Bio-Medical Materials and Engineering 23 (2013) 473–483 DOI 10.3233/BME-130772 IOS Press
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Preparation of size-controlled silver nanoparticles and chitosan-based composites and their anti-microbial activities Vinh Quang Nguyen a,b , Masayuki Ishihara b,∗ , Yasutaka Mori b,c , Shingo Nakamura d , Satoko Kishimoto b , Masanori Fujita b , Hidemi Hattori b , Yasuhiro Kanatani e , Takeshi Ono f , Yasushi Miyahira f and Takemi Matsui a a
Faculty of System Design, Tokyo Metropolitan University, Tokyo, Japan Research Institute, National Defense Medical College, Saitama, Japan c Aeromedical Laboratory, Japan Air Self-Defense Force, Saitama, Japan d Department of Surgery, National Defense Medical College, Saitama, Japan e Department of Health Crisis Management, National Institute of Public Health, Saitama, Japan f Department of Global Infectious Diseases and Tropical Medicine, National Defense Medical College, Saitama, Japan b
Received 18 January 2013 Accepted 25 April 2013 Abstract. We previously reported a simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) generated by autoclaving a mixture of silver-containing glass powder and glucose. The particle size is regulated by the glucose concentration, with concentrations of 0.25, 1.0 and 4.0 wt% glucose providing small (3.48 ± 1.83 nm in diameter), medium (6.53 ± 1.78 nm) and large (12.9 ± 2.5 nm) particles, respectively. In this study, Ag NP/chitosan composites were synthesized by mixing each of these three Ag NP suspensions with a 75% deacetylated (DAc) chitosan suspension (pH 5.0) at room temperature. The Ag NPs were homogeneously dispersed and stably embedded in the chitosan matrices. The Ag NP/chitosan composites were obtained as yellow or brown flocs. It was estimated that approximately 60, 120 and 360 µg of the small, medium and large Ag NPs, respectively, were maximally embedded in 1 mg of chitosan. The bactericidal and anti-fungal activities of the Ag NP/chitosan composites increased as the amount of Ag NPs in the chitosan matrix increased. Furthermore, smaller Ag NPs (per weight) in the chitosan composites provided higher bactericidal and anti-fungal activities. Keywords: Silver-nanoparticles, chitosan, composites, bactericidal, anti-fungal
1. Introduction Silver nanoparticles (Ag NPs) attract great interest because of their potential in various applications such as catalysts, photonic devices, biosensors, anti-microbials and drug delivery systems [1–4]. Many preparative processes have been proposed for controlling the physical and/or chemical characteristics of Ag NPs [5–9]. *
Address for correspondence: Masayuki Ishihara, PhD, National Defense Medical College, Research Institute, 3-2 Namiki, Tokorazawa, Saitama, 359-8513 Japan. Tel.: +81 429 95 1211; Fax: +81 429 91 1611; E-mail: [email protected]. 0959-2989/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
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Environmentally friendly processes using harmless materials are often used to prepare Ag NPs since complicated purification is then not required for biomedical and environmental applications. Raveendran et al. produced Ag NPs with diameters less than 10 nm using a process that employs D-glucose as the reducing agent and soluble starch as the stabilizing agent [10]. The particle sizes of Ag NPs are usually controlled by modifying reaction system parameters such as pH, temperature and reactant concentrations. The choice of stabilizing agent is an important factor for controlling the particle size of Ag NPs since Ag+ is reduced within the nanoscopic templates of the stabilizing agent [11]. The particle size of Ag NPs is one of the most fundamental parameters that affects their optical [12], anti-microbial [13–15] and anti-virus properties [16]. Sondi et al. reported that the anti-microbial activity of Ag NPs towards Gram-negative bacteria was dependent on the concentration of Ag NPs, and that the Ag NPs formed “pits” in the cell wall of bacteria [14]. Sondi et al. speculated that a similar mechanism may cause the degradation of the membrane structure of Escherichia coli (E. coli) during treatment with Ag NPs [14]. Ag NPs also exhibited potent anti-fungal effects, probably through the destruction of membrane integrity [17]. On the other hand, there are some concerns about the biological and environmental risks of Ag NPs. It is known that Ag NPs have adverse effects on some aquatic organisms, e.g., cytotoxicity and genotoxicity to fish [18] and the inhibition of photosynthesis in algae [19]. In mammals, a significant decline in mouse spermatogonial stem cells was observed following dosing with Ag NPs [20]. Therefore, methods for preventing the diffusion of Ag NPs into the environment and their uptake by living organisms are necessary before designed anti-microbial materials containing Ag NPs can be widely used [21–25]. In a previous study, we developed an environmentally friendly process for tightly controlling the size distribution of Ag NPs [22]. This process uses only three materials: AgNO3 -containing glass powder, glucose and water. The AgNO3 -containing glass powder is commonly used in environmental, osteal and dental applications as an anti-microbial agent since it releases silver ions (Ag+ ) into aqueous environments in a sustained manner. Glucose has the advantages of being environmentally friendly and a mild reducing agent, which enables the reaction kinetics to be easily controlled. Synthesis of Ag NPs was performed in an aqueous medium using an autoclave at 121◦ C and 200 kPa for 20 min. Caramel, which is formed from glucose during autoclaving, in turn functions as the stabilizing agent for Ag NPs in this system [22]. However, it was difficult to remove the caramel from the generated Ag NPs suspension without agglomeration and precipitation of the Ag NPs. Chitosan is the collective name for a family of de-N -acetylated chitin with different degrees of deacetylation [23,24]. In general, when the number of N -acetyl-glucosamine units exceeds 50%, the biopolymer is termed chitin, whereas the term “chitosan” is used to describe the polymer when the N -acetylglucosamine content is less than 50%. Chitosan has been studied as a natural cationic biopolymer because of its excellent biocompatibility, biodegradability, non-toxicity, anti-microbial capability and stimulation of wound healing [25]. These properties of chitosan are dependent on the molecular weight and degree of deacetylation (DAc) [25]. The DAc affects the solubility, hydrophobicity and ability of chitosan to interact electrostatically with polyanions via its protonated amino groups. In fact, chitosan with >70% DAc strongly interacts with Ag NPs, which are negatively charged due to halogenation and oxidization. In this work, low molecular weight chitosan with 75% DAc was added as a stabilizer to the Ag NP suspension to remove the generated caramel and to prevent agglomeration and precipitation of the Ag NPs. The Ag NPs in the Ag NP/chitosan composites are substantially stabilized compared to in the absence of chitosan. The size-controlled Ag NP/chitosan composites were evaluated for their bactericidal (against E. coli (strain DH5α)) and anti-fungal (against Aspergillus (A.) niger) activities.
