Biomacromolecules 2001, 2, 1133-1136
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Biological Activities of Carbohydrate-Branched Chitosan Derivatives Minoru Morimoto,† Hiroyuki Saimoto,† Hikaru Usui,† Yoshiharu Okamoto,‡ Saburo Minami,‡ and Yoshihiro Shigemasa*,† Department of Materials Science, Faculty of Engineering, Tottori University, Koyama, Tottori 680-8552, Japan; and Department of Veterinary Surgery, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan Received March 26, 2001; Revised Manuscript Received July 16, 2001
Two types of biological activities of the carbohydrate-branched chitosan derivatives were investigated. One is the specific interaction with lectins and bacterium. The other is activation of canine polymorphonuclear leukocyte (PMN) cells. The specific bindings of the L-fucose-branched chitosan derivative with Ulex europaeus agglutinin I (UEA-I) and the N-acetyl-D-glucosamine-branched chitosan derivative with Concanavalin A (Con A) were confirmed by a surface plasmon resonance technique. The specific aggregation of the fluorescence-labeled L-fucose-branched chitosan derivative with Pseudomonas aeruginosa was observed by fluorescent microscopic observation. The aggregation would be attributed to the specific binding between the L-fucose-branched chitosan derivative and PA-II receptor on the cell surface of P. aeruginosa. The influence of the chitosan derivatives on the active oxygen species generation from canine PMN cells was also investigated by the luminol-aided chemiluminescence method. The chemiluminescence responses depended on the degree of substitution and water solubility of the chitosan derivatives. The water-insoluble chitosan derivatives would stimulate the PMN cells by a phagocytosis mechanism, and the water-soluble ones would sensitize the PMN cells by a priming mechanism. Introduction Chitosan, a polysaccharide of D-glucosamine (GlcN), has created significant interest in biomedical applications due to its biodegradability, biocompatibility, and bioactivities such as antimicrobial activity and wound-healing acceleration. Biomaterials of chitosan have been used in veterinary practice to activate host defenses in preventing infection and to accelerate the wound healing.1 Chitosan act as stimulants for polymorphonuclear leukocytes (PMN) inducing the migration of PMN cells into the wounds and then inducing active biodebridement by these cells. Canine PMN cells can interact with chitosan particles by opsonization with complements in serum. This interaction accelerates the phagocytic activity of PMN cells.2 During the phagocytosis process, bactericidal toxic reactive oxygen species (•OH, 1O2, O2-, and H2O2) are generated, which sterilize the wound portion.3,4 Chitosan is generally insoluble under physiological conditions because of a strongly hydrogen-bonding network structure. Thus, further investigation for the biological activities and development have been restricted. To improve the solubility property, we have synthesized various chitosan derivatives by chemical modification. The chitosan derivatives modified with poly(ethylene glycol) (PEG) were soluble in neutral water and interacted with acrylic polymer having carboxy groups, and gave a more stable emulsion.5,6 The N-carboxymethylated, N,O-sulfated, and N-trimethylated * Corresponding author:
[email protected]. † Faculty of Engineering, Tottori University. ‡ Faculty of Agriculture, Tottori University.
chitosan derivatives showed reasonable low electric resistance.7 Recently, we have synthesized carbohydrate-branched chitosan derivatives to get water solubility and novel biological activates.8,9 A carbohydrate is one constituent of organisms and is a key molecule in an intercellular recognition and adhesion. In the present study, we have focused on the specific interaction with lectins or bacterium and the activation of canine polymorphonuclear leukocyte (PMN) cells as biological activities of the carbohydrate-branched chitosan derivatives. Experimental Section Materials. Completely deacetylated chitosan (DAC-100) with 100% degree of deacetylation (DDA) was prepared by repeated deacetylation10 of commercial chitosan (Chitosan 10B) purchased from Funakoshi Co., Ltd. Partially deacetylated chitosan (DAC-85) with 85% of DDA (Flonac C) was purchased from Kyowa Technos. The DDA values were confirmed by the analysis of 1H NMR and IR spectra.11 The number-averaged molecular weight of DAC-100 and DAC85 determined by the gel permeation chromatography (GPC) method on the basis of pullulan standard were 83 000 and 40 000, respectively. Ulex europaeus agglutinin I (UEA I) and Concanavalin A (ConA, Type IV) were purchased from Seikagaku Kogyo Co., Ltd. and Sigma Chemical Co., respectively. Zymosan A and luminol were purchased from Sigma Chemical Co. Ltd. and Wako Pure Chemical Industries Co. Ltd., respectively. Unless otherwise noted, the other chemicals were of reagent grade quality and used without further purification.
