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Pharmaceutical Nanotechnology, 2013, 1, 78-82
Fabrication of Silver Nanoparticles with Cotton for Antibacterial Wound Dressing MubarakAli D1, ArunKumar J2, Rahuman Sheriff M3, Pandiaraj D4 SheikSyedIshack K.A5 and N. Thajuddin1,* 1
Department of Microbiology, Bharathidasan University, Tiruchirappalli, India; 2PG Research Department of Microbiology and Biotechnology, Presidency College, Chennai, India; 3Max Planck Institute for Molecular Biology, Dortmund, Germany; 4Department of Botany, Jamal Mohammed College, Tiruchirappalli, India; 5Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, India Abstract: Nosocomial infections are presently an important health issue which is caused by the transfer of infection by patient, fomites and surgical materials in hospital. In solving these problems, silver nanoparticles play an inevitable role in the development of antibacterial and dressings materials by nanofabrication to minimize the risk factor in nosocomial infections. Nanofabrication can be defined as a bottom-up process to fabricate the material of interest from ionic level to nanoscale level with superior property. In the present study, silver nanoparticles have been fabricated into cotton fabrics for antibacterial wound dressing materials. Fabrication of silver nanoparticles into cotton fabrics was analyzed using SEM, EDAX, FTIR and UV-Vis spectroscopy. It was found that 20-nm -sized fine particles incorporated in cotton fiber without damaging the texture of the fiber. Antibacterial property of fabrics was tested against clinically isolated pathogens, Escherichia coli and Staphyloccocus aureus by disc diffusion and colony forming count method. The results proved that antibacterial activity was found against tested pathogens. This technology may apply for the development of silvernanoparticle- incorporated surgical materials and wound dressing materials to overcome nosocomial infections, secondary infection and drug storing articles.
Keywords: Nanofabrication, Silver nanoparticles, Cotton fabrics, Antibacterial, Wound dressing. 1. INTRODUCTION Cotton is an important crop used for fabrication of cloths articles etc. Cotton fabrics are widely used in hospitals for wound dressing wiping etc. The antibacterial property of fabrics is a significant feature in many assortments of fabrics used not only in healthcare sector but also in sports housekeeping etc. [1, 2]. Microbial communities in the environment in particular pathogens residing in hospital environment are likely to grow on the fabrics and spread infections (so-called fomite transmission) under appropriate environmental conditions. Their multiplication is the reason for the development of odor on the areas close to the skin and also for the development of infections in the case of pathogenic organism. Fabrics containing metal nanoparticles can be prepared by several physical and chemical methods. One of the important methods is in situ reduction of salts or complexes in the fabrics. These nanofabrics have attracted a great deal of attention due to their biomedical applications [3]. Biomedical device used in this study is a nanocomposite polymer containing metallic nanoparticles which are mostly silver nanoparticles [4]. Biogenic silver nanoparticles have broad spectrum of antimicrobial property against human pathogens as reported previously [5, 6]. In the present study a cost effective and eco-friendly nanofabrication of silver *Address correspondence to this author at the Department of Microbiology, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India; Tel: 91-431-2407082; Fax: 91-431-2407042; E-mail:
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nanoparticles into cotton fabrics for the active wound dressing material is developed. 2. MATERIALS AND METHODS 2.1. Materials Bleached cotton, silver nitrate, sodium citrate, Triton X100, double distilled water, sodium hydroxide, HCl and other reagents were purchased from Sigma, HiMedia, SRL (India). 2.2. Fabrication of Silver Nanoparticles with Cotton Fabrics Fabrication of silver ions into cotton as silver nanoparticles was described earlier [7]. A slight modification has been made in this method to avoid the complex steps involved and the concern toward cost-effectiveness. Briefly, 20 ml of silver nitrate (1mM) was taken in glass tube which contains washed cotton fabrics (2x2cm) and different volume of sodium citrate (1M) solution (0.1 l, 1l, 10 l and 100 l) was added. These glass tubes were incubated in boiling water bath at 70°C for 6h until cotton turns to brown color. Then the cotton fabrics were evaluated for color changes. It was squeezed and dried in microwave oven for 10 mins. The dried fabrics were again rinsed twice with double distilled water. Fully dried fabrics were washed with non-ionic detergent, Triton X-100, at a liquor ratio of 6:1 for 80oC for 1h. The mixture was vortexed for 5 mins. Again, the fabrics were washed with distilled water for three times until the water became clear and without any foam. The fabrics were then dried again in microwave oven for 10 mins. The fabrics were © 2013 Bentham Science Publishers
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Fig. (1). UV–Vis spectrum of silver coated cotton fabrics: silver nanoparticles absorption peaks at 450 nm due to Plasmon resonance of silver nanoparticles (a) control (b).
