Physicochemical Characteristics of Self-Aggregates of

Physicochemical Characteristics of Self-Aggregates of. Hydrophobically Modified Chitosans. Kuen Yong Lee and Won Ho Jo. Department of Fiber and Polyme...
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Langmuir 1998, 14, 2329-2332

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Physicochemical Characteristics of Self-Aggregates of Hydrophobically Modified Chitosans Kuen Yong Lee and Won Ho Jo Department of Fiber and Polymer Science, Seoul National University, Seoul 151-742, Korea

Ick Chan Kwon, Yong-Hee Kim, and Seo Young Jeong* Biomedical Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Korea Received August 18, 1997. In Final Form: February 20, 1998 Chitosan derivatives containing 2.8-5.1 deoxycholic acid groups per 100 anhydroglucosamine units of chitosan were synthesized by an EDC-mediated coupling reaction. Physicochemical properties of selfaggregates of the hydrophobically modified chitosans in aqueous media were studied by the dynamic light scattering method and fluorescence spectroscopy. The mean diameter (dH) of DC4.2 self-aggregates in phosphate-buffered saline (PBS) solution (pH 7.2) was 159 nm with unimodal size distribution (variance ) 0.082). It seemed that the interparticle interaction between self-aggregates was almost negligible. The mean diameter decreased with increasing degree of substitution (DS), pH, or ionic strength of the medium. The critical aggregation concentration (cac) of self-aggregates was determined from the fluorescence emission spectra of pyrene. The cac value of DC4.2 in PBS solution (pH 7.2) was 2.6 × 10-2 mg/mL. The cac values decreased with increasing DS, pH, or ionic strength of the medium.

Introduction Hydrophobically associating polymers have shown unusual rheological features and high solubilization properties in aqueous media.1 These properties arise from the inter- or intramolecular interaction among hydrophobic groups providing hydrophobic microdomains in an isotropic aqueous solution. Self-assemblies of block copolymers2 or hydrophobically modified polymers3 have been extensively investigated in the field of biotechnology and pharmaceutics.4 The formation of self-assemblies of polymeric amphiphiles in aqueous media is generally considered to resemble that of low molecular weight amphiphiles. Polymeric amphiphiles form self-assemblies consisting of the inner core of hydrophobic segments and the outer shell of hydrophilic segments because of the preference for the formation of free energy-minimized structure. This core-shell structure of self-assemblies of * To whom correspondence should be addressed. Tel: +82-2958-5911. Fax: +82-2-958-5909. E-mail: [email protected]. (1) (a) Duleh, A. J.; Steiner, C. A. Macromolecules 1990, 23, 251. (b) Tanaka, R.; Meadows, J.; Williams, P. A.; Phillips, G. O. Macromolecules 1992, 25, 1304. (c) Strauss, U. P.; Gersfeld, N. L. J. Phys. Chem. 1954, 58, 747. (d) Landoll, L. M. J. Polym. Sci., Polym. Chem. Ed. 1982, 20, 443. (e) Valint, P. L.; Bock, J. Macromolecules 1988, 21, 175. (2) (a) Xu, R.; Winnik, M. A.; Hallett, F. R.; Riess, G.; Croucher, M. D. Macromolecules 1991, 24, 87. (b) Anton, P.; Ko¨berle, P.; Laschewsky, A. Makromol. Chem. 1993, 194, 1. (c) Chu, B.; Wu, G.; Schneider, D. K. J. Polym. Sci., Polym. Phys. Ed. 1994, 32, 2605. (3) (a) Chu, D.; Thomas, J. K. Macromolecules 1987, 20, 2133. (b) Akiyoshi, K.; Deguchi, S.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Macromolecules 1993, 26, 3062. (c) Deguchi, S.; Akiyoshi, K.; Sunamoto, J. Makromol. Rapid Commun. 1994, 15, 705. (d) Guenoun, P.; Davis, H. T.; Tirrell, M.; Mays, J. W. Macromolecules 1996, 29, 3965. (4) (a) Bader, H.; Ringsdorf, H.; Schmidt, B. Angew. Makromol. Chem. 1984, 123/124, 457. (b) Yokoyama, M. Crit. Rev. Therap. Drug Carriers Systems 1992, 9, 213. (c) Kwon, G.; Naito, M.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. Langmuir 1993, 9, 945. (d) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecules 1994, 27, 7654. (e) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. J. Am. Chem. Soc. 1996, 118, 6110. (f) Trubetskoy, V. S.; Gazelle, G. S.; Wolf, G. L.; Torchillin, V. P. J. Drug Targeting 1997, 4, 381. (g) Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich, K.; Nazarova, O.; Seymour, L. W. Hum. Gene Ther. 1996, 7, 2123.

