Synthesis and Characterization of Sugar-Bearing Chitosan Derivatives

acetylation of the sugar-bearing chitosan (SBC) was carried out to enhance the water solubility because the crystallinity of chitosan, responsible for...
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Biomacromolecules 2003, 4, 1087-1091

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Synthesis and Characterization of Sugar-Bearing Chitosan Derivatives: Aqueous Solubility and Biodegradability Jae Hyung Park, Yong Woo Cho, 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 Received March 26, 2003; Revised Manuscript Received May 19, 2003

The extended use of chitosan in biomedical fields has been limited by its insoluble nature in a biological solution. To endow the water solubility in a broad range of pH, chitosan derivatives were prepared by the covalent attachment of a hydrophilic sugar moiety, gluconic acid, through the formation of an amide bond. These sugar-bearing chitosans (SBCs) were further modified by the N-acetylation in an alcoholic aqueous solution. Thereafter, the effect of the gluconyl group and the degree of N-acetylation (DA) on the water solubility at different pHs and on the biodegradability of chitosan were investigated. The SBCs showed the water solubility in a broader range of pH than chitosan, whereas they were still insoluble at neutral and alkali pH. The N-acetylation of SBCs significantly affected the water solubility, for example, the SBCs with the DA, ranging from 29% to 63%, were soluble in the whole range of pH. This might result from the improved hydrophilicity by the gluconyl group, accompanied by the role of the N-acetyl group that disturbed the hydrogen bonding between amino groups of chitosan. From the biodegradation tests, determined by the decrease in the viscosity of a polymer solution exposed to lysozyme, it was evident that the gluconyl group attached to chitosan improved the biodegradability. Thus, it was possible to control the biodegradability of chitosan by adjusting the amounts of gluconyl and N-acetyl groups in the chitosan backbone. The N-acetylated SBCs, soluble in the broad range of pH, might be useful for various biomedical applications. Introduction Chitosan, primarily composed of 2-amino-2-deoxy-β-Dglucopyranose (D-glucosamine), is the deacetylated derivative of chitin, the second most abundant natural polysaccharide. Owing to its specific structure and property, chitosan has attracted significant interest in a broad range of scientific areas such as biomedical, agricultural, and environmental fields.1 In particular, chitosan and its derivatives have been considered as biomaterials because of their biocompatibility, biodegradability, low immunogenicity, and biological activities.2-4 The extended applications of chitosan, however, are frequently limited by its insoluble nature in a biological solution (pH 7.4): for example, chitosan is only soluble in an aqueous acidic solution below pH 6.5, in which the primary amino groups, belonging to the chitosan backbone, are protonated. The presence of rigid crystalline domains, formed by intra- and/or intermolecular hydrogen bonding, is considered to be responsible for the poor solubility of chitosan in high pH solutions.5 In an attempt to improve the water solubility of chitosan, many efforts have been made to introduce hydrophilic moieties by the covalent attachment to reactive amino groups at the C2 position of chitosan. A few examples include acylation,6-8 akylation,8,9 carboxymethylation,10 and quaternarization11 of chitosan. Recently, these water-soluble chitosan derivatives have been investigated in vivo for bio* To whom correspondence should be addressed. Phone: +82-2-9585912. Fax: +82-2-958-5909. E-mail: [email protected].

medical applications.12-15 However, it is of importance to emphasize that, in most synthetic routes, to prepare watersoluble chitosan derivatives, a large number of D-glucosamine units in chitosan (g 50%) should be modified to introduce hydrophilic moieties.6,12,15 Because the physicochemical and biochemical activities of chitosan originate from the primary amino groups in the fundamental skeleton, the significant modification of D-glucosamine units in chitosan is obviously undesirable. Thus, as an alternative strategy to improve the water solubility but at the same time to minimize the consumption of D-glucosamine units in chitosan, the covalent conjugation of hydrophilic polymer such as poly(ethylene oxide) (PEO) has been recently suggested by Sugimoto et al.16 The resulting chitosan derivatives were fully soluble over the whole range of pH when the degree of substitution (DS) was higher than 15%. Nevertheless, the intrinsic nature of chitosan might not be preserved because the weight percentage of PEO in the chitosan derivative should be higher than 60% to ensure the water solubility. Without chemical modification, Chenite et al.17,18 prepared neutral solutions based on chitosan/glycerophosphate combinations which could be held liquid below room temperature but form monolithic gels at body temperature, showing potential for tissue engineering applications. N-Trimethyl chitosan chloride, prepared by Junginger et al.,19 showed much higher aqueous solubility in a broader range of pH than chitosan which allowed its use as an intestinal absorption enhancer20 and an effective gene carrier.21

