Biomacromolecules 2003, 4, 1691-1697
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Characterization and Blood Coagulation Evaluation of the Water-Soluble Chitooligosaccharides Prepared by a Facile Fractionation Method Chia-Wen Lin and Jui-Che Lin* Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70101 Received April 28, 2003; Revised Manuscript Received August 4, 2003
Water-soluble chitooligosaccharides have been reported to have specific biological activities. In this study, the chitosan samples with different degree of acetylation were used separately to prepare chitooligosaccharide (COS) and highly deacetylated chitooligosaccharide (HDCOS) through the nitrous acid depolymerization. Rather than using the conventional fractionation schemes commonly employed, such as dialysis and ultrafiltration which require a large amount of deionized water as well as a fair long dwell time, an unique fractionation scheme is explored to recover and desalt these nitrous-acid depolymerized chitosan with different molecular weights. This fractionation scheme is based on the differential solubility variation of depolymerized products within the aqueous solutions that contain various ratios of methanol. It was noted that chitosan with different molecular weight can be successfully recovered and fractionated with methanol added sequentially up to a volume of four times of original depolmerized product. In addition, chemical characterization of the fractionated water-soluble COS and HDCOS by 1H NMR spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) indicated that the chitosan depolymerization reaction is greatly influenced by the degree of acetylation of the parental chitosan reactant. Moreover, the modified whole blood clotting time assay and the platelet coagulation test suggested that the 1:2 fractionated water-soluble COS and HDCOS obtained are much less procoagulant than their parental chitosan compound and can be of use in biomedical applications in which blood coagulation is not desired. Introduction Chitosan, a linear binary copolymer of (1f4)-linked 2-acetamido-2-deoxy-β-D-glucopyranose (GlcNAc unit) and 2-amino-2-deoxy-β-D-glucopyranose (GlcNH2 unit), is chemically prepared by N-deacetylation of naturally occurring chitin. Chitin is the second most abundant polysaccharide in nature; it is widely found in the exoskeletons of crustacea and insects as well as in the cell walls of most fungi and some algae.1,2 Chitin and chitosan are known to be biodegradable and biocompatible to human and most animals. They have been proposed for a wide range of applications, e.g., water clarification, cosmetic, biomedical, and pharmaceutical uses.3 Chitin has been shown to be insoluble in water, dilute acids, cold alkalis of any concentration, and most organic solvents while be soluble in strong mineral acids, formic acids, di- and trichloroacetic acids, methanesulfonic acid, and a few lithium chloride/amide systems, e.g., LiCl/ DMAc.4 Its deacetylated form, chitosan, can be soluble in dilute aqueous solutions of many organic acids, but inherently water-insoluble. In some applications, however, it is desirable to render chitosan water soluble and/or to change its molecular character by specific methods, e.g., chemical modification, salt formation with acid, and changing polymer morphology and molecular weight.5,6 However, the cytotox* To whom correspondence should be addressed. E-mail address:
[email protected]. Phone: 886-6-275-7575 ext 62665. Fax: 886-6234-4496.
icity evaluation and haemolytic properties characterization had shown that some kinds of chitosan derivatives and chitosans of high molecular weight and specific salt form are toxic.7 Recently, several studies on chitin and chitosan have been attracted to convert them to oligosaccharides in view of their distinctive chemical and biological properties. Chitooligosaccharides have low viscosity and low molecular weight and are soluble in neutral aqueous solutions. They exhibit various interesting biological activities, including antitumor activities, immuno-enhancing effects, increasing protective effect against pathogen infection, antimicrobial activities, proliferation inhibition of a human promyeloytic leukemia cell line HL60, and DNA complexation capability.8-12 The use of such oligosaccharides would seem advantageous because they are safer than polysaccharides as they lack antigenicity and are less prone to accumulation in the host animal, which have made them quite attractive as a special biomaterial. Chitosan can be chemically depolymerized by different mechanisms, mainly the acid hydrolysis, oxidative-reductive, and nitrous acid depolymerization. In general, because chitosan is quite resistant to hydrolysis by acids,13 a large amount of reagents under drastic conditions would have to be used for producing a series of low-molecular-weight oligomers. It would make the subsequent products recovery from such a strongly acid solution a difficult and costly procedure. Depolymerization of chitosan by the use of nitrous acid is an easy method. It is a homogeneous reaction where