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2. Materials and methods 2.1. Materials Silver-containing glass powder (BSP21, silver content: 1 wt%, average grain size: 10 µm) was obtained from Kankyo Science (Kyoto, Japan). Low molecular weight chitosan with 75% DAc was purchased from Sigma-Aldrich Japan Co. (Tokyo, Japan). D-glucose (>98%) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). All chemicals were used as received. 2.2. Preparation of Ag NPs A suspension of size-controlled Ag NPs was prepared as previously described [22]. Briefly, 0.50 g of silver-containing glass powder was dispersed in 50 ml of an aqueous solution of 0.25, 1 or 4.0 wt% glucose in a 100 ml glass vial. The mixture was autoclaved using an Ikemoto IMC-30L autoclave (Ikemoto Inc., Tokyo, Japan) at 121◦ C and 200 kPa for 20 min. The mixture was then gradually cooled to room temperature and centrifuged at 3000 rpm for 10 min. The supernatant containing the Ag NP suspension was removed and stored in the dark at 4◦ C. The average diameter of Ag NPs prepared with 0.25, 1 and 4 wt% glucose was 3.48 ± 1.83, 6.53 ± 1.78 and 12.9 ± 2.50 nm, respectively [22]. 2.3. Preparation of Ag NP/chitosan composites In this study, 10 mg of low molecular weight chitosan (low molecular weight, DAc: 75%) was added to 1 ml distilled water. The chitosan suspension (10 mg/ml) at pH 4.5 was prepared by adding 1 N HCl. One ml of each Ag NP suspension (about 60 µg/ml) was added into the chitosan suspension (100 µl) and mixed well (at pH 5.0). The insoluble Ag NP/chitosan composites were prepared at pH 7.2 by adding 1 N NaOH and mixing well on a shaker (MildMixer PR-36; TAITEC, Tokyo, Japan) for 30 min. The insoluble Ag NP/chitosan composites were centrifuged at 6000 rpm for 10 min. The supernatant was analyzed using UV-visible spectrometer (Jasco V-630, Tokyo, Japan) to measure the amount of unreacted Ag NPs. The centrifuged composites washed twice with distilled water by centrifugation at 6000 rpm for 10 min. The washed composites were dried up at 70◦ C on a block heater (EYELA/MG – 2200, Rikakikai Co., LTD, Tokyo, Japan) for 2 h and used in bactericidal or anti-fungal assays the same day. The Ag NPs were homogeneously dispersed and stably immobilized in the chitosan matrices. TEM specimens were prepared by casting 5 µl of a suspension of Ag NPs onto a carbon-coated copper grid; excess solution was then removed using filter paper and the specimens were dried at room temperature. TEM images were obtained using a JEOL JEM-1010 microscope (Nihon Electronics Inc., Tokyo, Japan) at 80 kV. 2.4. Bactericidal activity of the Ag NP/chitosan composites A culture of Escherichia coli (E. coli: strain DH5α, Takara Co., Kyoto, Japan) was stored in Luria– Bertani (LB) broth medium containing 50% sterile glycerol at −80◦ C. Overnight cultures were prepared by growing a single E. coli colony in 5 ml LB medium at 37◦ C. On the next day, 200 µl of cultured E. coli were inoculated in 2 ml of LB medium and grown at 37◦ C for 6 h until optical density at 600 nm (OD600 ) reached 0.260 to ensured to obtained good quality of E. coli cultures. The cultured was diluted
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fourfold with LB broth and then 50 µl of the diluted E. coli suspension was added to sterile 1.5 ml ClickFit polypropylene microcentrifuge tubes (TreffLab AG, Degersheim, Switzerland) containing dried Ag NP/chitosan composites with the indicated amount of each Ag NPs (30, 15, 7.5, 3.8 and 0 µg) adsorbed in 10 mg chitosan as Ag NP/chitosan composites, followed by incubation at 37◦ C for 18 h. After incubation, 1 ml of LB medium was added to the E. coli suspensions and mixed well. The suspensions were stood for 3 min to precipitate the Ag NP/chitosan composites. Viable cells were enumerated by plating the 50 µl of ten-fold serial dilutions of the suspensions onto LB agar (Formedium Ltd, Hunstanton, England) in a Petri dish (90 × 15 mm) followed by incubation at 37◦ C for 24 h. 2.5. Anti-fungal activity of the Ag NP/chitosan composites Aspergillus (A.) niger (NBRC105649) (Japan Collection of Microorganisms; Wako, Saitama, Japan) was maintained in molten potato dextrose agar (PDA) medium (Difco, Becton Dickinson & Co., Sparks, MD, USA). Twenty µl of A. niger spore suspension (6.35 × 104 spores/ml) was inoculated into each well of an agar plate (24-multiwell plate; well diameter: 17 mm (Sumitomo Bakelite Co., Ltd TD, Tokyo, Japan) containing 60, 30, 15, 7.5 and 3.8 µg/ml of each Ag NPs adsorbed in 1 mg chitosan/ml as Ag NP/chitosan composites. The plates were incubated in the dark at 25◦ C for 3 days, then the A. niger spores were recovered in 500 µl of 0.3% sterile Tween 80 solution using a platinum loop. The absorbance of each spore suspension after vortexing was measured at 550 nm with a Jasco V-630 spectrophotometer [26]. 