cut into small pieces and sterilized at 121°C for 15 mins. Fabrics were also sun-and shadow-dried in order to analyze its moisture content in the fabric after washing. Sterilized silver nanoparticles coated on cotton fabrics were cut into square shape (approximately 5 cm) and then immersed in 10 ml of the deionized water for 24 h at room temperature. After the incubation, water was collected and subjected to UV-Vis spectroscopy. Again, the fabrics were immersed in 10 ml of deionized water. The process was repeated for five times.
ized by autoclaving at 120 °C for 15 mins in the boiling tube, then 2 ml of medium was taken. Finally, 20 L of 16-h culture of pathogens were inoculated and incubated for 24 h and 48h at 37°C. An empty fabric was used as control in all the experiments. After incubation, 0.1 ml of sample was procured from the tubes and performed spread plate techniques. The plates were incubated at 37°C for 24 h. The tubes and plates were monitored for bacterial growth and documented for procuring data.
2.3. Characterization of Nanofabrics
3. RESULTS AND DISCUSSION
UV-Vis spectroscopy is used for quantitative determination of silver nanoparticles, in the range from 200 to 900 nm, periodically after every coating and also after washing process. Infra red spectroscopic analysis was also carried out for further characterization of coated and uncoated cotton fabrics. The samples were mixed with KBr to make a pellet and it was placed into the sample holder. The spectrum was recorded at a resolution of 4cm-1. The dried cotton fabrics were analyzed for the fabrication of silver nanoparticles on the surface of cotton fabrics. A small piece of fabric was placed on the carbon coated grid, and it was observed under vacuum with accelerated voltage of 20kV with different magnifications. The elemental composition of the fabrics was detected by EDAX, using Li drift Si detector (Thermo Elution Corporation, USA).
3.1. Nanofabrication of Silver with Cotton Fabrics
2.4. Antibacterial Activity Studies 2.4.1. Disc Diffusion Method This method was performed in Muller-Hinton agar (MHA) medium. Dried silver nanoparticles-coated cotton fabrics were cut into 5 cm sized squares, sterilized at 120°C for 15 mins and placed on plates that already inoculated with pathogens, E. coli and S. aureus, which were then incubated at 37 °C for 24h. The subsequent inhibition zone was measured and documented accordingly [8]. 2.4.2. Colony Forming Count Method Dried silver nanoparticles-coated cotton fabrics were cut into a square shape with 5 cm. The sample pieces were steril-
Structure of cotton cellulose is a three-dimensional nonwoven network and consists of large amount of pores. Thus, when cotton was immersed in the aqueous AgNO3, silver nanoparticle ions were readily attached into cotton. The absorbed Ag+ were bound to bacterial cellulose microfibrils probably via electrostatic interactions, because the electronrich oxygen atoms of polar hydroxyl and ether groups of bacterial cellulose are expected to interact with electropositive transition metal cations [9]. Silver ions were reduced to form silver nanoparticles in sodium citrate solution. The original white-colored fabrics turned to yellow and then to brown color. Finally, the fabricated materials kept for drying in microwave oven for 30 mins. The color of the silver nanoparticle-coated fabrics gradually changed from brown to dark brown then to black with increasing concentration of sodium citrate to AgNO3 from 1:1 to 10:1 to 100:1. During washing process, loss of unbounded silver nanoparticles is reported. After the third wash, there was no loss of nanoparticles. It has been believed that the nanoparticles were covalently bound with cotton fabrics and unbound nanoparticles were washed out through initial to few washing. UV-Vis spectral analysis revealed that a broad peak observed at 450 nm is because of plasmon resonance of silver nanoparticles, which were released during washing. It is confirmed that the silver ions have turned into metallic silver nanoparticles (Fig. 1).
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Fig. (2). Scanning electrons microscopic images of silver coated cotton fabrics: fabrics bundle (a) closer view of cotton fabrics (b) silver nanoparticles encrusted on the cotton fabrics (c) and fine image of silver coated fabrics (d).