Figure 1. Chemical structure of (a) a repeat unit of chitosan and (b) deoxycholic acid.

polymeric amphiphiles has served as a potential delivery carrier because the inner core may contain the hydrophobic bioactive agents.5 In this paper, chitosan was hydrophobically modified by deoxycholic acid. Chitosan, next to cellulose, is the second most plentiful biomass and has a repeated structure of (1,4) linked 2-amino-2-deoxy-β-D-glucan (Figure 1a). Chitosan has been known as a good candidate material for biomedical uses because it is biocompatible, biodegradable, and of low toxicity when administered via oral (5) (a) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakura, Y. J. Controlled Release 1993, 24, 119. (b) Yokoyama, M.; Okano, T.; Sakurai, Y.; Ekimoto, H.; Shibazaki, C.; Kataoka, K. Cancer Res. 1991, 51, 3229. (c) Kabanov, A. V.; Batrakova, E. V.; M.-Nubarov, N. S.; Fedoseev, N. A.; Dorodnich, T. U.; Alakhov, V. Y.; Chekhonin, V. P.; Nazarova, I. R.; Kabanov, V. A. J. Controlled Release 1991, 22, 141.

S0743-7463(97)00928-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/11/1998

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Table 1. Effect of Degree of Substitution or pH on the Properties of Chitosan Self-Aggregatesa sample

pH

dHb (nm)

varianceb

cacc × 102 (mg/mL)

DC2.8 DC4.2

7.2 5.0 7.2 9.0 7.2

180 187 159 156 161

0.079 0.294 0.082 0.012 0.069

4.1 7.5 2.6 1.3 1.7

DC5.1

a [polymeric amphiphile] ) 2.0 mg/mL, T ) 25 °C. b Mean diameter and variance determined by dynamic light scattering (θ ) 90°). c Critical aggregation concentration determined by fluorescence spectroscopy.

or parenteral routes.6,7 Deoxycholic acid is a carboxylic acid with a cyclopentenophenanthrene nucleus containing a branched side chain of carbon atoms ending in a carboxyl group and has hydroxyl groups at both 3R and 12R positions (Figure 1b). Deoxycholic acid is a main component of bile acid, which is biologically the most detergentlike molecule in the body.8 Since bile acid can form selfassemblies in water, it is expected that the chitosan modified by bile acid also self-associates to form selfaggregates. In this context, we report here the effects of degree of substitution, pH, or ionic strength of the medium on the formation of self-aggregates of the hydrophobically modified chitosans. The formation of self-aggregates and their physicochemical characteristics are examined by dynamic light scattering and fluorescence spectroscopy. Experimental Section Chitosan (Mv ) 7.0 × 104, degree of deacetylation ) 80%) was hydrophobically modified by deoxycholic acid in the presence of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC).9 Different amounts of deoxycholic acid per glucosamine residues of chitosan (0.17-0.34, mol/mol) were added to the chitosan solution and followed by the dropwise addition of EDC at room temperature. The mole ratio of EDC per deoxycholic acid used in this study was kept constant. After 24 h, the reaction mixture was poured into the methanol/ammonia solution (7/3, v/v). The precipitated chitosan derivatives were filtered off and washed thoroughly, followed by drying in a vacuum at room temperature. The degree of substitution (DS), defined as the number of deoxycholic acid groups per 100 anhydroglucosamine units of chitosan, was determined by elemental analysis. The DS of deoxycholic acid-modified chitosan (DC) was in the range 2.85.1. The number behind the sample code DC in Table 1 indicates the DS of the sample. Hydrophobically modified chitosan was suspended in distilled water or phosphate-buffered saline (PBS) solution under gentle shaking at 37 °C. After 48 h of swelling, the solution was sonicated using a probe type sonifier (Sigma Ultrasonic Processor, GEX-600) at 30 W for 2 min and repeated three times to get an optically clear solution. The pulse function was used to prevent the sample solution from the heat buildup during sonication (pulse on, 5.0 s; pulse off, 1.0 s). (6) (a) Hirano, S.; Seino, H.; Akiyama, Y.; Nonaka, I. In Progress in Biomedical Polymers; Gebelein, C. G., Dunn, R. L., Eds.; Plenum Press: New York, 1990; p 283. (b) Muzzarelli, R. A. A. Chitin; Pergamon: New York, 1977. (c) Sandford, P. A.; Steinnes, A. In Water-Soluble Polymers; Shalaby, S. W., McCormick, C. L., Butler, G. B., Eds.; ACS Symposium Series, Vol. 467; American Chemical Society: Washington, DC, 1991; p 430. (d) Brine, C. J.; Sandford, P. A.; Zikakis, J. P. Advances in Chitin and Chitosan; Elsevier Applied Science: New York, 1992. (7) (a) Bhaskara, S.; Sharma, C. P. J. Biomed. Mater. Res. 1997, 34, 21. (b) Li, Q.; Dunn, E. T.; Grandmaison, E. W.; Goosen, M. F. A. J. Bioact. Compat. Polym. 1992, 7, 379. (c) Lee, K. Y.; Ha, W. S.; Park, W. H. Biomaterials 1995, 16, 1211. (8) Small, D. M. In The Bile Acids, Chemistry, Physiology, and Metabolism; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; p 249. (9) (a) Lee, K. Y.; Jo, W. H.; Kwon, I. C.; Kim, Y.-H.; Jeong, S. Y. Macromolecules 1998, 31, 378. (b) Lee, K. Y.; Kwon, I. C.; Kim, Y.-H.; Jo, W. H.; Jeong, S. Y. J. Controlled Release 1998, 51, 213.