2.6. Statistical analysis Statistical analyses were carried out using StatMate III, Macintosh Version (ATMS Co., Tokyo, Japan). Statistical significance was assumed when p < 0.01. 3. Results 3.1. Characterization of Ag NP/chitosan composites Ag NP/chitosan composites were synthesized by mixing chitosan and Ag NP suspension at pH 5. Generally, chitosan is soluble only in strong acidic conditions (pH < 3) because of the protonation of the primary amines in the chitosan chains. Although the mixing of these solutions was performed under mildly acidic conditions (at pH 5), the Ag NP/chitosan composites were partially precipitated at all the mixing conditions used in this study. NaOH was used to adjust the pH of the mixture to 7.2 to precipitate the composite thoroughly. Typical TEM micrographs of the small (A) and large (B) Ag NPs are shown in Fig. 1. Ag NP/chitosan composites were obtained as a floc composed of aggregated spherical submicrometer particles. The color of the composite was yellow or brown: a darker composite was obtained when larger amounts of Ag NPs reacted with chitosan. It was estimated that approximately 60, 120 and 360 µg of the small, medium and large Ag NPs, respectively, maximally embedded into 1 mg of chitosan. Figure 2 shows the UV-vis spectra of the three sizes of the original Ag NPs in suspension and spectra of the supernatants of the post-reaction mixtures in which various amounts of chitosan were reacted with the Ag NPs. The peak at 390.5 nm is representative of the spherical Ag NPs used in this work [12,22]. There is a relationship between the absorbance at 390.5 nm and the concentration of each size of Ag NPs in the suspension (Fig. 3). The amount of Ag NPs remaining in the supernatant of the
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Fig. 1. (A) TEM micrographs (upper) and size distributions (lower) of Ag NP suspensions prepared with 1.0 wt% of silver– containing glass powder and 0.25 (left – small Ag NPs) or 4.0 wt% (right – large Ag NPs) glucose at 121◦ C for 20 min. Scale bar = 100 nm. (B) TEM micrograph of Large Ag NP/chitosan composites (15 µg Ag NPs in 1 mg chitosan).
post-reaction mixture decreased as the concentration of chitosan in the reaction mix increased (Fig. 2). Thus, it appears that Ag NPs reacted with chitosan, and the two components precipitated together upon centrifugation. 3.2. Bactericidal activity of Ag NP/chitosan composites The size-controlled Ag NP/chitosan composites and chitosan alone were evaluated for their bactericidal activities (against E. coli) in LB medium. Chitosan alone showed a weak bactericidal activity (Fig. 4). The additions of small, medium and large Ag NPs into chitosan exhibited strong concentrationdependent bactericidal activities (Fig. 4). Especially, >15, 30 and 30 µg of small, medium and large Ag NPs/chitosan composites, respectively exhibited complete bactericidal activities. Thus, smaller Ag NPs showed higher bactericidal activity. Furthermore, the Ag NP/chitosan composites exhibited higher bactericidal activity than chitosan alone. 3.3. Anti-fungal activity of Ag NPs/chitosan composites Size-controlled Ag NP/chitosan composites, chitosan alone, and Ag/NPs alone were also evaluated for their anti-fungal (against A. niger) activities. The fungi were incubated in a molten potato dextrose
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Fig. 2. UV-vis spectra of small (A), medium (B) and large (C) original Ag NPs in suspensions (original) and supernatants from the post-reaction mixture in which various amounts of chitosan were reacted with the Ag NPs. Excess Ag NPs in the supernatant of the post-reaction mixture decreased as the amount of chitosan added increased.
agar (PDA) with the test materials. Chitosan alone has only weak concentration-dependent anti-fungal activity, with half-growth inhibition of more than 5 mg/ml (data not shown). Small, medium and large Ag NPs alone have concentration-dependent anti-fungal activity with half-growth inhibition of about 10, 15 and 30 µg/ml (data not shown). When composites with various amounts of small, medium and large Ag NPs in 1 mg/ml chitosan were added to the fungal cultures in PDA, the Ag NP/chitosan composites showed an strong anti-fungal activity in a concentration-dependent manner of each Ag NPs adsorbed in the chitosan with half-growth inhibition of about 3.8, 7.5 and 15 µg/ml in 1 mg chitosan/ml (Fig. 5). Thus, smaller Ag NPs have higher anti-fungal activity. Furthermore, the Ag NP/chitosan composites exhibit higher anti-fungal activity than Ag NPs or chitosan alone.