10.1021/bm034129n CCC: $25.00 © 2003 American Chemical Society Published on Web 09/05/2003
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the number of glycosidic bonds broken is roughly stoichiometric to the amount of nitrous acid used.14 The mechanism has been found to be specific in the sense that nitrous acid (HNO2) attacks the amino group of GlcNH2 units, with subsequent cleavage of the following glycosidic linkage and a formation of 2,5-anhydro-D-mannose (M-unit) at the new reducing end. Because the M-residue is unstable, it could be reduced to 2,5-anhydro-D-mannitol with NaBH4. However, it has some advantages over a normal reducing end. The aldehyde group of 2,5-anhydro-D-mannose could be available for reactions that could make the obtained chitooligosaccharides an interesting precursor in organic synthesis (e.g., glycodendrimers which have potential biomedical applications as antitumor and antiinfective compounds).15,16 In this paper, a simple separation and fractionation method was pursued to recover the water-soluble chitooligosaccharides from the reaction solutions prepared by the nitrous acid degradation of chitosan. The chitosan samples of different degree of acetylation, %DA ) 18 and %DA < 4, were used separately to form chitooligosaccharides (COS) and highly deacetylated chitooligosaccharides (HDCOS). The feasibility for product fractionation utilizing the solubility variation among the different nitrous-acid-degraded products within the mixtures of various volumetric ratios of methanol and reaction solution was also evaluated. This fractionation approach is different from the methods of dialysis and ultrafiltration that would use a series of membranes with different molecular weight cutoffs (MWCO) to collect and/ or desalt the water-soluble chitooligosaccharides. In addition, these two conventional fractionation treatments would require a large amount of deionized water and lengthy separation time. Therefore, a large-scale production of chitooligosaccharides based on this relatively simple separation and fractionation process proposed here is very likely to be economically viable and technologically acceptable. The water-soluble portion of the fractionated products was characterized by 1H NMR spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and gel permeation chromatography (GPC). The preliminary blood compatibility of the water-soluble chitooligosaccharides was also determined. Materials and Method Deacetylation of Chitosan. Chitosan (Koyo Chemical Co. Ltd., Tokyo, Japan) (ca. 30 g) was first purified by a successive extraction with 500 mL of acetone and ethanol in a Soxhlet extractor, then washed with 250 mL of diethyl ether, and finally dried under vacuum at 50 °C. A suspension of 10 g of purified chitosan in 100 mL of 50%(w/w) aqueous sodium hydroxide was heated at 110 °C for 10 h. After filtration, the solid was washed with deionized water until the pH of the filtrate was neutral and then dried. The obtained sample was further treated with alkaline solution under the same conditions as above. The resulted highly deacetylated chitosan (HD-chitosan) was collected with filtration, washed with water until neutral and then with acetone, and dried finally.17 The original chitosan and HD-chitosan were then characterized by 1H NMR (AMX-400, Bruker) to determine the degree of acetylation.
Lin and Lin
Depolymerization of Chitosan/HD-Chitosan with NaNO2. A solution was prepared by adding 6 g of chitosan/HDchitosan to 200 mL of a 2%(v/v) aqueous acetic acid solution. Then sodium nitrite (NaNO2, FW ) 69), corresponding to 1/2 mol equivalent to the amino groups (-NH2) on the GlcNH2 units, was slowly added into the solution. The mixture was vigorously stirred at room temperature for 3 h and then adjusted to pH ) 7.0∼7.05 with 9 N NaOH(aq.).15,16,18 Excess water was evaporated under vacuum with an Eyela rotary evaporator (Tokyo Rikakikai Co., Ltd.) at 50 °C. The final concentrated solution was about 20 mL. Fractionation. The methanol, with a volume equal to the concentrated depolymerized chitosan or HD-chitosan prepared above, was gradually poured into the depolymerized solution with mixing. The precipitate, known as 1:1 (volumetric ratio of reacted solution to the methanol added) fraction, was then