10.1021/bm034094r CCC: $25.00 © 2003 American Chemical Society Published on Web 06/18/2003

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Figure 1. Synthesis of sugar-bearing chitosan derivatives with different degrees of N-acetylation.

In this study, we attempted to develop novel chitosan derivatives that are water-soluble over the whole range of pH and contain a significant number of D-glucosamine units, accounting for the original physicochemical and biological activities of chitosan. To improve the hydrophilicity of chitosan, D-gluconic acid as a representative hydrophilic sugar derivative was covalently attached because it may provide further biological functionality.22-24 Also, the Nacetylation of the sugar-bearing chitosan (SBC) was carried out to enhance the water solubility because the crystallinity of chitosan, responsible for the poor solubility in water, is known to be dependent on the degree of N-acetylation (DA).13,25,26 The chitosan derivatives, synthesized in this study, were then characterized with regard to the solubility in the water with different pHs. Because biodegradation kinetics are of great importance for various biomedical applications, the biodegradability of the derivatives was also estimated by the decrease in the viscosity of polymer solutions exposed to lysozyme. Experimental Section Materials. Chitosan (degree of deacetylation ) 95%, Mn ) 100 000) was purchased from Synyoung Chitosan Co., Korea. It was dissolved in 5% aqueous acetic acid solution to give a polymer concentration of 2 w/v%, filtered through a 0.45 µm membrane, and precipitated in 1 N NaOH solution. The precipitate was washed repeatedly with distilled water until a neutral pH was achieved. The product was lyophilized to obtain a white chitosan powder and used for further experiments. D-Gluconic acid, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimid (NHS), acetic anhydride, and lysozyme were obtained from Sigma and used as received. Chitosan Modification. The overall synthetic route for the modification of chitosan is summarized in Figure 1. Gluconic acid was covalently attached to chitosan, followed by N-acetylation. The detailed experimental procedure is as follows:

First, chitosan was modified by the chemical conjugation of gluconic acid in the presence of an active ester intermediate, EDC. Chitosan (1 g) and gluconic acid (0.1 or 0.2 equiv/[-NH2]) were dissolved in 0.1 N HCl solution (100 mL). After the addition of EDC (1.5 equiv/[gluconic acid]) and NHS (0.25 equiv/[EDC]), the pH of the solution was adjusted to 5.0 with 1 N NaOH/1 N HCl solutions. The resulting solution was stirred for 24 h at room temperature. After an increase in pH to 9 using 1 N NaOH solution, the solution was dialyzed for 5 days against the excess amount of distilled water and was lyophilized so that the SBC could be obtained. Second, the N-acetylation of the SBC was performed under homogeneous condition as reported by Vachoud et al.27 with slight modification. The SBC (0.5 g) was dissolved in 0.5% aqueous acetic acid solution (50 mL). The solution was mixed with a 40 mL of 1,2-propanediol and degassed by staying it at room temperature without stirring for 1 day. To this solution, the acetylating solution, composed of 1,2propanediol (10 mL) and the desired amount of acetic anhydride, was slowly added and stirred at room temperature for 5 h. The resulting solution was adjusted to pH 9 with the addition of 1 N NaOH solution. After being dialyzed against distilled water and being lyophilized, the N-acetylated SBCs were obtained. Characterization. 1H NMR spectra were obtained on an UnityPlus 300 (Varian) which was operated at 300 MHz. The samples were dissolved in 5% CD3COOD/D2O solution to give a polymer concentration of 1 w/v%. The NMR instrument was performed at 70 °C in order to shift the signal of HOD to a higher field, which allowed identifying and quantifying the H1 peak at 4.87 ppm of glucosamine residue in chitosan derivatives. The chemical shifts were expressed in PPM, based on the signal for sodium 3-(trimethylsilyl)propionate-d4 as an internal reference. The DS of gluconic acid was determined using a colloidal titration method that is based on the reaction between the positively charged polyelectrolytes and the negatively charged ones.28 In brief, the sample (∼4 mg) was dissolved in 2%

