2000, 16, 4792-4797. (12) Huh, K. M.; Lee, K. Y.; Kwon, I. C.; Kim, Y.; Kim, C.; Jeong, S. Y. Langmuir 2000, 16, 10566-10568. (13) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. J. Am. Chem. Soc. 1996, 118, 6110-6115. (14) Lee, K. Y.; Jo, W. H.; Kwon, I. C.; Kim, Y.; Jeong, S. Y. Macromolecules 1998, 31, 378-383. (15) Nichifor, M.; Lopes, A.; Carpov, A.; Melo, E. Macromolecules 1999, 32, 7078-7085. (16) Gref, R.; Rodrigues, J.; Couvreur, P. Macromolecules 2002, 35, 9861-9867. (17) Duval-Terrie´, C.; Cosette, P.; Molle, G.; Muller, G.; De´, E. Protein Sci. 2003, 12, 681-689. (18) Duval-Terrie´, C.; Huguet, J.; Muller, G. Colloids Surf., A 2003, 220, 105-115. (19) Li, Q.; Lunn, E. T.; Grandmaison, E. W.; Goosen, M. F. A. In Applications of Chitin and Chitosan; Goosen, M. F. A, Ed.; Technomic Publishing Co., Inc.: Lancaster, 1997; p 3. (20) Lee, K. Y.; Kim, J. H.; Kwon, I. C.; Jeong, S. Y. Colloid. Polym. Sci. 2000, 278, 1216-1219.
10.1021/la0350608 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/28/2003
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Experimental Section
Figure 1. Chemical structure of HGCs.
derivatives by chemical modification.27-29 For example, our group developed deoxycholic acid-modified chitosan derivatives that form self-aggregates in an aqueous solution.14,30 The self-aggregetes showed a promising potential in vitro as a carrier for the hydrophobic drug20 and gene.23,25 Nevertheless, clinical trials using the selfaggregates were not available because the insoluble nature of chitosan in an aqueous phase resulted in the precipitation of self-aggregates within a few days, when exposed to biological solution. Of chitosan derivatives with improved hydrophilicity, glycol chitosan is emerging as a novel carrier of drugs because of its solubility in water and biocompatibility.31-33 In this study, to obtain novel amphiphilic polymers that provide potential applications in biotechnology and medicine, hydrophobically modified glycol chitosans (HGCs) were prepared by covalent attachment of 5β-cholanic acid to glycol chitosan through amide formation. The detailed chemical structure of the HGCs, prepared in this study, is shown in Figure 1. Bile acids such as deoxycholic acid and 5β-cholanic acid are known to form micelles in water because of their amphiphilicity, which plays a vital role in the emulsification, solubilization, and absorption of cholesterol, fats, and liphophilic vitamins in the body.34 Thus, it was expected that the introduction of 5β-cholanic acid into glycol chitosan would induce self-association to form self-aggregates. Herein, we investigated in detail the effect of 5β-cholanic acid attached to glycol chitosan on the formation and physicochemical characteristics of self-aggregates. (21) Ruel-Gariepy, E.; Leclair, G.; Hildgen, P.; Gupta, A.; Leroux, J. C. J. Controlled Release 2002, 82, 373-383. (22) Zhang, Y.; Zhang, M. J. Biomed. Mater. Res. 2002, 62, 378-386. (23) Lee, K. Y.; Kwon, I. C.; Kim, Y. H.; Jo, W. H.; Jeong, S. Y. J. Controlled Release 1998, 51, 213-220. (24) Roy, K.; Mao, H. Q.; Huang, S. K.; Leong, K. W. Nat. Med. 1999, 5, 380-381. (25) Kim, Y. H.; Gihm, S. H.; Park, C. R.; Lee, K. Y.; Kim, T. W.; Kwon, I. C.; Chung, H.; Jeong, S. Y. Bioconjugate Chem. 2001, 12, 932938. (26) Koping-Hoggard, M.; Tubulekas, I.; Guan, H.; Edwards, K.; Nilsson, M.; Varum, K. M.; Artursson, P. Gene Ther. 2001, 8, 11081121. (27) Yalpani, M.; Hall, L. Macromolecules 1984, 17, 272-281. (28) Sugimoto, M.; Morimoto, M.; Sashiwa, H.; Saimoto, H.; Shigemasa, Y. Carbohydr. Polym. 1998, 36, 49-59. (29) Sashiwa, H.; Shigemasa, Y. Carbohydr. Polym. 1999, 39, 127138. (30) Lee, K. Y.; Jo, W. H.; Kwon, I. C.; Kim, Y.; Jeong, S. Y. Langmuir 1998, 14, 2329-2332. (31) Dufes, C.; Scha¨tzlein, A. G.; Tetley, L.; Gray, A. I.; Watson, D. G.; Olivier, J.; Couet, W.; Uchegbu, I. F. Pharm. Res. 2000, 17, 12501258. (32) Uchegbu, I. F.; Sadiq, L.; Arastoo, M.; Gray, A. I.; Wang, W.; Waigh, R. D.; Scha¨tzlein, A. G. Int. J. Pharm. 2001, 224, 185-199. (33) Martin, L.; Wilson, C. G.; Koosha, F.; Tetley, L.; Gray, A. I.; Senel, S.; Uchegbu, I. F. J. Controlled Release 2002, 80, 87-100. (34) Enhsen, A.; Kramer, W.; Wess, G. Drug Discov. Today 1998, 3, 409-418.