Dynamic light scattering (DLS) experiments were carried out with an argon ion laser system (Lexel Laser Model 95) tuned at 488 nm. The scattering angle was varied from 30 to 135°. The intensity autocorrelation was measured with a Brookhaven BI9000AT digital autocorrelator, and the temperature was controlled to 25 ( 0.1 °C. When the difference between the measured and the calculated baselines was less than 0.1%, the correlation function was accepted. A nonlinear regularized inverse Laplace transformation technique (CONTIN)10 was used to obtain the distribution of decay constant (Γ). The mean diameter (dH) was evaluated by the Stokes-Einstein relationship. 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). The concentrations of sample solutions were varied from 2.5 × 10-4 to 1.0 mg/mL. For measurement of the intensity ratio of the first and the third highest energy bands 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/1 nm.

Results and Discussion Synthesis of Hydrophobically Modified Chitosans. EDC is a so-called “zero-length” cross-linker, which gives an amide linkage without leaving a spacer molecule.11 EDC reacts with a carboxyl group of the deoxycholic acid to form an active ester intermediate, which reacts with a primary amino group of the chitosan to form an amide bond.9 The degree of substitution (DS) increases as the added amount of deoxycholic acid and EDC increases. The DS is in the range from 2.8 to 5.1 per 100 anhydroglucosamine units of chitosan in this experiment. Because of the limited solubility of deoxycholic acid in the acidic reaction medium, the derivatives with higher DS cannot be obtained. In the FTIR spectra, the drastic increase of the amide I band at 1655 cm-1 with increasing DS indicates the formation of amide bonds between chitosan and deoxycholic acid (data not shown).9 Size and Its Distribution of Self-Aggregates. The size of self-aggregates and their size distribution in aqueous media were measured by DLS. Since the unmodified chitosan does not form self-aggregates in the medium used, it cannot be used as a control. Figure 2a shows a typical intensity autocorrelation function, g(2)(τ), of DC4.2 self-aggregates in PBS solution (pH 7.2) at θ ) 90° and T ) 25 °C. Under the assumption of a polydisperse system, the CONTIN algorithm was adopted to calculate the mean diameter and size distribution of self-aggregates (Figure 2b). The DC4.2 self-aggregate in PBS solution (pH 7.2) has a mean diameter of 159 nm with unimodal size distribution (variance ) 0.082). A plot of Γ vs q2 for DC4.2 self-aggregates in PBS solution (pH 7.2) is shown in Figure 3. q is defined as a scattering vector [)(4πn/λ0) sin(θ/2)], where n is the refractive index of the scattering medium and λ0 is the wavelength of the incident light in a vacuum. Since a linear relationship is obtained, the measured Γ can be predominantly attributed to a diffusive mode. From the transmission electron micrograph of self-aggregates that were negatively stained by a uranyl acetate solution, it could be confirmed that self-aggregates had nearly spherical shapes.9b Figure 4 shows the change of the mean diameter of DC4.2 self-aggregates in PBS solution (pH 7.2). It seems that the size of self-aggregates is scarcely affected by the (10) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213. (11) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. In Immobilized Affinity Ligand Techniques; Academic Press: San Diego, 1992; p 81.