collected by filtration. Following that, additional methanol, with a volume same as the one used in the first precipitation, was added into the filtrate prepared above to form the 1:2 fraction. The same treatment scheme was repeated to prepare the 1:3 and 1:4 fractions. These precipitates, prepared by adding methanol sequentially, were washed with methanol for several times. After drying, these precipitates were added into 10 mL of deionized water separately. The fractionated products that can be dissolved in water were finally collected with lyophilization for 1 day after filtration to isolate the dissolved molecules. Sample Characteristics. The intrinsic viscosity [η] of the chitosan and HD-chitosan was measured in 0.2 M acetic acid/ 0.1 M sodium acetate by using a Ubbelohde capillary viscometer (Ø ) 0.58 mm) at 30 ( 0.1 °C. The viscosityaverage molecular weights of chitosan and HD-chitosan were then calculated by using the Mark-Houwink equation. The molecular weight distribution of the water-soluble products prepared from the original chitosan and HD-chitosan were analyzed by gel permeation chromatography (GPC) with Shodex standard P-82 series (Pullulan, Showa Denko, Tokyo, Japan) and PEG standards (MW ) 4660 and 1500 from Aldrich, MW ) 400 from TCI, Japan) on a VISCOTEK T60A apparatus, equipped with a Waters 410 differential refractometer. Two columns (Waters Ultrahydrogel 120 and 250 Milford, MA) were connected in series at the operational temperature of 30 °C. The eluate used was deionized water and the flow rate was set at 0.7 mL‚min-1. The water-soluble chitooligosaccharides were characterized by the diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) using a BioRad Model FTS40 FTIR spectrometer (Cambridge, MA) and 1H NMR (AMX-400, Bruker). The conditions for the acquisition of 1H NMR spectra were as follows: 400.13 MHz; 27 °C; size of spectral window, 8930 Hz; acquisition time, 2 s; actual pulse repetition time, 3 s; number of scans, 80 and a 30° excitation pulse-angle was used; data size, 32 K. Whole Blood Clotting Time. Whole blood clotting time (WBCT) assay was carried out as described by Hougie19 with modification to determine the preliminary blood compatibility of the water-soluble chitooligosaccharides. Venous blood was collected from a human donor who was not on any medication. A venipuncture was performed, and a stopwatch
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was started as soon as the blood entered the plastic syringe with no anticoagulant. A total of 1 mL of blood was delivered immediately to each glass tube (10×100 mm) containing 100 µL of test specimen that was dissolved in phosphate buffer solution (PBS, pH ) 7.4) at the concentration of 10 wt %. PBS of the same volume (100 µL) was used as the control. The tubes were placed steady in a water bath at 37 °C. After 4 min, each tube was gently tilted every 30 s at the angle of about 45° until a solid clot was formed and the coagulation time was recorded. The differences in clotting time among the different water-soluble chitooligosaccharides and PBS control were examined by the two-tailed t test. Platelet Aggregation Test. Whole blood was drawn by veinpuncture from normal, healthy adult volunteers who had not taken aspirin within 8 days prior to the test and collected into a plastic tube containing buffered sodium citrate (1:9). Platelet-rich plasma (PRP) was obtained by centrifugation of the anticoagulated blood at 100× g for 15 min at room temperature. The remaining blood specimen was recentrifuged at 2000× g for 15 min at room temperature, and platelet-poor plasma (PPP) was collected from the supernatant. A total of 50 µL of test specimen that was dissolved in Tris-buffered saline (0.05 M Tris buffer and 2.20 g/L sodium chloride, pH 7.4) at the concentration of 10 wt % was added into the plastic cuvette containing 450 µL of PRP with stirring. Adenosine diphosphate (ADP), 20 µM, of the same volume (50 µL) was used as the positive control. The platelet coagulation was measured and recorded by using a turbidimetric aggregation monitoring device (Helena PACKS-4).
Table 1. Fractionation of Depolymerized Chitosan and HD-Chitosan (10 g)
chitosan
HD-chitosan
fractiona
yield (g)
1:1 1:2 1:3 1:4 1:1 1:2 1:3 1:4
1.21 3.13 0.48 0.44 0 3.30 0.90 0.32
wt.% of water-soluble productb 0 63.9 (2.00 g) 100 100 76.4 (2.52 g) 100 100
a The volume ratio of original depolymerized products to the total amount of methanol added. b The weight percentage of the water-soluble products after dissolving the collected fraction of methanol precipitate in 10 mL of deionized water.
Figure 1. GPC chromatograms for water-soluble fractions of chitooligosaccharides (COS).