Materials. Glycol chitosan (Mn ) 2.5 × 105, degree of deacetylation ) 88%) was purchased from Sigma. It was dissolved in distilled water, filtered to remove insoluble impurities, and dialyzed against distilled water. 5β-Cholanic acid, 1-ethyl-3(3dimethylaminopropyl)carbodiimide hydrochloride (EDC), Nhydroxysuccinimid (NHS), pyrene, and cetylpyridinium chloride (CPC) were obtained from Sigma and used without further purification. The water, used for synthesis and characterization, was purified by distillation, deionization, and reverse osmosis (Milli-Q Plus). All other chemicals were analytical grades and used as received. Modification of Glycol Chitosan with 5β-Cholanic Acid. Glycol chitosan (0.5 g) was dissolved in distilled water (60 mL), followed by dilution with methanol (60 mL or 180 mL). Different amounts of 5β-cholanic acid per sugar residues of glycol chitosan (0.0115-0.345 mol/mol) were added and stirred until the solutions were optically transparent. To activate carboxylic acid pertaining to 5β-cholanic acid, equal amounts (1.5 equiv/[5β-cholanic acid]) of EDC and NHS were added into the polymer solution, which allowed formation of the amide linkage by the reaction with primary amino groups in glycol chitosan. The resulting solution was stirred for 24 h at room temperature, dialyzed for 3 days against the excess amount of water/methanol mixture (1v/4v), and lyophilized to obtain HGCs. The HGCs were analyzed using 1H NMR (Inova 600, Varian) which was operated at 600 MHz. The degree of substitution (DS), defined as the number of 5βcholanic acid per 100 sugar residues of glycol chitosan, was determined by the colloidal titration method being based on the reaction between positively charged polyelectrolytes and negatively charged ones.35 In brief, HGC (5 mg) was dissolved in 2% aqueous acetic acid solution (10 mL). After adding 20 µL of the indicator, 0.1 w/v% toluidine blue, HGC solution was titrated with N/400 potassium polyvinyl sulfate solution. Consequently, the HGCs synthesized in this study were coded depending on the DS of 5β-cholanic acid, for example, HGC12 indicates the glycol chitosan bearing 5β-cholanic acid with the DS value of 12. Preparation of Self-Assembled Nanoparticle. The HGCs were suspended in a phosphate-buffered saline (PBS, pH 7.4) under gentle shaking for 6 h. The solution was then sonicated three times using a probe-type sonifier (Sigma Ultrasonic Processor, GEX-600) at 90 W for 2 min each, in which the pulse was turned off for 1 s with the interval of 5 s to prevent the increase in temperature. The solution of self-aggregates was passed through membrane filter (pore size: 0.45 µm, Millipore) and stored at room temperature. Measurement of Dynamic Light Scattering. To determine the particle size of the self-aggregates, dynamic light scattering measurements were performed using the helium ion laser system (Spectra Physics Laser Model 127-35) which was operated at 633 nm and 25 ( 0.1 °C. The scattered light was measured at an angle of 90° and was collected with BI-9000AT autocorrelator. The concentration of self-aggregates was kept constant at 1 mg/ mL. The hydrodynamic diameter of self-aggregates was calculated by the Stokes-Einstein equation. The polydispersity factor, represented as µ2/Γ2, was evaluated from the cumulant method,36,37 where µ2 is the second cumulant of the decay function and Γ is the average characteristic line width. Measurement of Fluorescence Spectroscopy. The pyrene solution (3.0 × 10-2 M in THF), which had been stored at 4 °C prior to use, was added to the distilled water to give a pyrene concentration of 12 × 10-7 M, and THF was removed using a rotary evaporator at 30 °C for 2 h. This solution was mixed with the solution of HGC to obtain polymer concentrations from 1.0 × 10-4 to 5.0 mg/mL, resulting in a pyrene concentration of 6.0 × 10-7 M. Pyrene fluorescence spectra were obtained by using an ISS K2 multifrequency phase and modulation fluorometer (ISS, Champaign, IL). To obtain pyrene excitation spectra, the slit widths for emission and excitation were set at 2 and 0.5 mm, respectively; on the other hand, the slit widths were set at 0.5 and 2 mm, respectively, for the measurement of pyrene emission spectra. The excitation (λex) and emission (λem) wavelengths were (35) Ueno, K.; Kina, K. J. Chem. Educ. 1985, 62, 627-629. (36) Harada, A.; Kataoka, K. Macromolecules 1995, 28, 5294-5299. (37) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288-294.
