In vitro biological properties and characterization of nanosilver coated cotton fabrics – An application for antimicrobial textile finishing

International Biodeterioration & Biodegradation 107 (2016) 48e55

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In vitro biological properties and characterization of nanosilver coated cotton fabrics e An application for antimicrobial textile finishing M.D. Balakumaran a, *, R. Ramachandran a, S. Jagadeeswari b, P.T. Kalaichelvan a a b

Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India Department of Microbiology, D.G. Vaishnav College, Chennai 600 106, Tamil Nadu, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 July 2015 Received in revised form 7 November 2015 Accepted 7 November 2015 Available online xxx

The aim of this study was to evaluate the antibacterial and antifungal activities of cotton fabrics coated with silver nanoparticles (AgNPs). Stable AgNPs synthesized using Aspergillus terreus were characterized using UVeVis. spectrophotometer, HR-TEM, XRD, and EDX. The synthesized AgNPs were 8e20 nm in size. In agar based diffusion and parallel streak methods, AgNPs coated cotton fabrics exhibited broad spectrum antibacterial activity against all pathogens tested. Importantly, AgNPs coated fabrics provided excellent laundering durability with pronounced antibacterial activity even after 15 wash cycles. The extent of release of silver ions from AgNPs coated fabrics into artificial sweat was studied using four different formulations (AATCC, ISO, and EN standards) with a pH range of 4.0e8.0. The maximum release of silver ions (31.71 mg/kg) was observed at pH 4.3 (AATCC formulation) while the minimum level of release (12.27 mg/kg) occurred at pH 8.0 (ISO formulation). HR-SEM micrographs of cotton fabrics coated with AgNPs exhibited a rough surface and the presence of nanoparticles on the cotton surface was also observed. Thus, the results of the present study clearly suggest that AgNPs synthesized using a green chemical technology could be considered in the development of antimicrobial textile finishes. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Aspergillus terreus Silver nanoparticles Cotton fabrics Antibacterial activity Laundering durability Antifungal activity

1. Introduction Natural fibers such as linen or cotton are more susceptible to microbial attack than the man-made fibers. Microbial growth on fabrics and other textile products becomes evident as surface changes, discoloration, and unpleasant odors (Szostak-Kotowa, 2004). The chemical changes occurring as a result of the growth of microorganisms will reduce the tensile strength of the fabric leading to partial or total destruction of the material. Thus, the increased exposure of textiles to microbial attack has the potential to cause cross infection, transfer of diseases, allergic reactions, and odor on humans (Thilagavathi et al., 2006). Recently, it has been reported that microorganisms may stay alive on fabric materials from days to months in a hospital environment (Neely and Maley, 2000; Koca et al., 2012). The high survival rate of methicillinresistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) on commonly used hospital items such as scrub suits, lab coats, and hospital privacy drapes underlined the importance of careful disinfection of hospital fabrics (Neely and

* Corresponding author. E-mail address: [email protected] (M.D. Balakumaran). http://dx.doi.org/10.1016/j.ibiod.2015.11.011 0964-8305/© 2015 Elsevier Ltd. All rights reserved.

Maley, 2000). In order to reduce the bacterial population and to minimize pathogenic infections, the development of antimicrobials is a possible alternative and promising approach. Therefore, it is imperative that specific agents must be used in order for the textile materials to provide them with an antimicrobial finish (Gao and Cranston, 2008). Chitosan and silver nanoparticles are one of the most frequently studied and described agents for finishing of cellulosic textiles (Goren
sek and Recelj, 2007; Dastjerdi and Montazer, 2010; Gouda and Keshk, 2010; Ali et al., 2011; Silva et al., 2011; Ibrahim et al., 2012). Unfortunately, many chemicals and the methods of imparting antimicrobial activity into textiles are toxic to humans and bacteria will develop resistance over such chemicals or antibiotics (Rajendran et al., 2013; Ul-Islam et al., 2013). Because of these drawbacks, it has been advised that materials manufactured using a green chemistry approach should be used since they are highly biocompatible. As the specific surface area of nanoparticles increases, their biological efficacy can also be increased proportionately. Physical characteristics such as size, shape, and stability are essential for boosting the biological properties of nanoparticles (Rajan et al., 2015). In this fashion, silver nanoparticles (AgNPs) synthesized through green routes have been found to be attractive