4. Discussion In this work, low molecular weight chitosan with 75% DAc was added as a stabilizer to the Ag NP suspension to remove the produced caramel and prevent agglomeration and precipitation of the Ag NPs. The Ag NPs in the Ag NP/chitosan composites are substantially stabilized compared to Ag NPs alone. The size and shape of each Ag NPs in Ag NP/chitosan composites were similar to those of the original Ag NPs used for synthesizing the composites.
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Fig. 3. The relationship between the peak at 390.5 nm arising from small (A), medium (B) and large (C) spherical Ag NPs and the concentration of each size of Ag NPs.
The Ag NP/chitosan composites were evaluated for their bactericidal (against E. coli) and anti-fungal (against A. niger) activities. For all three sizes of Ag NPs used in this work, the anti-microbial activity of Ag NP/chitosan composites increased as the amount of Ag NPs increased. On a % weight basis, stronger anti-microbial activity was generally evident with Ag NP/chitosan composites containing smaller Ag NPs, suggesting that the embedded Ag NPs reinforced the anti-microbial activity of chitosan matrices. Although little work has been conducted on the mechanism by which Ag NPs act against bacteria and fungi, it has been reported that the anti-microbial activity of Ag NPs on Gram-negative bacteria is dependent on the concentration of Ag NPs and is tied to the formation of “pits” in the cell wall of bacteria [14]. The authors speculated that a similar mechanism may degrade the membrane structure of E. coli during treatment with Ag NPs [14]. The bactericidal activity of Ag NPs (against E. coli) is likely due to direct binding of the Ag NPs to the microbial envelope glycoproteins, thereby destroying membrane integrity. Ag NPs also exhibited potent anti-fungal effects (against A. niger), probably by destroying membrane integrity [17]. The effect of size of the Ag NPs on their bactericidal and anti-fungal activity was investigated in the present study. It appears that the binding of Ag NPs to the microorganisms tested depends on the surface area available for the interaction. Smaller particles have a larger surface area available for the interaction, thus providing stronger anti-microbial activity than larger particles. Furthermore, the increased number of small particles is present per unit weight of the Ag NPs compared to large particles. In the Ag NP/chitosan composites, spatial restriction due to the chitosan matrix was expected to prevent or weaken the interaction between microorganisms and Ag NPs. However, the present study indicates that both the bactericidal and anti-fungal activities of the composites are higher than those of Ag NPs or chitosan alone. When embedded Ag NPs can interact with microorganisms, the interaction increases with increasing number of Ag NPs in the Ag NP/chitosan composites. This is supported by the experimental results showing the relationship between anti-microbial activity and the amount of Ag NPs added to the cultures.
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Fig. 4. The size-controlled Ag NP/chitosan composites were evaluated for their bactericidal (against E. coli) activities in LB medium. The composites contained various amounts of small (A), medium (B) and large (C) Ag NPs in 10 mg chitosan. The Ag NP/chitosan composites exhibited strong bactericidal activity in a concentration-dependent manner. Data are mean value ± standard deviation, n = 6. ND means “not detected”. The asterisk (∗ ) represents statistical significant difference (p < 0.01) using two-samples t-test.
5. Conclusion The Ag NPs in chitosan composites were homogeneously dispersed and stably embedded in the chitosan matrices. Approximately 60, 120 and 360 µg of the small, medium and large Ag NPs, respectively, were maximally embedded in 1 mg of chitosan. Although the bactericidal and anti-fungal activities of
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Fig. 5. The size-controlled Ag NP/chitosan composites were evaluated for their anti-fungal (against A. niger) activity by incubating with the fungi in molten potato dextrose agar (PDA). When composites with various amounts of small (A), medium (B) and large (C) Ag NPs in 1 mg chitosan/ml were added in PDA, the Ag NP/chitosan composites exhibited anti-fungal activity in a concentration-dependent manner. The upper figure is a representative picture for growth of A. niger on Ag NP/chitosan composite-containing PDA. The lower figure shows spore-concentrations in the recovered suspension from the above plates on day 3. Data are mean value ± standard deviation, n = 6. The asterisk (∗ ) represents statistical significant difference (p < 0.01) using two-samples t-test. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-130772.)
the chitosan alone were low, those of Ag NP/chitosan composites increased as the amount of Ag NPs in the chitosan matrix increased. Furthermore, smaller Ag NPs (per weight) in the chitosan composites provided higher bactericidal and anti-fungal activities. Those results show the potential for wide application of Ag NP/chitosan composites as a novel approach for imparting strong anti-microbial activity to chitosan, decreasing concerns about the safety for preventing the diffusion of Ag NPs into the environment and their uptake by living organisms.
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Acknowledgements The authors would like to thank Ms. Y. Ichiki at Laboratory Center of National Defense Medical College (Tokorozawa, Japan) for her helping with electron microscopy experiment.