Results and Discussion Fractionation. The viscosity-average molecular weights, which were calculated by using the Mark-Houwink equation parameters of a ) 0.96 and 0.81 and K ) 1.424 × 10-3 (mL g-1), 16.800 × 10-3 (mL g-1),20 of the original chitosan and HD-chitosan used in this study were 6.3 × 105 and 2.2 × 105, respectively. The degree of acetylation (%DA) determined by 1H NMR was 18% for chitosan and 0.05). Platelet aggregation test of the water-soluble chitooligosaccharides was also performed in our study. The principle of this test is that PRP is a turbid suspension that at a given wavelength transmits a certain amount of light relative to
PPP blank. When the platelet aggregation was induced by adding the test specimen into PRP, a change of light transmittance could be rapidly detected because of the change in shape (the platelets change from disks to spheres) and formation of clumps. To take the different extent of aggregation in comparison, the percentage of platelet aggregation was defined and calculated as shown in eq 1, and PRP and PPP were set as 0% and 100% aggregation, respectively. The data from the aggregation test, shown in Figure 5, indicated that both water-soluble 1:2 fractions of COS and HDCOS have no significant effect on the platelet aggregation as compared with ADP (platelet aggregation ) 89.5% after 550 s) % aggregation ) (O.D.Initial - O.D.t)/(O.D.Initial) × 100% (1) where O.D.t ) optical density of the specimen mixed with PRP at time ) t seconds O.D.initial) optical density of the specimen mixed with PRP at time ) 0 s. In these two studies, these low-molecular-weight, watersoluble chitooligosaccharides showed no hemostatic effect, on the other hand, they might possess a marginal anticoagulation property when exposed to whole blood (Table 3). These blood-contacting properties are quite different from those of their parental polysaccharide, chitosan. Malette et al. have reported that chitosan solution formed a coagulum in contact with whole blood.24 Rao et al. found that the coagulum also formed as whole blood contact with the chitosan film, and the platelets showed distinct adhesion to the polymer surface within a short time.25 They concluded that the hemostatic property was attributed to the polycationic property of chitosan and its nonspecific binding to cell membranes.
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Figure 5. Platelet aggregation percentage of 10 wt % water-soluble 1:2 fraction COS in Tris-buffered saline (a), 10 wt % water-soluble 1:2 fraction HDCOS in Tris-buffered saline (b), and 20 µM of ADP in Tris-buffered saline (c), which was recorded by turbidimetric aggregation monitoring device (Helena PACKS-4) within 600 s.
With the decrease in the molecular chain length by nitrous acid depolymerization through the initial attack of amino group of GlcNH2 as indicated earlier, it would be expected that the density of cationic amino group would be decreased in the low-molecular-weight chitooligosaccharides than the parental chitosans. In addition, the reaction between the amino group of GlcNH2 residue and aldehyde group of the reducing M-unit (as shown in IR analysis) could lead to a decrease of amine functionality in these chitooligosaccharides as well. Therefore, the chitooligosaccharides will exhibit a better blood compatibility than chitosan. This unique property will be of great use for intravenous injection of chitooligosaccharides for potential medications. We are further exploring this unique application. Summary In this study, the nitrous acid depolymerized chitosan and highly deacetylated chitosan with different average molecular weight could be sequentially collected and fractionated by using this methanol fractionation/precipitation method. Four consecutive methanol precipitation operations would be sufficient to recover the most part of methanol-precipitable products. In addition, the DPn of resulting water-soluble depolymerized products and the extent of depolymerization reaction are directly related to the distribution of N-acetyl group (GlcNAc) in the biomacromolecular backbone. Moreover, for the water-soluble 1:2 fractions obtained, the weight percentage of HDCOS with n ) 3∼10, in which high biological activities have been suggested of, was higher than COS. From the preliminary blood compatibility test, these water-soluble chitooligosaccharides exhibited no hemostatic
effect but are marginally more anticoagulative than chitosans. This unique nonhemostatic property may be useful to some chitooilgosaccharide-related biomedical applications in which intravenous injection or extensive blood contact is needed. Acknowledgment. We thank Professor Tsun-Mei Lin and her laboratory members in the Department of Medical Technology of National Cheng Kung University for helping the blood compatibility study. Professor Chuh-Yung Chen in the Department of Chemical Engineering of National Cheng Kung University is acknowledged for generously lending the GPC instrumentation. References and Notes (1) Tolaimate, A.; Desbrie`res, R. M.; Alagui, A.; Vincedon, M.; Vottero, P. On the influence of deacetylation process on the physicochemical characteristics of chitosan from squid chitin. Polymer 2000, 41, 2463-2469. (2) Muzzarelli, R. A. A. In Natural Chelating Polymers, Alginic Acid, Chitin and Chitosan; Belcher, R., Freiser, H., Eds.; Pergamon: Oxford, 1973; pp 83-141. (3) Brugnerotto, J.; Desbrie`res, J.; Roberts, G.; Rinaudo, M. Characterization of chitosan by steric exclusion chromatography. Polymer 2001, 42, 9921-9927. (4) Austin, P. R. In Biomass. Part B, Lignin, pectin, and chitin; Wood, W. A., Kellogg, S. T., Eds.; Academic Press: San Diego, 1988; pp 403-407. (5) Kubota, N.; Eguchi, Y. Facile preparation of water-soluble Nacetylated chitosan and molecular weight dependence of its watersolubility. Polym. J. 1997, 29, 123-127. (6) Felse, T. P. Studies on applications of chitin and its derivatives. Bioprocess Eng. 1999, 20, 505-512. (7) Carren˜o-Go´mez, B.; Duncan, R. Evaluation of the biological properties of soluble chitosan and chitosan microspheres. Int. J. Pharm. 1997, 148, 231-240. (8) Suzuki, K.; Mikami, T.; Okawa, Y.; Tokoro, A.; Suzuki, S.; Suzuki M. Antimutor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohydr. Res. 1986, 151, 403-408.