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aqueous acetic acid solution (10 mL). After the addition of an indicator (20 µL), 0.1% toluidine blue, the polymer solution was titrated with N/400 potassium polyvinyl sulfate (PVSK, Wako Pure Chemical Ind., Ltd.). The DA of SBC was estimated by an identical method. The chitosan derivatives were then coded depending on the DS and DA. For example, SBC-X-Y indicates the chitosan derivative whose DS and DA are X% and Y%, respectively. Estimation of Water Solubility. The chitosan derivative (10 mg) was dissolved in 0.1 N HCl (5 mL), the solution was placed into a quartz cell with an optical path length of 1 cm, and the transmittance of the solution was recorded on a UV/vis spectrophotometer (Perkin-Elmer, Lambda 18, Germany) at 600 nm. The pH of the polymer solution was adjusted with the addition of 6 N NaOH solution, by which the solubility in aqueous solutions was determined at pH 1.0-12.0. The polymer was considered as an insoluble one when the transmittance of the polymer solution was lower than 50%, compared to that of a control solution (0.1 N HCl). In Vitro Biodegradation. The biodegradation of the chitosan derivatives was estimated by a decrease in the viscosity of a polymer solution in the presence of lysozyme. The sample was dissolved in 0.1 M acetate buffer (pH 5) to produce a 0.2 w/v% polymer solution and warmed to 37 °C. Thereafter, lysozyme was added to give a final concentration of 18 µg/mL. The solution was immediately incubated at 37 °C, and the viscosity of 1 mL aliquots was measured with an Ubbelohde viscometer as a function of time. Results and Discussion Synthesis and Characterization of SBCs. To improve water solubility and biodegradability, chitosan was modified with hydrophilic gluconic acid, followed by the N-acetylation (Figure 1). Gluconic acid was covalently attached to the primary amino groups of chitosan through the amide formation in the presence of EDC and NHS.29 Because EDC is known to activate effectively carboxyl groups in a relatively low pH range, the reaction was performed in aqueous acidic solution (pH 5).30 However, the DS, determined by colloidal titration, was not sufficiently high; for example, although significant amount of gluconic acid (0.1 or 0.2 equiv/[-NH2]) was added to the reaction mixture, the DS was only 3% and 5%, respectively. In acidic solution, most primary amino groups of chitosan are protonated, when considering its pKa (∼6.5). Thus, the lack of uncharged amino groups which can react with carboxylic acids might result in the low DS.27 It should be noted, however, that the DS, when considering the molar feed ratio of gluconic acid to amino group, was comparable to those of other sugar-modified chitosans which were prepared by N-acylation8 or N-alkylation9,31 method. These SBCs prepared are expected to have a wide utility in the field of drug/gene delivery because sugar groups can be used as targeting moieties to the cells expressing their receptors.23,24 The N-acetylation of SBC-5-5 was carried out in an acetic acid-water-propanediol solution by varying the initial molar ratio of acetic anhydrid to glucosamine residue. Furthermore, four samples with different DAs ranging from 30% to 65%

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Figure 2. Relation between the degree of N-acetylation and molar ratio of acetic anhydride to D-glucosamine residue (the error bar is for standard deviation, n ) 3).