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Preparation of size-controlled silver nanoparticles and chitosan-based composites and their anti-microbial activities Vinh Quang Nguyen a,b , Masayuki Ishihara b,∗ , Yasutaka Mori b,c , Shingo Nakamura d , Satoko Kishimoto b , Masanori Fujita b , Hidemi Hattori b , Yasuhiro Kanatani e , Takeshi Ono f , Yasushi Miyahira f and Takemi Matsui a a
Faculty of System Design, Tokyo Metropolitan University, Tokyo, Japan Research Institute, National Defense Medical College, Saitama, Japan c Aeromedical Laboratory, Japan Air Self-Defense Force, Saitama, Japan d Department of Surgery, National Defense Medical College, Saitama, Japan e Department of Health Crisis Management, National Institute of Public Health, Saitama, Japan f Department of Global Infectious Diseases and Tropical Medicine, National Defense Medical College, Saitama, Japan b
Received 18 January 2013 Accepted 25 April 2013 Abstract. We previously reported a simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) generated by autoclaving a mixture of silver-containing glass powder and glucose. The particle size is regulated by the glucose concentration, with concentrations of 0.25, 1.0 and 4.0 wt% glucose providing small (3.48 ± 1.83 nm in diameter), medium (6.53 ± 1.78 nm) and large (12.9 ± 2.5 nm) particles, respectively. In this study, Ag NP/chitosan composites were synthesized by mixing each of these three Ag NP suspensions with a 75% deacetylated (DAc) chitosan suspension (pH 5.0) at room temperature. The Ag NPs were homogeneously dispersed and stably embedded in the chitosan matrices. The Ag NP/chitosan composites were obtained as yellow or brown flocs. It was estimated that approximately 60, 120 and 360 µg of the small, medium and large Ag NPs, respectively, were maximally embedded in 1 mg of chitosan. The bactericidal and anti-fungal activities of the Ag NP/chitosan composites increased as the amount of Ag NPs in the chitosan matrix increased. Furthermore, smaller Ag NPs (per weight) in the chitosan composites provided higher bactericidal and anti-fungal activities. Keywords: Silver-nanoparticles, chitosan, composites, bactericidal, anti-fungal
1. Introduction Silver nanoparticles (Ag NPs) attract great interest because of their potential in various applications such as catalysts, photonic devices, biosensors, anti-microbials and drug delivery systems [1–4]. Many preparative processes have been proposed for controlling the physical and/or chemical characteristics of Ag NPs [5–9]. *
Address for correspondence: Masayuki Ishihara, PhD, National Defense Medical College, Research Institute, 3-2 Namiki, Tokorazawa, Saitama, 359-8513 Japan. Tel.: +81 429 95 1211; Fax: +81 429 91 1611; E-mail: [email protected]. 0959-2989/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
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Environmentally friendly processes using harmless materials are often used to prepare Ag NPs since complicated purification is then not required for biomedical and environmental applications. Raveendran et al. produced Ag NPs with diameters less than 10 nm using a process that employs D-glucose as the reducing agent and soluble starch as the stabilizing agent [10]. The particle sizes of Ag NPs are usually controlled by modifying reaction system parameters such as pH, temperature and reactant concentrations. The choice of stabilizing agent is an important factor for controlling the particle size of Ag NPs since Ag+ is reduced within the nanoscopic templates of the stabilizing agent [11]. The particle size of Ag NPs is one of the most fundamental parameters that affects their optical [12], anti-microbial [13–15] and anti-virus properties [16]. Sondi et al. reported that the anti-microbial activity of Ag NPs towards Gram-negative bacteria was dependent on the concentration of Ag NPs, and that the Ag NPs formed “pits” in the cell wall of bacteria [14]. Sondi et al. speculated that a similar mechanism may cause the degradation of the membrane structure of Escherichia coli (E. coli) during treatment with Ag NPs [14]. Ag NPs also exhibited potent anti-fungal effects, probably through the destruction of membrane integrity [17]. On the other hand, there are some concerns about the biological and environmental risks of Ag NPs. It is known that Ag NPs have adverse effects on some aquatic organisms, e.g., cytotoxicity and genotoxicity to fish [18] and the inhibition of photosynthesis in algae [19]. In mammals, a significant decline in mouse spermatogonial stem cells was observed following dosing with Ag NPs [20]. Therefore, methods for preventing the diffusion of Ag NPs into the environment and their uptake by living organisms are necessary before designed anti-microbial materials containing Ag NPs can be widely used [21–25]. In a previous study, we developed an environmentally friendly process for tightly controlling the size distribution of Ag NPs [22]. This process uses only three materials: AgNO3 -containing glass powder, glucose and water. The AgNO3 -containing glass powder is commonly used in environmental, osteal and dental applications as an anti-microbial agent since it releases silver ions (Ag+ ) into aqueous environments in a sustained manner. Glucose has the advantages of being environmentally friendly and a mild reducing agent, which enables the reaction kinetics to be easily controlled. Synthesis of Ag NPs was performed in an aqueous medium using an autoclave at 121◦ C and 200 kPa for 20 min. Caramel, which is formed from glucose during autoclaving, in turn functions as the stabilizing agent for Ag NPs in this system [22]. However, it was difficult to remove the caramel from the generated Ag NPs suspension without agglomeration and precipitation of the Ag NPs. Chitosan is the collective name for a family of de-N -acetylated chitin with different degrees of deacetylation [23,24]. In general, when the number of N -acetyl-glucosamine units exceeds 50%, the biopolymer is termed chitin, whereas the term “chitosan” is used to describe the polymer when the N -acetylglucosamine content is less than 50%. Chitosan has been studied as a natural cationic biopolymer because of its excellent biocompatibility, biodegradability, non-toxicity, anti-microbial capability and stimulation of wound healing [25]. These properties of chitosan are dependent on the molecular weight and degree of deacetylation (DAc) [25]. The DAc affects the solubility, hydrophobicity and ability of chitosan to interact electrostatically with polyanions via its protonated amino groups. In fact, chitosan with >70% DAc strongly interacts with Ag NPs, which are negatively charged due to halogenation and oxidization. In this work, low molecular weight chitosan with 75% DAc was added as a stabilizer to the Ag NP suspension to remove the generated caramel and to prevent agglomeration and precipitation of the Ag NPs. The Ag NPs in the Ag NP/chitosan composites are substantially stabilized compared to in the absence of chitosan. The size-controlled Ag NP/chitosan composites were evaluated for their bactericidal (against E. coli (strain DH5α)) and anti-fungal (against Aspergillus (A.) niger) activities.