Self-Aggregates of Modified Chitosans

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Figure 4. Size change of DC4.2 self-aggregates as a function of concentration of polymeric amphiphiles in PBS solution (pH 7.2; θ ) 90°; T ) 25 °C). Table 2. Effect of Ionic Strength on the Properties of Chitosan Self-Aggregatesa ionic strength

dHb (nm)

varianceb

cacc × 102 (mg/mL)

0 0.01 0.1 1.0

193 185 172 168

0.128 0.016 0.019 0.056

6.6 4.3 2.2 1.9

a [DC4.2] ) 2.0 mg/mL, T ) 25 °C. b Mean diameter and variance determined by dynamic light scattering (θ ) 90°). c Critical aggregation concentration determined by fluorescence spectroscopy.

Figure 2. (a) Intensity autocorrelation function, g(2)(τ) and (b) size distribution of DC4.2 self-aggregates in PBS solution (pH 7.2; [DC4.2] ) 2.0 mg/mL; θ ) 90°; T ) 25 °C).

Figure 3. Γ vs q2 for the diffusive mode of DC4.2 self-aggregates in PBS solution (pH 7.2; [DC4.2] ) 2.0 mg/mL; T ) 25 °C).

concentration of polymeric amphiphiles. The mean diameter remains almost constant up to ca. 100-fold critical aggregation concentration. Therefore, it may be concluded that the interparticle interaction between self-aggregates is almost negligible, even in the medium of high ionic strength. If we consider the biomedical uses of selfaggregates as a drug delivery system in the body, it is very important that the size of self-aggregates in PBS solution (pH 7.2) is not dependent on the concentration of polymeric amphiphiles. The mean diameter of self-aggregates and its distribution in PBS solution are listed in Table 1. The increase of the DS may enhance the chances of hydrophobic interactions between deoxycholic acid groups attached to

the chitosan backbone, resulting in the formation of more complete hydrophobic cores. When the DS reaches about 4.2 or higher, however, the size of self-aggregates seems to be independent of the DS. Since chitosan is a cationic polyelectrolyte, the effects of pH or ionic strength of the medium on the aggregation of the modified chitosans are important. At a low pH, amino groups of chitosan are well-protonated and interor intramolecular electrostatic repulsion becomes predominant. Consequently, this electrostatic repulsion may counteract the hydrophobic interaction, resulting in the formation of larger self-aggregates and broader size distribution (Table 1). Self-aggregates of the modified chitosan in PBS solution of pH 5.0 have a larger mean diameter and broader size distribution than those in PBS solution of higher pH. The mean diameter of selfaggregates and their size distribution decrease with increasing pH; however, there is no significant change in size above pH 7.2. When self-aggregates are formed in distilled water, the mean diameter is 193 nm (Table 2). The mean diameter decreases with increasing concentration of sodium chloride in the medium; however, the size seems to be scarcely affected by the ionic strength of the medium above 0.1 M NaCl. It may be considered that the electrolytes screen the electrostatic repulsion of the positive charges in the chitosan backbone, resulting in the reduction of size. Since the interparticle interaction is almost negligible, as shown in Figure 4, the decrease of size in Table 2 may be attributed to the intraparticle interaction of self-aggregates. Critical Aggregation Concentration of Self-Aggregates. The aggregation behavior of deoxycholic acidmodified chitosans in aqueous media was monitored by fluorometry in the presence of pyrene as a fluorescence probe. Figure 5 shows the fluorescence emission spectra