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Table 1. Effect of Solvent and Feed Ratio on Degree of Substitution
Table 2. Composition and Mean Diameter of Self-Aggregates
solventa
feed ratiob (× 102)
DSc (%)
samplea
Mn b
DS
xc
dd (nm)
µ2/Γ2
50% methanol
1.15 5.76 11.50 23.00 34.50 1.15 5.76 11.50 23.00
1.10 3.67 5.21 6.42 6.98 0.78 0.92 2.64 8.65
HGC1 HGC5 HGC12
255 000 272 000 299 000
1.10 5.21 11.50
0.019 0.082 0.165
850 302 210
0.043 0.015 0.005
75% methanol
a Glycol chitosan bearing 5β-cholanic acid, in which the number indicates the DS of 5b-cholanic acid. b Number-average molecular weight, estimated from the colloidal titration result. c Weight fraction of 5β-cholanic acid. d Mean diameter in PBS (pH 7.4) measured by dynamic light scattering.
a Glycol chitosan was dissolved in water, followed by dilution with desired amount of methanol. b Mole ratio of 5β-cholanic acid to sugar residues of glycol chitosan. c Degree of substitution of 5βcholanic acid, determined by colloidal titration.
336 and 390 nm, respectively. The spectra were accumulated with an integration time of 3 s/nm. The aggregation number of 5β-cholanic acid groups per one hydrophobic domain was determined by using the steady-state fluorescence quenching method,38 where CPC was used as a fluorescence quencher for pyrene. For microheterogeneous system such as an aqueous micellar solution, the steady-state quenching data is known to fit in the quenching kinetics as follows:39-41
ln(I0/I) ) [Q]/[M]
(1)
where I0 and I are the fluorescence intensity in the absence and presence of a quencher, [Q] is the concentration of the quencher, and [M] is the concentration of hydrophobic microdomains in self-aggregates. Thus, [M] can be obtained from the slope of ln(I0/I) ) f([Q]) and the aggregation number per one hydrophobic microdomain (Nbile) given by eq 2.
Nbile ) [5β-cholanic acid]/[M]
(2)
Transmission Electron Microscopy (TEM). The size and distribution of self-aggregates were observed by using a Philips CM 30 which was operated at an accelerating voltate of 200 kV. A drop of sample solution (∼5 µL) with the concentration of 2 mg/mL was placed onto a copper grid coated with carbon, taped with a filter paper to remove surface water, and air-dried for 5 min. The self-aggregates, deposited on the grid, were then negatively stained by 2 wt % uranyl acetate solution.
Results and Discussion Synthesis and Characterization of HGCs. 5βCholanic acid was covalently attached to glycol chitosan in the presence of EDC and NHS, thus producing polymeric amphiphiles. Formation of the amide linkage between glycol chitosan and 5β-cholanic acid was confirmed by the increase in the amide I band at 1655 cm-1 of the FT-IR spectra. Also, the presence of 5β-cholanic acid in HGCs was demonstrated by the characteristic peaks of bile acid appearing at 0.6-2.5 ppm in the 1H NMR spectra. Various HGCs with different amounts of 5β-cholanic acid were prepared by changing the feed ratio of 5β-cholanic acid to glycol chitosan, as listed in Table 1. Since 5β-cholanic acid is well soluble in methanol but shows poor solubility in distilled water because of its hydrophobicity, we used methanol/water mixture solvent for the chemical modification of glycol chitosan. The results indicated that the DS was significantly dependent on the solvent system (composition of methanol/water mixture) and increased with the increase in the feed ratio of 5β-cholanic acid. The (38) Akiyoshi, K.; Deguchi, S.; Tajima, H.; Nishikawa, T.; Sunamoto, J. Macromolecules 1997, 30, 857-861. (39) Turro, N. J.; Yekta, A. J. J. Am. Chem. Soc. 1978, 100, 59515952. (40) Chu, D. Y.; Thomas, J. K. Macromolecules 1987, 20, 2133-2138. (41) Alami, E.; Almgren, M.; Brown, W. Macromolecules 1996, 29, 2229-2243.
Figure 2. 1H NMR spectra of HGC12 in (a) D2O/CD3OD (1v/ 4v) and (b) D2O at 25.0 °C. Arrows indicate characteristic peaks of 5β-cholanic acid attached to glycol chitosan.
effective solvent at the low DS region (