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2. Materials and methods 2.1. Materials Silver-containing glass powder (BSP21, silver content: 1 wt%, average grain size: 10 µm) was obtained from Kankyo Science (Kyoto, Japan). Low molecular weight chitosan with 75% DAc was purchased from Sigma-Aldrich Japan Co. (Tokyo, Japan). D-glucose (>98%) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). All chemicals were used as received. 2.2. Preparation of Ag NPs A suspension of size-controlled Ag NPs was prepared as previously described [22]. Briefly, 0.50 g of silver-containing glass powder was dispersed in 50 ml of an aqueous solution of 0.25, 1 or 4.0 wt% glucose in a 100 ml glass vial. The mixture was autoclaved using an Ikemoto IMC-30L autoclave (Ikemoto Inc., Tokyo, Japan) at 121◦ C and 200 kPa for 20 min. The mixture was then gradually cooled to room temperature and centrifuged at 3000 rpm for 10 min. The supernatant containing the Ag NP suspension was removed and stored in the dark at 4◦ C. The average diameter of Ag NPs prepared with 0.25, 1 and 4 wt% glucose was 3.48 ± 1.83, 6.53 ± 1.78 and 12.9 ± 2.50 nm, respectively [22]. 2.3. Preparation of Ag NP/chitosan composites In this study, 10 mg of low molecular weight chitosan (low molecular weight, DAc: 75%) was added to 1 ml distilled water. The chitosan suspension (10 mg/ml) at pH 4.5 was prepared by adding 1 N HCl. One ml of each Ag NP suspension (about 60 µg/ml) was added into the chitosan suspension (100 µl) and mixed well (at pH 5.0). The insoluble Ag NP/chitosan composites were prepared at pH 7.2 by adding 1 N NaOH and mixing well on a shaker (MildMixer PR-36; TAITEC, Tokyo, Japan) for 30 min. The insoluble Ag NP/chitosan composites were centrifuged at 6000 rpm for 10 min. The supernatant was analyzed using UV-visible spectrometer (Jasco V-630, Tokyo, Japan) to measure the amount of unreacted Ag NPs. The centrifuged composites washed twice with distilled water by centrifugation at 6000 rpm for 10 min. The washed composites were dried up at 70◦ C on a block heater (EYELA/MG – 2200, Rikakikai Co., LTD, Tokyo, Japan) for 2 h and used in bactericidal or anti-fungal assays the same day. The Ag NPs were homogeneously dispersed and stably immobilized in the chitosan matrices. TEM specimens were prepared by casting 5 µl of a suspension of Ag NPs onto a carbon-coated copper grid; excess solution was then removed using filter paper and the specimens were dried at room temperature. TEM images were obtained using a JEOL JEM-1010 microscope (Nihon Electronics Inc., Tokyo, Japan) at 80 kV. 2.4. Bactericidal activity of the Ag NP/chitosan composites A culture of Escherichia coli (E. coli: strain DH5α, Takara Co., Kyoto, Japan) was stored in Luria– Bertani (LB) broth medium containing 50% sterile glycerol at −80◦ C. Overnight cultures were prepared by growing a single E. coli colony in 5 ml LB medium at 37◦ C. On the next day, 200 µl of cultured E. coli were inoculated in 2 ml of LB medium and grown at 37◦ C for 6 h until optical density at 600 nm (OD600 ) reached 0.260 to ensured to obtained good quality of E. coli cultures. The cultured was diluted
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fourfold with LB broth and then 50 µl of the diluted E. coli suspension was added to sterile 1.5 ml ClickFit polypropylene microcentrifuge tubes (TreffLab AG, Degersheim, Switzerland) containing dried Ag NP/chitosan composites with the indicated amount of each Ag NPs (30, 15, 7.5, 3.8 and 0 µg) adsorbed in 10 mg chitosan as Ag NP/chitosan composites, followed by incubation at 37◦ C for 18 h. After incubation, 1 ml of LB medium was added to the E. coli suspensions and mixed well. The suspensions were stood for 3 min to precipitate the Ag NP/chitosan composites. Viable cells were enumerated by plating the 50 µl of ten-fold serial dilutions of the suspensions onto LB agar (Formedium Ltd, Hunstanton, England) in a Petri dish (90 × 15 mm) followed by incubation at 37◦ C for 24 h. 2.5. Anti-fungal activity of the Ag NP/chitosan composites Aspergillus (A.) niger (NBRC105649) (Japan Collection of Microorganisms; Wako, Saitama, Japan) was maintained in molten potato dextrose agar (PDA) medium (Difco, Becton Dickinson & Co., Sparks, MD, USA). Twenty µl of A. niger spore suspension (6.35 × 104 spores/ml) was inoculated into each well of an agar plate (24-multiwell plate; well diameter: 17 mm (Sumitomo Bakelite Co., Ltd TD, Tokyo, Japan) containing 60, 30, 15, 7.5 and 3.8 µg/ml of each Ag NPs adsorbed in 1 mg chitosan/ml as Ag NP/chitosan composites. The plates were incubated in the dark at 25◦ C for 3 days, then the A. niger spores were recovered in 500 µl of 0.3% sterile Tween 80 solution using a platinum loop. The absorbance of each spore suspension after vortexing was measured at 550 nm with a Jasco V-630 spectrophotometer [26]. 