were obtained. Because the reaction was performed under the homogeneous condition, the N-acetyl groups should be distributed randomly along the chitosan backbone.32 Figure 2 shows the relationship between the DA and the initial amount of acetic anhydrid. The results indicated that the DA increased as the initial amount of acetic anhydride increased. It is known that, in an aqueous acetic acid solution, a large amount of acetic anhydride is needed for the preparation of the chitosan derivative with the desired DA; for example, for 50% acetylation, 8-9 equiv of acetic anhydride is needed based on the primary amino groups of chitosan.6 In our solvent system, containing alcohol (1,2-propanediol), 50% acetylation was achieved by adding only 0.6 equiv of acetic anhydrid, which was consistent with other reports.7,27 The presence of alcohol also helps to avoid side reactions such as the O-acetylation occurring between the primary alcohol of the glucosidic ring and acetic anhydrid.33 It is noteworthy that, although the DA of SBC-5-5 increased with the initial amount of acetic anhydrid, the effectiveness of the reaction was attenuated in the high DA region, as shown in Figure 2. This fact can be attributed to the difficulty of access to the amine functionality, as the N-acetylation proceeds. Also, the increased fraction of protonated primary amines by the consumption of uncharged ones in chitosan that can react with acetic anhydride may play a critical role, as discussed earlier. Figure 3 shows 1H NMR spectra for chitosan and SBC5-63 as a representative chitosan derivative, synthesized in this study. For chitosan, the small peaks at 2.05 and 4.58 ppm appeared because of the presence of -CH3 and H1′ of N-acetylglucosamine residues. On the other hand, the relatively large peak at 4.87 ppm was observed because of H1 of glucosamine residues, indicating the low DA of chitosan. The DA (5%) of chitosan, estimated from the ratio of integral intensity of the N-acetyl proton to the sum of integral intensities of the H2-H6, was in agreement with that reported by the manufacturer. The chemical modification of chitosan by gluconic acid resulted in the appearance of a characteristic peak at 4.24 ppm (Ha of gluconyl group), as shown in Figure 3b. Other peaks appearing from the gluconyl group in the region of 3.4-4.0 ppm were overlapped with the peaks of H3-H6 in

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Figure 3. 1H NMR spectra of (a) chitosan and (b) SBC-5-63 in D2O with 5 v/v% CD3COOD. Table 1. Solubility of N-Acetylated Chitosan Derivativesa sampleb

DWc

AAd

pHe

chitosan SBC-3-5 SBC-5-5 SBC-5-29 SBC-5-48 SBC-5-63

+ -

+ + + + + +

6.5 ( 0.1 6.6 ( 0.1 6.8 ( 0.1 12.0 ( 0.0 12.0 ( 0.0 12.0 ( 0.0

a The solubility was estimated by the transmittance of the polymer solution, measured by UV/vis spectrophotometer. b SBC-X-Y represents the chitosan derivative whose DS and DA, determined by colloidal titration, are X% and Y%, respectively. c Distilled water. d 2% aqueous acetic acid. e The highest pH that can dissolve the polymer; the pH of the polymer solution in 0.1 N HCl was adjusted by adding stepwise 6N NaOH. +, soluble; -, insoluble.

the chitosan backbone. Also, the significant increase in the integral intensities of H1′ and -CH3 was observed, following N-acetylation. Water Solubility of SBCs. The water solubility of chitosan and SBCs with different DA was evaluated in various pH solutions. Chitosan and its derivatives, synthesized in this study, were soluble in an aqueous acidic solution below pH 6.5 (Table 1). This was caused by the protonation of primary amino groups, thus indicating that a significant amount of D-glucosamine residues still remained for the unique solubility of chitosan. In particular, even for SBC5-63 which possesses only 32 D-glucosamines per 100 sugar units of the chitosan backbone, its solubility in acidic solution was comparable to chitosan and other derivatives. This was expected because, if the DA is less than 80%, the random distribution of N-acetyl groups is considered to inhibit the formation of the ordered structure via inter- and/or intramolecular hydrogen bonding between acetamide bonds.26,27 In our case, the enhanced hydrophilicity, attributed to the gluconyl group, may further improve the solubility in aqueous solution. It was evident that the SBCs were soluble in the slightly broader range of pH than chitosan, as shown in Table 1. The pH range, dissolving SBCs, was extended as the DS

