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Antioxidant-potentiality of gold–chitosan nanocomposites
Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123 www.elsevier.com/locate/colsurfb
Antioxidant-potentiality of gold chitosan nanocomposites /
Kunio Esumi *, Naoko Takei, Tomokazu Yoshimura Department of Applied Chemistry and Institute of Colloid and Interface Science, Tokyo University of Science, Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan Received 17 April 2003; accepted 23 May 2003
Abstract Gold nanoparticles were prepared in the presence of chitosan via reduction of HAuCl4 with sodium borohydride. The average particle size of gold nanoparticles was significantly affected with the concentration of chitosan added and was ranged between 6 and 16 nm. The gold /chitosan nanocomposites are formed by adsorbing chitosan molecules on the gold nanoparticles. The catalytic activity of gold /chitosan nanocomposites upon elimination of hydroxyl radicals formed in an H2O2/FeSO4 system was examined using a spin-trapping method. The catalytic activity increased with the concentration of chitosan added and showed a maximum and then decreased with a further concentration of chitosan added, although a strict correlation between the average diameter of gold and the catalytic activity was not found. In addition, the activity of gold /chitosan was 80 times higher than that of ascorbic acid, which is well known as an antioxidant. # 2003 Elsevier B.V. All rights reserved. Keywords: Gold /chitosan nanocomposite; Hydroxyl radical; Antioxidant; ESR
1. Introduction Preparation of noble metal nanoparticles by reducing metal salts has been extensively studied [1]. To stabilize dispersion of nanoparticles, it is necessary to use protective agents, such as polymers, surfactants, and chelating agents. These
* Corresponding author. Tel.: /81-332-604-271x2454; fax: /81-332-352-214. E-mail address: [email protected] (K. Esumi).
nanoparticles have widely been studied with a view to improve the quality of catalysts. All living organisms are suffered from the damage caused by the free-radical oxygen species. Free-radical oxygen species damage cells by attacking unsaturated fatty acids in the cell membrane. Fortunately, a protective enzyme, superoxide dismutase, completely converts these free-radical oxygen species into two water molecules and oxygen [2,3]. However, it is known that superoxide dismutase tissue levels decrease with aging. So, it is a challenging task to develop new
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00151-6
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catalysts for the elimination of active radical species. Chitin, poly-B-(1,4)-n -acetyl-D-glucosamine, is a cellulose-like biopolymer widely distributed in nature, especially in marine invertebrates, insects, fungi, and yeasts. Chitin also has unique properties, including toughness, bioactivity, and biodegradability. Recently, chitosan, which is deacetylated chitin, has been attractive [4,5], because the free amino groups in this modified product contribute polycationic, chelating, and film-forming properties, along with ready solubility in dilute acetic acid. In particular, partially deacetylated chitin is water soluble, and is similar in behavior to hydrophilic polymers that adsorb on metal particle surfaces and form complexes. In this study, gold /chitosan nanocomposites were prepared and their catalytic activities were evaluated from elimination of hydroxyl radicals using a spin trapping technique.
2. Experimental
the solutions under stirring and left stirring for 30 min.
2.3. Measurements The gold nanoparticles obtained were analyzed by TEM and UV /vis absorption spectroscopy. TEM observation was performed for the samples dried on carbon-coated copper grids. A Hitachi H900 NAR transmission electron microscope was operated at accelerating voltage of 300 kV and direct magnification of 200,000 /. The size distribution of the gold nanoparticles was determined from about 100 particles. The catalytic activities of gold nanoparticles upon elimination of hydroxyl radicals were estimated by using a spin trapping technique [6]. In a typical experiment, solutions containing hydrogen peroxide, ferric sulfate, DMPO (5,5-dimethyl-1pyrroline-N -oxide), and gold /chitosan nanocomposites were measured with ESR; stable DMPO/ OH adduct can be detected by competitive reaction [7] of OH radicals with DMPO or the gold particles. The measurements were carried out at 1
2.1. Materials Chitosan was obtained from Wako Pure Chemicals Co. Chloroauric acid was obtained from Wako Pure Chemicals Co. Milli-Q water (Millipore Co.) was used in all experiments. The other chemicals were of analytical grade.