2.6. Statistical analysis Statistical analyses were carried out using StatMate III, Macintosh Version (ATMS Co., Tokyo, Japan). Statistical significance was assumed when p < 0.01. 3. Results 3.1. Characterization of Ag NP/chitosan composites Ag NP/chitosan composites were synthesized by mixing chitosan and Ag NP suspension at pH 5. Generally, chitosan is soluble only in strong acidic conditions (pH < 3) because of the protonation of the primary amines in the chitosan chains. Although the mixing of these solutions was performed under mildly acidic conditions (at pH 5), the Ag NP/chitosan composites were partially precipitated at all the mixing conditions used in this study. NaOH was used to adjust the pH of the mixture to 7.2 to precipitate the composite thoroughly. Typical TEM micrographs of the small (A) and large (B) Ag NPs are shown in Fig. 1. Ag NP/chitosan composites were obtained as a floc composed of aggregated spherical submicrometer particles. The color of the composite was yellow or brown: a darker composite was obtained when larger amounts of Ag NPs reacted with chitosan. It was estimated that approximately 60, 120 and 360 µg of the small, medium and large Ag NPs, respectively, maximally embedded into 1 mg of chitosan. Figure 2 shows the UV-vis spectra of the three sizes of the original Ag NPs in suspension and spectra of the supernatants of the post-reaction mixtures in which various amounts of chitosan were reacted with the Ag NPs. The peak at 390.5 nm is representative of the spherical Ag NPs used in this work [12,22]. There is a relationship between the absorbance at 390.5 nm and the concentration of each size of Ag NPs in the suspension (Fig. 3). The amount of Ag NPs remaining in the supernatant of the
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Fig. 1. (A) TEM micrographs (upper) and size distributions (lower) of Ag NP suspensions prepared with 1.0 wt% of silver– containing glass powder and 0.25 (left – small Ag NPs) or 4.0 wt% (right – large Ag NPs) glucose at 121◦ C for 20 min. Scale bar = 100 nm. (B) TEM micrograph of Large Ag NP/chitosan composites (15 µg Ag NPs in 1 mg chitosan).
post-reaction mixture decreased as the concentration of chitosan in the reaction mix increased (Fig. 2). Thus, it appears that Ag NPs reacted with chitosan, and the two components precipitated together upon centrifugation. 3.2. Bactericidal activity of Ag NP/chitosan composites The size-controlled Ag NP/chitosan composites and chitosan alone were evaluated for their bactericidal activities (against E. coli) in LB medium. Chitosan alone showed a weak bactericidal activity (Fig. 4). The additions of small, medium and large Ag NPs into chitosan exhibited strong concentrationdependent bactericidal activities (Fig. 4). Especially, >15, 30 and 30 µg of small, medium and large Ag NPs/chitosan composites, respectively exhibited complete bactericidal activities. Thus, smaller Ag NPs showed higher bactericidal activity. Furthermore, the Ag NP/chitosan composites exhibited higher bactericidal activity than chitosan alone. 3.3. Anti-fungal activity of Ag NPs/chitosan composites Size-controlled Ag NP/chitosan composites, chitosan alone, and Ag/NPs alone were also evaluated for their anti-fungal (against A. niger) activities. The fungi were incubated in a molten potato dextrose
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Fig. 2. UV-vis spectra of small (A), medium (B) and large (C) original Ag NPs in suspensions (original) and supernatants from the post-reaction mixture in which various amounts of chitosan were reacted with the Ag NPs. Excess Ag NPs in the supernatant of the post-reaction mixture decreased as the amount of chitosan added increased.
agar (PDA) with the test materials. Chitosan alone has only weak concentration-dependent anti-fungal activity, with half-growth inhibition of more than 5 mg/ml (data not shown). Small, medium and large Ag NPs alone have concentration-dependent anti-fungal activity with half-growth inhibition of about 10, 15 and 30 µg/ml (data not shown). When composites with various amounts of small, medium and large Ag NPs in 1 mg/ml chitosan were added to the fungal cultures in PDA, the Ag NP/chitosan composites showed an strong anti-fungal activity in a concentration-dependent manner of each Ag NPs adsorbed in the chitosan with half-growth inhibition of about 3.8, 7.5 and 15 µg/ml in 1 mg chitosan/ml (Fig. 5). Thus, smaller Ag NPs have higher anti-fungal activity. Furthermore, the Ag NP/chitosan composites exhibit higher anti-fungal activity than Ag NPs or chitosan alone.