10.1021/bm010063p CCC: $20.00 © 2001 American Chemical Society Published on Web 09/22/2001
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Figure 1. Chemical structures of the carbohydrate-branched chitosan derivatives.
Carbohydrate-Branched Chitosan Derivative. The carbohydrate-branched chitosan derivatives with various DS values were synthesized according to the method of Holme and Hall.12 Carbohydrates used in this study were L-fucose (L-Fuc), D-fucose (D-Fuc), N-acetyl-D-glucosamine (DGlcNAc), D-mannose (D-Man), and β-D-lactose (D-Lac). Poly(ethylene glycol) (PEG, Mn 2000) was also used as a side chain. The chemical structure of these chitosan derivatives is shown in Figure 1. All of the chitosan derivatives were characterized by 1H- and 13C NMR. The degree of substitution (DS) of the chitosan derivatives was determined by 1H NMR. Fluorescence-Labeled Chitosan Derivative. A 40 mg sample of L-Fuc- or D-Fuc-branched chitosan derivative was dissolved in 12 mL of distilled water. To the solution was added 1 mL of a suspension of fluorescein isothiocyanate (FITC) in distilled water (1.2 mg/mL). The mixture was stirred at room temperature for 48 h in the dark, dialyzed for 6 days, filtered, and lyophilized to give the fluorescencelabeled chitosan derivatives. The yields were 39 and 35 mg for the L-Fuc- and D-Fuc-branched chitosan derivatives, respectively. Their purities were checked by the GPC method with a fluorescence detector. SPR Analysis. The specific bindings of the carbohydratebranched chitosan derivatives with lectins were analyzed by a BIAcore 2000 biosensor based on surface plasmon resonance (SPR). The chitosan derivative was diluted to 33 µg/mL in 10 mmol/L acetate buffer (pH 5) and then injected over a sensor chip (CM5, research grade) at a flow rate of 5 µL/min for 7 min. The sensor chip was washed out with the running buffer (10 mmol/L HEPES, 150 mmol/L NaCl, 0.005% Tween 20, pH 7.4). A solution of UEA-I or Con A in the running buffer at various concentrations (92-1060 µmol/L) was injected over the immobilized sensor chip at a flow rate of 2 µL/min. The binding constants of the ligand were calculated by the standard BIAcore software. Microscopic Observation. Pseudomonas aeruginosa B17 (P. aeruginosa), a standard laboratory strain, was stored in our laboratory. Five loopfuls of the pre-cultured P. aeruginosa (37 °C, 24 h) on an agar plate (Nissui Pharmaceutical Co., Ltd.) was suspended in 0.5 mL of physiological saline. To this suspension, 1 mL of a solution of the fluorescencelabeled L-Fuc- and D-Fuc-branched chitosan derivatives in a physiological saline (5 mg/mL) was added. After incubation of the mixture at 37 °C for 4 h in the dark, the P. aeruginosa
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cells were collected by centrifugation at 3000 rpm for 15 min. The collected cells were resuspended in 0.1 mL of a physiological saline. The suspension was dropped on a slide glass and a cover glass was set on it. Microscopic image of the suspensions was observed under a fluorescence microscope. CL Analysis. The relative concentration of active oxygen species generated from canine PMN was estimated from a luminol-aided chemiluminescence (CL) method.13 The CL response was measured with a Luminat LB 9510 (Berthold Co., Ltd). (a) In the Absence of Zymosan. Heparinized peripheral blood (100 µL) of a healthy adult dog was diluted with Hanks’ balanced salt