Evaluation of the Water-Soluble COSs (9) Tokoro, A.; Tatewaki, N.; Suzuki, K.; Mikami, T.; Suzuki, S.; Suzuki, M. Growth-inhibition effect of hexa-N-acetylchitohexaose and chitohexaose against Meth-A solid tumor. Chem. Pharm. Bull. 1988, 36, 784-790. (10) Jeon, Y. J.; Park, P. J.; Kim, S. K. Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydr. Polym. 2001, 44, 71-76. (11) Pae, H. O.; Seo, W. G.; Kim, N. Y.; Oh, G. S.; Kim, G. E.; Kim, Y. H.; Kwak, H. J.; Yun, Y. G.; Jun, C. D.; Chung, H. T. Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer. Leuk. Res. 2001, 25, 339-346. (12) Richardson, S. C. W.; Kolbe, H. V. J.; Duncan, R. Potential of low molecular mass chitosan as a DNA delivery system: Biocompatibility, body distribution and ability to complex and protect DNA. Int. J. Pharm. 1999, 178, 231-243. (13) Barker, S. A.; Foster, A. B.; Stacey, M.; Webber, J. M. Amino-sugars and related compounds. Part IV. Isolation and properties of oligosaccharides obtained by controlled fragmentation of chitin. J. Am. Chem. Soc. 1958, 80, 8-2227. (14) Peniston, Q. P.; Johnson, E. L. Process for depolymerization of chitosan. United States Patent, 3,922,260, 1975. (15) Va˚rum, K. M.; Ottøy, M. H.; Smidsrød, O. Acid hydrolysis of chitosans. Carbohydr. Polym. 2001, 46, 89-98. (16) Tømmeraas, K.; Va˚rum, K. M.; Christensen, B. E.; Smidsrød, O. Preparation and characterization of oligosaccharides produced by nitrous acid depolymerisation of chitosans. Carbohydr. Res. 2001, 333, 137-144.
Biomacromolecules, Vol. 4, No. 6, 2003 1697 (17) Mima, S.; Miya, M.; Iwamoto, R.; Yoshikawa, S. Highly deacetylated chitosan and its properties. J. Appl. Polym. Sci. 1983, 28, 19091917. (18) Baumann, H.; Faust, V. Concepts for improved regioselective placement of O-sulfo, N-sulfo, N-acetyl, and N-carboxymethyl groups in chitosan derivatives. Carbohydr. Res. 2001, 331, 43-57. (19) Hougie, C. In Hematology; Williams, W. J., Beutler, E., Marshall Lichtman, A., Erslev, A. J., Eds.; McGraw-Hill Information Services Co.: New York, 1990; p 1765. (20) Wang, W.; Bo, S.; Li, S.; Qin, W. Determination of the MarkHouwink equation for chitosans with different degrees of deacetylation. Int. J. Biol. Macromol. 1991, 3, 281-285. (21) Signini, R.; Campana Filho, S. P. On the preparation and characterization of chitosan hydrochloride. Polym. Bull. 1999, 42, 159166. (22) Sashiwa, H.; Saimoto, H.; Shigemasa, Y.; Tokura, S. N-Acetyl group distribution in partially deacetylated chitins prepared under homogeneous conditions. Carbohydr. Res. 1993, 242, 167-172. (23) Hirano, S. A facile method for the preparation of novel membranes from N-arylidene-chitosan gels. Agric. Biol. Chem. 1978, 42, 19391940. (24) Malette, W. G.; Quigley, H.; Gaines, R. D.; Johnson, N. D.; Rainer, G. Chitosan: A new hemostatic. Ann. Thoracic. Surg. 1983, 36, 5558. (25) Rao, S. B.; Sharma, C. P. Use of chitosan as a biomaterial: Studies on its safety and hemostatic potential. J. Biomed. Mater. Res. 1997, 34, 21-28.
BM034129N