2.2. Preparation of gold /chitosan nanocomposites Preparation of gold nanoparticles in aqueous solution was conducted by chemical reduction of HAuCl4 /chitosan mixtures with sodium borohydride. For a typical experiment, 0.2 cm3 of freshly prepared 20 mmol dm 3 HAuCl4 solution was added to 19.7 cm3 of chitosan of various concentrations, and the solutions were stirred for 1 h. Then, 0.1 cm3 of 0.4 mol dm 3 freshly prepared ice-cold sodium borohydride was quickly added to
Fig. 1. UV /vis spectra of HAuCl4 aqueous solutions in the presence of chitosan after addition of sodium borohydride.
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
119
Fig. 2. Transmission electron micrographs and size distribution of gold nanoparticles; [chitosan] /(a) 0.0005; (b) 0.01; (c) 0.05; (d) 0.1; (e) 0.2; (f) 0.5; and (g) 1.0 wt%.
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Fig. 2 (Continued)
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
121
Fig. 2 (Continued)
min after mixing of the solutions. The rate constant (Ks) of the catalytic activity of gold nanoparticles can be evaluated by following equations: F =(1 F ) R [S]=[DMPO]
(1)
R Ks =Kl F [DMPO]=(1 F )[S]
(2)
where R is the degree of reaction, F is the degree of decrease of ESR signal intensity, Kl and Ks are rate constants of DMPO and the gold nanoparticles, and [DMPO] and [S] are the concentrations of DMPO and the gold nanoparticles. Here, the value for Kl by DMPO is taken as 3.4 /109 M 1 s 1 [8].
3. Results and discussion Fig. 1 shows UV spectra of HAuCl4 aqueous solutions in the presence of chitosan after addition of sodium borohydride. All the spectra exhibit an absorption band at around 510 /520 nm which is a typical plasmon band, suggesting the formation of gold nanoparticles [9]. When the concentration of chitosan added is increased, the intensity of the absorption band decreases and then increases again with a further increase of the chitosan concentration. This result indicates that the size of gold nanoparticles formed is altered with the
concentration of chitosan, which operates as controller of nucleation as well as stabilizer. To check the particle size of gold particles obtained TEM measurements were performed. Fig. 2 shows several TEM images of gold particles obtained and their size distributions. It is apparent that the size of gold particles increases with an increase of the chitosan concentration and shows a maximum, and then decreases with a further concentration of chitosan. At 0.0005 wt% of chitosan, very small gold particles are formed. This is that since the interaction site of chitosan and AuCl 4 is very few, the number of nucleation is large, resulting in small gold particles which might be stabilized by adsorption of chloride ions. In the concentration region between 0.0006 and 0.001 wt%, the gold nanoparticles obtained were unstable and sedimented, probably due to a bridging effect by chitosan molecule between gold nanoparticles. When the concentration of chitosan increases from 0.01 to 0.1 wt%, the nucleation and growth rate of gold particles will be slowed due to the interaction between chitosan and AuCl 4 ; resulting in large gold particles. By addition of a further concentration of chitosan, the size of gold particles decreases. This decrement is due to a protective action by chitosan; chitosan may prevent the growth of gold particles by adsorbing their surfaces.
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The catalytic activity upon elimination of hydroxyl radicals with gold /chitosan nanocomposites obtained was investigated using a spin trapping technique. Fig. 3 shows a typical ESR signal change of DMPO/OH adduct in the presence of gold/chitosan nanocomposites with var-
Fig. 3. Change of ESR spectra of DMPO-OH ESR signal with gold nanoparticles; [Au] /(a) 0; (b) 0.00625; (c) 0.0125; (d) 0.025; (e) 0.033 mmol dm 3.
ious concentrations. The intensity of the ESR signal decreases with the concentration of gold / chitosan nanocomposites, indicating that the gold /chitosan nanocomposites have an ability to depress the activity of hydroxyl radicals. It should be mentioned here that the action by chitosan itself is negligible compared to that by the gold / chitosan nanocomposites. Using the equations described in the Section 2, the rate constant (Ks) by the gold/chitosan nanocomposites was obtained. Fig. 4 gives plots of the rate constant/ particle diameter of gold versus the concentration of chitosan added. The rate constant increases with the concentration of chitosan added and attains a maximum, and then decreases with a further increase of the chitosan concentration. Although the change in the rate constant with the concentration of chitosan added is not strictly correlated with that in the particle size of gold, the mechanism for elimination activity of hydroxyl radicals by the gold/chitosan nanocomposites may be proposed as follows. At a very low concentration of chitosan, the rate constant is very small. This is a possibility that chloride ions adsorbing on the gold particles may inhibit the activity for the elimination of hydroxyl radicals. With an increase of chitosan concentration added, the nucleation and growth of gold particles are prevented and large particles are obtained whose surfaces are covered by chitosan to some extent. The gold/chitosan nanocomposites obtained between 0.01 and 0.1 wt% of chitosan show high rate constant. However, with a further concentration of chitosan, the gold particles are covered fully with chitosan molecules so that the elimination effect would be lowered by the chitosan molecules on the gold particles. In addition, it is interesting to compare the rate constant by the gold /chitosan nanocomposites with that by ascorbic acid which is known as an antioxidant agent; since the rate constant by ascorbic acids is obtained as 1.98 / 1011 M 1 s 1 the rate constant at the maximum in this study is about 80 times that by ascorbic acid. Thus, it is found that the gold /chitosan nanocomposites have a high activity upon the elimination of hydroxyl radicals.