4. Discussion In this work, low molecular weight chitosan with 75% DAc was added as a stabilizer to the Ag NP suspension to remove the produced caramel and prevent agglomeration and precipitation of the Ag NPs. The Ag NPs in the Ag NP/chitosan composites are substantially stabilized compared to Ag NPs alone. The size and shape of each Ag NPs in Ag NP/chitosan composites were similar to those of the original Ag NPs used for synthesizing the composites.
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Fig. 3. The relationship between the peak at 390.5 nm arising from small (A), medium (B) and large (C) spherical Ag NPs and the concentration of each size of Ag NPs.
The Ag NP/chitosan composites were evaluated for their bactericidal (against E. coli) and anti-fungal (against A. niger) activities. For all three sizes of Ag NPs used in this work, the anti-microbial activity of Ag NP/chitosan composites increased as the amount of Ag NPs increased. On a % weight basis, stronger anti-microbial activity was generally evident with Ag NP/chitosan composites containing smaller Ag NPs, suggesting that the embedded Ag NPs reinforced the anti-microbial activity of chitosan matrices. Although little work has been conducted on the mechanism by which Ag NPs act against bacteria and fungi, it has been reported that the anti-microbial activity of Ag NPs on Gram-negative bacteria is dependent on the concentration of Ag NPs and is tied to the formation of “pits” in the cell wall of bacteria [14]. The authors speculated that a similar mechanism may degrade the membrane structure of E. coli during treatment with Ag NPs [14]. The bactericidal activity of Ag NPs (against E. coli) is likely due to direct binding of the Ag NPs to the microbial envelope glycoproteins, thereby destroying membrane integrity. Ag NPs also exhibited potent anti-fungal effects (against A. niger), probably by destroying membrane integrity [17]. The effect of size of the Ag NPs on their bactericidal and anti-fungal activity was investigated in the present study. It appears that the binding of Ag NPs to the microorganisms tested depends on the surface area available for the interaction. Smaller particles have a larger surface area available for the interaction, thus providing stronger anti-microbial activity than larger particles. Furthermore, the increased number of small particles is present per unit weight of the Ag NPs compared to large particles. In the Ag NP/chitosan composites, spatial restriction due to the chitosan matrix was expected to prevent or weaken the interaction between microorganisms and Ag NPs. However, the present study indicates that both the bactericidal and anti-fungal activities of the composites are higher than those of Ag NPs or chitosan alone. When embedded Ag NPs can interact with microorganisms, the interaction increases with increasing number of Ag NPs in the Ag NP/chitosan composites. This is supported by the experimental results showing the relationship between anti-microbial activity and the amount of Ag NPs added to the cultures.
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Fig. 4. The size-controlled Ag NP/chitosan composites were evaluated for their bactericidal (against E. coli) activities in LB medium. The composites contained various amounts of small (A), medium (B) and large (C) Ag NPs in 10 mg chitosan. The Ag NP/chitosan composites exhibited strong bactericidal activity in a concentration-dependent manner. Data are mean value ± standard deviation, n = 6. ND means “not detected”. The asterisk (∗ ) represents statistical significant difference (p < 0.01) using two-samples t-test.
5. Conclusion The Ag NPs in chitosan composites were homogeneously dispersed and stably embedded in the chitosan matrices. Approximately 60, 120 and 360 µg of the small, medium and large Ag NPs, respectively, were maximally embedded in 1 mg of chitosan. Although the bactericidal and anti-fungal activities of
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Fig. 5. The size-controlled Ag NP/chitosan composites were evaluated for their anti-fungal (against A. niger) activity by incubating with the fungi in molten potato dextrose agar (PDA). When composites with various amounts of small (A), medium (B) and large (C) Ag NPs in 1 mg chitosan/ml were added in PDA, the Ag NP/chitosan composites exhibited anti-fungal activity in a concentration-dependent manner. The upper figure is a representative picture for growth of A. niger on Ag NP/chitosan composite-containing PDA. The lower figure shows spore-concentrations in the recovered suspension from the above plates on day 3. Data are mean value ± standard deviation, n = 6. The asterisk (∗ ) represents statistical significant difference (p < 0.01) using two-samples t-test. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-130772.)
the chitosan alone were low, those of Ag NP/chitosan composites increased as the amount of Ag NPs in the chitosan matrix increased. Furthermore, smaller Ag NPs (per weight) in the chitosan composites provided higher bactericidal and anti-fungal activities. Those results show the potential for wide application of Ag NP/chitosan composites as a novel approach for imparting strong anti-microbial activity to chitosan, decreasing concerns about the safety for preventing the diffusion of Ag NPs into the environment and their uptake by living organisms.
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Acknowledgements The authors would like to thank Ms. Y. Ichiki at Laboratory Center of National Defense Medical College (Tokorozawa, Japan) for her helping with electron microscopy experiment.
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