solution (HBSS, 400 µL) in a cuvette. After incubation at 37 °C for 15 min, 20 µL of aqueous luminol solution (2 mg/mL) was added to the blood suspension. After measurement of baseline for 1 min, 50 µL of sample solution in HBSS (1 mg/mL) was added to the blood suspension. (b) In the Presence of Zymosan. To the canine blood (100 µL) diluted with HBSS (350 µL) was added 50 µL of sample solution in HBSS (1 mg/mL). After incubation at 37 °C for 15 min, 20 µL of aqueous luminol solution (2 mg/ mL) was added to the blood suspension. After measurement of baseline for 1 min, 50 µL of zymosan solution in HBSS (1 mg/mL) was added to the blood suspension. (c) CL Measurement. The CL response for the chitosan derivatives was continuously measured for 25 min. Zymosan was used as the reference material. The CL intensity for zymosan was defined as 100%. The peak CL count and relative CL intensity for the samples were calculated with the following equation: relative CL intensity (%) )
CL intensity for sample × 100 CL intensity for zymosan
Results and Discussion Specific Binding with Lectin. UEA-I lectin binds specifically to L-fucose and Con A lectin binds to D-mannose and D-glucose type carbohydrates including N-acetyl-D-glucosamine. The specific binding of the carbohydrate-branched chitosan derivatives and lectins was investigated by a SPR technique. The sensorgrams for the binding of the chitosan derivatives (L-Fuc-chitosan, DS ) 0.3, and D-GlcNAcchitosan, DS ) 0.4) and lectins (UEA-I and Con A) are shown in Figure 2. As expected, significant bindings of UEA-I to L-Fuc-chitosan and Con A to D-GlcNAc-chitosan were observed. Maximum values of the resonance unit (RU) were approximately 2900 for L-Fuc-chitosan (solid line) with UEA-I and 3300 for D-GlcNAc-chitosan (dotted line) with Con A. The apparent binding constants for the interactions were 1.2 × 108 and 0.53 × 108 L/mol, respectively. No interaction between UEA-I and D-GlcNAc-chitosan was observed. Small response on the sensorgram for the interaction between Con A and L-Fuc-chitosan was detected. This response would be attributed to the binding of Con A to glucosamine moiety at the nonreducing terminal end of chitosan main chain. No significant interaction was observed on the sensorgram for D-Fuc-chitosan (DS ) 0.3) with both
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Carbohydrate-Branched Chitosan Derivatives
Table 1. Water Solubility and Chemiluminescence Response for the Carbohydrate-Branched Chitosan Derivatives
Figure 2. Sensorgrams for the interactions of the carbohydratebranched chitosan derivatives with UEA-I and Con A lectins. Immobilized ligand: L-Fuc-chitosan (solid line) or D-GlcNAc-chitosan (dotted line) on CM5 sensor chips. Injected analyte: UEA-I (left) or Con A (right) in 10 mM HEPES buffer (pH 7.4).
sample
DS
water solubilitya
DAC-100 DAC-85 PEG D-Lac D-Lac PEG D-Man D-Man PEG D-Man D-Lac D-Lac D-Man
0 0.15 0.05 0.1 0.2 0.2 0.3 0.4 0.4 0.5 0.5 0.7 0.8
+ + + + + + + + +
relative CL intensityb (%) without zymosan
with zymosan
41.6 ( 12.0 26.2 ( 5.0 13.0 ( 2.0 16.9 ( 6.5 7.0 ( 3.5 5.0 ( 2.8 4.3 ( 2.1 7.5 ( 3.5 4.4 ( 2.1 0.7 ( 0.7 2.3 ( 1.0 1.0 ( 0.7 0.6 ( 0.3
98 ( 8 100 ( 15 101 ( 10 101 ( 5 190 ( 18 178 ( 15 139 ( 11 114 ( 5 146 ( 21 98 ( 16 124 ( 14 106 ( 7 92 ( 7
a Key: (-) insoluble; (+) soluble at pH 7. b The CL measurement was repeated six times. The relative intensity shows mean value ( standard deviation.