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
123
Fig. 4. Plots of rate constant of catalytic activity and diameter of gold nanoparticles with the concentration of chitosan added.
References [1] G. Schmid (Ed.), Clusters and Colloids from Theory to Applications, Weinheim, 1994. Publisher VCH. [2] D. Kristensen, L.H. Skibsted, J. Agric. Food Chem. 47 (1999) 3099. [3] H. Taniguchi, K.P. Madden, J. Am. Chem. Soc. 121 (1999) 11 875. [4] J.P. Zikakis (Ed.), Chitin, Chitosan, and Related Enzymes, Academic Press, London, 1984.
[5] R.A.A. Muzzarelli, C. Jeuniaux, G.W. Gooday (Eds.), Chitin in Nature and Technology, Plenum Press, New York, 1985. [6] E. Finkelstein, G.M. Rosen, E.J. Rauckman, Mol. Pharmacol. 16 (1979) 676. [7] G.M. Rosen, B.A. Freeman, Proc. Natl. Acad. Sci. USA 81 (1984) 7269. [8] E. Finkelstein, G.M. Rosen, E.J. Rauckman, J. Am. Chem. Soc. 102 (1980) 4994. [9] N. Ishizuki, K. Torigoe, K. Esumi, K. Meguro, Colloids Surf. 55 (1991) 15.
Antioxidant-potentiality of gold chitosan nanocomposites /
Kunio Esumi *, Naoko Takei, Tomokazu Yoshimura Department of Applied Chemistry and Institute of Colloid and Interface Science, Tokyo University of Science, Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan Received 17 April 2003; accepted 23 May 2003
Abstract Gold nanoparticles were prepared in the presence of chitosan via reduction of HAuCl4 with sodium borohydride. The average particle size of gold nanoparticles was significantly affected with the concentration of chitosan added and was ranged between 6 and 16 nm. The gold /chitosan nanocomposites are formed by adsorbing chitosan molecules on the gold nanoparticles. The catalytic activity of gold /chitosan nanocomposites upon elimination of hydroxyl radicals formed in an H2O2/FeSO4 system was examined using a spin-trapping method. The catalytic activity increased with the concentration of chitosan added and showed a maximum and then decreased with a further concentration of chitosan added, although a strict correlation between the average diameter of gold and the catalytic activity was not found. In addition, the activity of gold /chitosan was 80 times higher than that of ascorbic acid, which is well known as an antioxidant. # 2003 Elsevier B.V. All rights reserved. Keywords: Gold /chitosan nanocomposite; Hydroxyl radical; Antioxidant; ESR
1. Introduction Preparation of noble metal nanoparticles by reducing metal salts has been extensively studied [1]. To stabilize dispersion of nanoparticles, it is necessary to use protective agents, such as polymers, surfactants, and chelating agents. These
* Corresponding author. Tel.: /81-332-604-271x2454; fax: /81-332-352-214. E-mail address: [email protected] (K. Esumi).