increased, indicating the role of the gluconyl group. Similarly, Yang et al.9 reported that the chitosan derivatives, prepared by the reductive N-alkylation using monosaccharides such as glucose and galactose, exhibited the water solubility in a slightly broader range of pH than chitosan. However, those derivatives were not soluble at neutral or alkali pH, although a large quantity of monosaccharides was covalently attached (the DS was higher than 35%). In this study, we assumed that the N-acetylation of SBC further improves the water solubility because N-acetyl groups, randomly distributed along the chitosan backbone, are known to effectively disturb the formation of the crystalline domain and the hydrogen bond among the amino groups of chitosan.26 This assumption was demonstrated from the solubility of N-acetylated SBCs; for example, three derivatives synthesized were soluble in the whole region of pH. The soluble nature of SBC-5-48 at neutral pH was reasonable because 50% N-acetylated chitosan is known to be soluble at neutral pH. It has been, however, reported that 50% N-acetylated chitosan is not directly soluble in distilled water but shows the solubility at neutral pH adjusted by the addition of alkaline solution after being dissolved in acidic solution,34 indicating the importance of the hydration by acidic solution. Also, a high molecular weight of 50% N-acetylated chitosan, larger than 78k, exhibits the precipitation at high pH (> ∼7.5) because of the strong intermolecular interactions and low pKa (6.5) of D-glucosamine, though tested after being dissolved in acidic solution.6 Interestingly, despite its high molecular weight (ca. 120k), SBC-5-48 was directly soluble in distilled water and there was no significant decrease in the solubility over the whole range of pH. This result might be attributed to the improved hydrophilicity because of the presence of the gluconyl group, in addition to the role of the N-acetyl group. The significance of the gluconyl group was further observed from the solubility of SBC-5-29 and SBC-5-63 in a broad range of pH, although both of them were not directly soluble in distilled water. In the case of SBC-5-29, the remaining D-glucosamine residues are as high as 66 per 100 sugar units of the chitosan backbone, which may allow further modification to give a specific functionality in biological (pH 7.4) or basic solutions. Enzymatic Susceptibility of SBCs. Chitosan is considered as a biodegradable polymer because of its susceptibility to various enzymes such as chitinase, chitosanase, lysozyme, β-glucosidase, and proteases.35,36 Of various enzymes, lysozyme has been widely used to estimate in vitro degradation behavior of chitosan because lysozyme is known to be ubiquitous in the body and to produce the results correlated with in vivo degradation.13,37 Thus, in this study, chitosan and its derivatives were incubated with lysozyme-containing acetate buffer (pH 5) at 37 °C, and the decrease in the viscosity of the polymer solution, caused by the enzymatic degradation, was investigated as a function of time (Figure 4). No significant decrease in the viscosity was observed in the chitosan solution within 90 min, whereas the solutions of other derivatives exhibited the rapid decrease in the viscosity just after 10 min because of the hydrolysis in the presence of lysozyme. The preservation of the viscosity of

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References and Notes

Figure 4. Enzymatic degradation of the chitosan derivatives: (b) chitosan; (9) SBC-3-5; (2) SBC-5-5; (O) SBC-5-29; (0) SBC-5-48; (4) SBC-5-63. Each polymer solution (0.2 w/v% in 0.1 M acetate buffer) was exposed to lysozyme (18 µg/mL).

the chitosan solution can be attributed to the fact that chitosan (DA ) 5%) has a small amount of N-acetylglucosamine residues that are susceptible to lysozyme. It was of interest that the SBCs with a higher DS showed a higher degradation rate, indicating that the gluconyl moieties in chitosan enhanced the susceptibility to lysozyme; for example, after 90 min, the viscosities of SBC-3-5 and SBC-5-5 solutions decreased to 78 and 72% of initial viscosity, respectively. Lee et al.38 also reported that the N-acylated chitosans with bulky side groups can be degraded readily in the presence of lysozyme. The biodegradability of SBCs was further improved by the N-acetylation, as shown in Figure 4. Also, the rate of degradation was correlated with the DA, which was consistent with the results obtained by other groups.25,39 Overall, it is to be suggested that the degradability of the N-acetylated SBCs, soluble in the broad range of pH, could be controlled by adjusting the DA for specific applications. Conclusion This study explored the aqueous solubility and biodegradability of SBCs with different DS and DA. The solubility of chitosan was improved by the modification with hydrophilic gluconic acid, by which SBCs exhibited a soluble nature in a broader range of pH compared to chitosan. The Nacetylation of SBCs further improved the solubility, thereby dissolving in a whole range of pH. The biodegradability study demonstrated that chitosan modification with gluconic acid enhanced the susceptibility to lysozyme. As a consequence, by simple modification with gluconic acid and acetic anhydride, it was possible to control the aqueous solubility and biodegradability of chitosan, which may provide a potential for various biomedical applications. Acknowledgment. This study was supported by Gant No. 2I19190 from REGEN Biotech, Inc., Korea.

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