Figure 3. Fluorescence microscopic images of P. aeruginosa preincubated with the fluorescence labeled L-Fuc-branched chitosan derivative (left) and D-Fuc-branched chitosan derivative (right).
lectins (data not shown). These results clearly indicate that the carbohydrate-branched chitosan derivative can bind to its corresponding lectins specifically. Specific Binding with P. aeruginosa. Specific binding of the carbohydrate-branched chitosan derivative to P. aeruginosa was investigated by fluorescence microscopic observation. P. aeruginosa is known to have a L-fucose specific receptor protein (PA-II) on the cell surface.14 Thus, L-Fuc-chitosan could bind with P. aeruginosa specifically. A location of the fluorescence-labeled chitosan derivative was visualized as a fluorescence image. Some fluorescent areas were observed in the fluorescence microscopic image for the interaction of L-Fuc-chitosan with P. aeruginosa (Figure 3, left). These fluorescent areas agreed with the location of aggregated P. aeruginosa cells. On the other hand, little fluorescent area was observed in the florescence microscopic image for D-Fuc-chitosan (Figure 3, right), whereas many P. aeruginosa cells were observed in the same magnitude of normal microscopic image. The aggregation of P. aeruginosa cells suggested L-Fuc-chitosan bound specifically to P. aeruginosa and acted as a somatic agglutinin. This specific binding would be mediated by the interaction of the L-Fuc-chitosan with PA-II receptor on the cell surface of P. aeruginosa. PMN Activation. In response to an appropriate stimulation such as phagocytosis of the infective bacteria, PMN generate active oxygen species. The active oxygen is involved in the killing of phagocytosed microorganisms. Concentration of the generated active oxygen can be estimated by the CL
Figure 4. Chemiluminescence responses of PMN with various chitosan derivatives in the presence (upper) and absence (lower) of zymosan. Branched molecules of the chitosan derivatives are D-Man (open circle), D-Lac (closed circle), PEG (closed square), and acetyl group (open triangle). Chitosan derivatives in the hatched area are insoluble in neutral water.
method based on a CL of luminol excited by the active oxygen. Zymosan, a complex of proteoglycans obtained from cell membrane of Saccharomyces cereVisiae, is phagocytosed by PMN and used as a positive control for the PMN activation. Chitosan induced active oxygen generation from canine PMN with 50% CL intensity of that induced by zymosan.2 Results of water-solubility and CL response for the carbohydrate-branched chitosan derivatives are summarized in Table 1. The water-solubility of the carbohydrate-branched chitosan derivatives depended on the DS values. The chitosan derivatives with less than 0.15 of DS were insoluble (hatched area in Figure 4) and those with more than 0.2 were soluble in neutral water (HBSS, pH 7). The solubility of the watersoluble ones was over 10 mg/mL. The water solubility of the chitosan derivatives remarkably affected on the CL response. For the CL response in the absence of zymosan, the relative CL intensities for the waterinsoluble chitosan derivatives (DS < 0.2) were about 1342%, whereas those for the water-soluble ones (DS ) 0.20.8) were a few percent (lower section in Figure 4). These results suggested that the water-insoluble chitosan derivatives
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would be phagocytosed, but the water-soluble ones were not done. For the CL response in the presence of zymosan, the relative CL intensity of the water-soluble chitosan derivatives were markedly enhanced and the values were varied from 190% to 92%, while no influence on the CL response for zymosan was observed and those were the same as that of zymosan only (upper section in Figure 4). The active oxygen generation by the water-soluble chitosan derivatives decreased with the increase of DS by carbohydrate onto chitosan and the decrease of the amino group amount. In this assay, no significant effect of branched carbohydrate was detected. This result suggested that the water-soluble chitosan derivatives sensitized the PMN cells membrane and activated phagocytosis of PMN cells for zymosan by a priming effect15 and that the number of amino groups of glucosamine residues of the chitosan derivatives would be important for the priming effect of the water-soluble chitosan derivatives. We previously reported that glucosamine oligomers induced enhancement of CL response of canine PMN cells to zymosan by the priming effect, but N-acetylglucosamine oligomers did not change the CL response.16 In the present study, it is observed that the amount of amino groups in chitosan main chain of the water-soluble chitosan derivatives might play a significant role on the generation of reactive oxygen species by PMN cells. Conclusion The carbohydrate-branched chitosan derivatives bound to its corresponding lectins specifically. P. aeruginosa was aggregated by L-Fuc-branched chitosan derivative with 0.3 of DS through the specific binding of the L-Fuc residues of the chitosan derivatives to PA-II receptor protein on the cell surface.