nanoparticles have widely been studied with a view to improve the quality of catalysts. All living organisms are suffered from the damage caused by the free-radical oxygen species. Free-radical oxygen species damage cells by attacking unsaturated fatty acids in the cell membrane. Fortunately, a protective enzyme, superoxide dismutase, completely converts these free-radical oxygen species into two water molecules and oxygen [2,3]. However, it is known that superoxide dismutase tissue levels decrease with aging. So, it is a challenging task to develop new
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00151-6
118
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
catalysts for the elimination of active radical species. Chitin, poly-B-(1,4)-n -acetyl-D-glucosamine, is a cellulose-like biopolymer widely distributed in nature, especially in marine invertebrates, insects, fungi, and yeasts. Chitin also has unique properties, including toughness, bioactivity, and biodegradability. Recently, chitosan, which is deacetylated chitin, has been attractive [4,5], because the free amino groups in this modified product contribute polycationic, chelating, and film-forming properties, along with ready solubility in dilute acetic acid. In particular, partially deacetylated chitin is water soluble, and is similar in behavior to hydrophilic polymers that adsorb on metal particle surfaces and form complexes. In this study, gold /chitosan nanocomposites were prepared and their catalytic activities were evaluated from elimination of hydroxyl radicals using a spin trapping technique.
2. Experimental
the solutions under stirring and left stirring for 30 min.
2.3. Measurements The gold nanoparticles obtained were analyzed by TEM and UV /vis absorption spectroscopy. TEM observation was performed for the samples dried on carbon-coated copper grids. A Hitachi H900 NAR transmission electron microscope was operated at accelerating voltage of 300 kV and direct magnification of 200,000 /. The size distribution of the gold nanoparticles was determined from about 100 particles. The catalytic activities of gold nanoparticles upon elimination of hydroxyl radicals were estimated by using a spin trapping technique [6]. In a typical experiment, solutions containing hydrogen peroxide, ferric sulfate, DMPO (5,5-dimethyl-1pyrroline-N -oxide), and gold /chitosan nanocomposites were measured with ESR; stable DMPO/ OH adduct can be detected by competitive reaction [7] of OH radicals with DMPO or the gold particles. The measurements were carried out at 1
2.1. Materials Chitosan was obtained from Wako Pure Chemicals Co. Chloroauric acid was obtained from Wako Pure Chemicals Co. Milli-Q water (Millipore Co.) was used in all experiments. The other chemicals were of analytical grade.
2.2. Preparation of gold /chitosan nanocomposites Preparation of gold nanoparticles in aqueous solution was conducted by chemical reduction of HAuCl4 /chitosan mixtures with sodium borohydride. For a typical experiment, 0.2 cm3 of freshly prepared 20 mmol dm 3 HAuCl4 solution was added to 19.7 cm3 of chitosan of various concentrations, and the solutions were stirred for 1 h. Then, 0.1 cm3 of 0.4 mol dm 3 freshly prepared ice-cold sodium borohydride was quickly added to
Fig. 1. UV /vis spectra of HAuCl4 aqueous solutions in the presence of chitosan after addition of sodium borohydride.
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
119
Fig. 2. Transmission electron micrographs and size distribution of gold nanoparticles; [chitosan] /(a) 0.0005; (b) 0.01; (c) 0.05; (d) 0.1; (e) 0.2; (f) 0.5; and (g) 1.0 wt%.
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K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
Fig. 2 (Continued)
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
121
Fig. 2 (Continued)
min after mixing of the solutions. The rate constant (Ks) of the catalytic activity of gold nanoparticles can be evaluated by following equations: F =(1 F ) R [S]=[DMPO]
(1)
R Ks =Kl F [DMPO]=(1 F )[S]
(2)
where R is the degree of reaction, F is the degree of decrease of ESR signal intensity, Kl and Ks are rate constants of DMPO and the gold nanoparticles, and [DMPO] and [S] are the concentrations of DMPO and the gold nanoparticles. Here, the value for Kl by DMPO is taken as 3.4 /109 M 1 s 1 [8].
3. Results and discussion Fig. 1 shows UV spectra of HAuCl4 aqueous solutions in the presence of chitosan after addition of sodium borohydride. All the spectra exhibit an absorption band at around 510 /520 nm which is a typical plasmon band, suggesting the formation of gold nanoparticles [9]. When the concentration of chitosan added is increased, the intensity of the absorption band decreases and then increases again with a further increase of the chitosan concentration. This result indicates that the size of gold nanoparticles formed is altered with the
concentration of chitosan, which operates as controller of nucleation as well as stabilizer. To check the particle size of gold particles obtained TEM measurements were performed. Fig. 2 shows several TEM images of gold particles obtained and their size distributions. It is apparent that the size of gold particles increases with an increase of the chitosan concentration and shows a maximum, and then decreases with a further concentration of chitosan. At 0.0005 wt% of chitosan, very small gold particles are formed. This is that since the interaction site of chitosan and AuCl 4 is very few, the number of nucleation is large, resulting in small gold particles which might be stabilized by adsorption of chloride ions. In the concentration region between 0.0006 and 0.001 wt%, the gold nanoparticles obtained were unstable and sedimented, probably due to a bridging effect by chitosan molecule between gold nanoparticles. When the concentration of chitosan increases from 0.01 to 0.1 wt%, the nucleation and growth rate of gold particles will be slowed due to the interaction between chitosan and AuCl 4 ; resulting in large gold particles. By addition of a further concentration of chitosan, the size of gold particles decreases. This decrement is due to a protective action by chitosan; chitosan may prevent the growth of gold particles by adsorbing their surfaces.