3.2. Characterization of Nanofabrics Highly condensed cotton with three-dimensional nonwoven structures of microfibrils, which are highly crosslinked, slightly damaged structures, is observed (Fig. 2). It turns little harder after the treatment. Silver nanoparticles of 20 nm size were encrusted on the surface of the cotton fabrics. It has been reported that the cotton fabric was made by in situ coating of silver nanoparticles using coating bath containing silver nitrate precursor [9]. In bacterial cellulosic based preparation of silver impregnated wound dresser, it was found that the intact bacteria and debris were not observed on the membrane after treatment, whereas in untreated membrane showed intact bacteria and debris by SEM analysis [10]. EDAX spectrum of silver-coated cotton fabrics showed two series of elements where it was found that higher percentage of metallic silver signals (Fig. 3). The elemental weight of 5.90% and atomic level of 0.92% are reported in the present study. When compared to previous report that the elemental weight and the atomic values are increased by 1.91% and 0.25% respectively [9]. This confirmed the presence of silver in the cotton fabrics. In FTIR analysis, characteristics peaks of cotton such as O-H stretch-
ing, C-H stretching, C-H wagging, C-H bending and C-O stretching were observed [11]. In silver-coated cotton fabrics showed that broadening of peaks is due to functionalized cotton with silver ions. The presence of peaks at 1543 cm-1, 1364 cm-1, 1223 cm-1 and 1034 cm-1 due to –CH2 and –CH3 bending vibrations, C-H bending and C-O stretching in the silver-coated cotton fabrics (Fig. 4). 3.3. Antibacterial Property Antibacterial activity of silver-coated cotton fabrics was analyzed. It is found that clear zone was observed around the cotton fabrics by disc diffusion method. Silver-coated cotton fabrics showed 15 mm of zone of inhibition on E.coli, whereas in S.aureus, 13 mm of inhibition. In control experiment, growth was observed on the cotton, which may be due to the deterioration of cellulosic material for its growth. The silver-impregnated cellulosic materials showed that the zone of inhibition on E.coli and S.aureus was 2 mm and 3.5 mm respectively [10]. It has been reported that the repeated washing has improved the effectiveness of the silver coated cotton fabrics against different microorganisms [9]. Comparatively, the present results showed greater percentage of
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Fig. (3). Energy dispersive spectrum of cotton coated with silver showing silver signal in higher percentage (a) quantitative analysis of EDS (b). 100.0
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Fig. (4). FTIR spectrum of silver coated cotton fabrics.
antimicrobial property. It is clearly indicating that the antimicrobial activity is only due to the silver nanoparticles-coated cotton fabrics. In colony forming count method, no bacterial growth was observed in tube contained silver coated cotton fabrics, whereas in uncoated cotton fabrics, it was decayed due to bacterial growth (Fig. 5). No colony was observed on media
contained silver-coated cotton fabrics and nearly, 142 colonies were observed on the tube contained uncoated silver nanoparticles after 24 h of incubation. Prolonged incubation led to complete cotton decay. It is also stated that bacterial inhibition is due to silver nanoparticles binding to the substrate by bonding on the surface. An attached agent disrupts the cell membrane of the bacterial cells by physical and ionic
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CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS
a
b
The authors wish to thank Council for Scientific Industrial Research (09/475/0167/2012-EMR-I) and Department of Biotechnology, Govt. of India (BT/PR11316/PBD/26/164/2008) for their grant for Senior Research Fellowship. REFERENCES
1
[1]
[2] [3]
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[4]
b [5]
[6]
2 Fig. (5). Antibacterial activity studies: Silver coated fabrics (a) test fabrics control (b) in the broth contains E.coli (1) and S. aureus (2).
[7]
phenomenon [12]. Silver ions interact with the thiol group of enzymes and proteins that are important for the bacterial respiration and transport of important substances across the membrane and within the cell. Moreover, silver ions alter the functions of the bacterial cell membrane, thus silver metal and its fabrication were effectively preventing infection of the wound [13-15].
[8]
4. CONCLUSIONS
[11]
Silver ions were successfully coated on the cotton fabrics in the form of nanoparticles by the optimized protocols. It is found to be spherical-shaped nanoparticles with an average size range of 20 nm. They are formed as thin films on the surface of the cotton fabrics. The processed fabrics were tested against clinically isolated multi-drug resistant pathogens such as E. coli and S. aureus for antibacterial property and found that maximal inhibitory pattern was observed against isolated pathogens. The technology is useful to manufacture the cotton and wound dressing materials with antibiosis property and self-protective feature. Received: August 11, 2012
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Revised: September 17, 2012
Accepted: October 16, 2012