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The water-solubility of the carbohydrate-branched chitosan derivatives depended on the DS values. The water-insoluble carbohydrate-branched chitosan derivatives (DS < 0.2) activated canine PMN cells by a phagocytosis mechanism, and the water-soluble ones (DS > 0.2) would sensitize the PMN cells by a priming mechanism. References and Notes (1) Shigemasa, Y.; Minami, S. Biotechnol. Genetic Eng. ReV. 1995, 13, 383-420. (2) Minami, S.; Okamoto, Y.; Tanioka, S. I.; Sashiwa, H.; Saimoto, H.; Matsuhashi, A.; Shigemasa, Y. In Carbohydrates and Carbohydrate Polymers; Yalpani, M., Ed.; ATL Press: Mount Prospect, IL, 1993; pp 141-152. (3) Allen, R. C.; Stjernholm, R. L.; Steele, R. H. Biochem. Biophys. Res. Commun. 1972, 47, 679-684. (4) Babior, B. M. Blood 1984, 64, 959-966. (5) Sugimoto, M.; Morimoto, M.; Sashiwa, H.; Saimoto, H.; Shigemasa, Y. Carbohydr. Polym. 1998, 36, 49-59. (6) Sugimoto, M.; Shigemasa, Y. Chitin Chitosan Res. 1998, 4 (2), 8596. (7) Suzuki, K.; Saimoto, H.; Shigemasa, Y. Carbohydr. Polym. 1999, 39, 145-150. (8) Li, X.; Morimoto, M.; Sashiwa, H.; Saimoto, H.; Okamoto, Y.; Minami, S.; Shigemasa, Y. Polym. AdV. Technol. 1999, 10, 455458. (9) Li, X.; Tushima, Y.; Morimoto, M.; Saimoto, H.; Okamoto, Y.; Minami, S.; Shigemasa, Y. Polym. AdV. Technol. 2000, 11, 176179. (10) Horton, D.; Lineback, D. R. In Methods in Carbohydrate Chemistry; Whistler, R. L., Ed.; Academic Press: New York, 1965; Vol. 5, pp 403-406. (11) Shigemasa, Y.; Matsuura, H.; Sashiwa, H.; Saimoto, H. Int. J. Biol. Macromol. 1996, 18, 237-242. (12) Holme, K.; Hall, L. Carbohydrate Res. 1992, 225, 291-306. (13) Makimura, S.; Sawaki, M. J. Vet. Med. Sci. 1992, 54 (1), 63-67. (14) Garber, N.; Guempel, U.; Giboa-Garber, N.; Doyel, R. J. FEMS Microbiol. Lett. 1987, 48, 331-336. (15) Pabst, M. J.; Johnson, R. B., Jr. J. Exp. Med. 1980, 151, 101-114. (16) Usami, Y.; Okamoto, Y.; Takayama, T.; Shigemasa, Y.; Minami, S. Carbohydr. Polym. 1998, 36, 137-141.
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