122
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
The catalytic activity upon elimination of hydroxyl radicals with gold /chitosan nanocomposites obtained was investigated using a spin trapping technique. Fig. 3 shows a typical ESR signal change of DMPO/OH adduct in the presence of gold/chitosan nanocomposites with var-
Fig. 3. Change of ESR spectra of DMPO-OH ESR signal with gold nanoparticles; [Au] /(a) 0; (b) 0.00625; (c) 0.0125; (d) 0.025; (e) 0.033 mmol dm 3.
ious concentrations. The intensity of the ESR signal decreases with the concentration of gold / chitosan nanocomposites, indicating that the gold /chitosan nanocomposites have an ability to depress the activity of hydroxyl radicals. It should be mentioned here that the action by chitosan itself is negligible compared to that by the gold / chitosan nanocomposites. Using the equations described in the Section 2, the rate constant (Ks) by the gold/chitosan nanocomposites was obtained. Fig. 4 gives plots of the rate constant/ particle diameter of gold versus the concentration of chitosan added. The rate constant increases with the concentration of chitosan added and attains a maximum, and then decreases with a further increase of the chitosan concentration. Although the change in the rate constant with the concentration of chitosan added is not strictly correlated with that in the particle size of gold, the mechanism for elimination activity of hydroxyl radicals by the gold/chitosan nanocomposites may be proposed as follows. At a very low concentration of chitosan, the rate constant is very small. This is a possibility that chloride ions adsorbing on the gold particles may inhibit the activity for the elimination of hydroxyl radicals. With an increase of chitosan concentration added, the nucleation and growth of gold particles are prevented and large particles are obtained whose surfaces are covered by chitosan to some extent. The gold/chitosan nanocomposites obtained between 0.01 and 0.1 wt% of chitosan show high rate constant. However, with a further concentration of chitosan, the gold particles are covered fully with chitosan molecules so that the elimination effect would be lowered by the chitosan molecules on the gold particles. In addition, it is interesting to compare the rate constant by the gold /chitosan nanocomposites with that by ascorbic acid which is known as an antioxidant agent; since the rate constant by ascorbic acids is obtained as 1.98 / 1011 M 1 s 1 the rate constant at the maximum in this study is about 80 times that by ascorbic acid. Thus, it is found that the gold /chitosan nanocomposites have a high activity upon the elimination of hydroxyl radicals.
K. Esumi et al. / Colloids and Surfaces B: Biointerfaces 32 (2003) 117 /123
123
Fig. 4. Plots of rate constant of catalytic activity and diameter of gold nanoparticles with the concentration of chitosan added.
References [1] G. Schmid (Ed.), Clusters and Colloids from Theory to Applications, Weinheim, 1994. Publisher VCH. [2] D. Kristensen, L.H. Skibsted, J. Agric. Food Chem. 47 (1999) 3099. [3] H. Taniguchi, K.P. Madden, J. Am. Chem. Soc. 121 (1999) 11 875. [4] J.P. Zikakis (Ed.), Chitin, Chitosan, and Related Enzymes, Academic Press, London, 1984.
[5] R.A.A. Muzzarelli, C. Jeuniaux, G.W. Gooday (Eds.), Chitin in Nature and Technology, Plenum Press, New York, 1985. [6] E. Finkelstein, G.M. Rosen, E.J. Rauckman, Mol. Pharmacol. 16 (1979) 676. [7] G.M. Rosen, B.A. Freeman, Proc. Natl. Acad. Sci. USA 81 (1984) 7269. [8] E. Finkelstein, G.M. Rosen, E.J. Rauckman, J. Am. Chem. Soc. 102 (1980) 4994. [9] N. Ishizuki, K. Torigoe, K. Esumi, K. Meguro, Colloids Surf. 55 (1991) 15.