2332 Langmuir, Vol. 14, No. 9, 1998

Figure 5. Effect of polymer concentration on the fluorescence emission spectra of pyrene (6.0 × 10-7 M) in PBS solution (pH 7.2) in the presence of DC4.2 at 25 °C: [DC4.2] ) (a) 1 mg/mL, (b) 0.5, (c) 0.25, (d) 0.1, (e) 0.01, (f) 0.001. The excitation wavelength was 336 nm, and the spectra were accumulated with an integration time of 5 s/1 nm.

of pyrene incorporated into self-aggregates of DC4.2 in PBS solution (pH 7.2) at 25 °C. If micelles or other hydrophobic microdomains are formed in an aqueous solution, the pyrene preferably lies close to (or inside) these microdomains and strongly emits,12 while it is quenched in polar media. When the pyrene coexists with DC4.2 self-aggregates, the total emission intensity increases and especially the intensity of the third highest vibrational band at 383 nm (I3) starts to drastically increase at a certain concentration of polymeric amphiphiles. This concentration is defined as a critical aggregation concentration (cac), meaning the threshold concentration of self-aggregation of polymeric amphiphiles. The cac can be determined by measuring the intensity ratio (I1/I3) of the first and the third highest energy bands in the emission spectra of pyrene. The change of the intensity ratio (I1/I3) is shown in Figure 6. At low concentrations of polymeric amphiphiles, the I1/I3 values are close to the value13 (1.87) for pyrene in water followed by a linear decrease with the addition of polymeric amphiphiles above cac. The cac is determined by the interception of two straight lines. The cac values of deoxycholic acid-modified chitosans (Tables 1 and 2) are roughly 2 orders of magnitude lower than the critical micelle concentration (cmc) of low molecular weight surfactants, e.g., 2.3 mg/mL for sodium dodecyl sulfate (SDS) in water14 and 1.0 mg/mL for deoxycholic acid in water.15 The lower cac values of the chitosan derivatives as compared with low molecular weight surfactants can be one of the important characteristics of polymeric amphiphiles, indicating the stability of self-aggregates at dilute conditions. The increase of hydrophobicity by introduction of a large amount of hydrophobic groups reduces the cac values (Table 1). (12) Magny, B.; Iliopolous, I.; Zana, R.; Audebert, R. Langmuir 1994, 10, 3180. (13) Dong, D. C.; Winnik, M. A. Can. J. Chem. 1984, 62, 2560. (14) Phillips, J. N. Trans. Faraday Soc. 1955, 51, 561. (15) Kratohvil, J. P.; Hsu, W. P.; Kwok, D. I. Langmuir 1986, 2, 256.

Lee et al.

Figure 6. Intensity ratio (I1/I3) for pyrene in PBS solution (pH 7.2) of the hydrophobically modified chitosan as a function of concentration: (O) DC2.8; (0) DC4.2; (4) DC5.1.

The cac values decrease with increasing pH or ionic strength of the medium due to the effective hydrophobic interaction arising from the relative increase of hydrophobic character of the modified chitosan (Tables 1 and 2). Unlike nonionic amphiphiles, the aggregation behavior of ionic amphiphiles in water is influenced by the addition of electrolytes. For most of the ionic amphiphiles, the effect of added electrolytes can be empirically expressed by

log cmc ) -a log c + b

(1)

where a and b are constant for a given amphiphile at a given temperature and c is the concentration of monovalent electrolyte (mol/L).16 In the case of DC4.2, the cac values linearly decrease with the addition of sodium chloride (Table 2) and the values of a ) 0.179 and b ) -1.764 are obtained from eq 1. Conclusions Hydrophobically modified chitosans by deoxycholic acid provide colloidally stable self-aggregates in aqueous media above the critical aggregation concentration. Chitosan self-aggregates have mean diameters of less than 200 nm with unimodal size distribution. The formation of selfaggregates and their physicochemical properties depend on the DS, pH, or ionic strength of the medium. It seems that the interparticle interaction between self-aggregates is negligible. The mean diameter and the cac value decrease with increasing DS, pH, or ionic strength of the medium. The study of physicochemical properties may suggest the potential applicability of self-aggregates to the pharmaceutical and biomedical fields, especially to the delivery of bioactive agents. Acknowledgment. This work was supported by the KIST-2000 project. LA970928D (16) Meyers, D. Surfaces, Interfaces, and Colloids; VCH Publishers: New York, 1991; p 323.