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materials for antibacterial coatings on leather and fabric surfaces (Velmurugan et al., 2014). Silver exerts its antimicrobial activity by several different mechanisms. It is worth mentioning that resistance development against silver is rare compared to antibiotics. This is most probably due to the multiple antimicrobial mechanisms mediating silver bactericidal activity; whereas, antibiotics have specific mechanisms (Taglietti et al., 2014). Recently, AgNPs have been employed as a new generation of antimicrobials and are being researched as coating materials (Chaloupka et al., 2010; Taglietti et al., 2014). n et al. (2007) have observed excellent antibacterial activity Dura against S. aureus when Fusarium oxysporum synthesized AgNPs were incorporated into cotton fabrics. El-Rafie et al. (2010) have shown the antibacterial activity of AgNPs treated cotton fabrics against S. aureus and Escherichia coli. Natural plant compounds or herbal extracts have been used to impart antibacterial activity to the cotton fabrics. Reddy et al. (2013) have shown the antibacterial activity of curcumin-treated cotton fabrics against E. coli and S. aureus. More recently, Velmurugan et al. (2014) have demonstrated the antibacterial activity of AgNPs treated cotton fabrics against Gram-positive odor causing bacteria Brevibacterium linens and Staphylococcus epidermidis. In addition, cotton fabrics finished with herbal extracts loaded nanoparticles have also shown better laundering durability besides superior antimicrobial activity (Rajendran et al., 2013). Very recently, in our laboratory, highly stable AgNPs were synthesized using Aspergillus terreus Bios PTK 6. These nanoparticles have shown broad spectrum antimicrobial activity against human pathogenic bacteria including MRSA and yeast strains (Balakumaran et al., 2016). Hence, in this study, we aimed to test whether these AgNPs could impart antimicrobial activity to cotton fabrics. 2. Material and methods 2.1. Preparation of silver nanoparticles treated cotton fabrics The dried cotton fabrics of required length were washed with double distilled water and sterilized by autoclaving at 121
C for 15 min at 15 lbs pressure. A. terreus (Bios PTK 6) was grown in potato dextrose broth (PDB) and was incubated at 27
C for 7 days. After incubation, the profusely grown fungal mat was washed extensively using sterile double distilled water to remove the traces of medium components. Typically, 10 g (wet weight) of fungal mat was brought in contact with 100 mL sterile double distilled water in an ErlenMeyer flask and was kept under shaker condition (120 rpm) for 48 h at 27
C. Then, the mycelial free filtrate was obtained by passing it through Whatman No. 1 filter paper. The filtrate was reacted with known quantity of silver nitrate to yield an overall Agþ ion concentration of 103 M and the reaction was carried out in dark at room temperature. In addition, the reaction parameters such as fungal biomass, silver nitrate, pH, and temperature were optimized for the better synthesis of AgNPs (Balakumaran, 2015; Balakumaran et al., 2016). The change in color was observed for 48 h and the synthesized AgNPs were characterized using UVeVis. spectrophotometer, high resolutiontransmission electron microscopy (HR-TEM), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDX). The results of the above studies were briefly discussed here. The very first evidence for nanoparticle synthesis is color change; the colorless solution turned into brown color, which indicates the formation of AgNPs in the medium. The color change was due to the excitation of surface plasmon vibrations, which is a characteristic feature of synthesized nanoparticles (Song et al., 2009). The peak observed at 430 nm in the UVeVis. spectrophotometer further confirmed the

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formation of AgNPs. HR-TEM image depicted spherical shaped AgNPs with a size of 8e20 nm. The XRD data showed face cubic centered silver and the EDX spectrum represented peaks at silver region, thus, confirming the successful synthesis of AgNPs (Balakumaran, 2015; Balakumaran et al., 2016). The sterilized fabrics were dried, immersed in Erlenmeyer flask (250 mL) containing mycosynthesized AgNPs (100 ppm) and were continuously agitated at 37
C using rotary shaker incubator for 24 h at 120 rpm. The treated fabrics were squeezed using laboratory padder at constant pressure for 1 min and then allowed to dry at 70
C for 3 min in hot air oven. Finally, the fabrics were kept at 150
C for 2 min for curing process and were stored at room temperature in a container (ElRafie et al., 2010). 2.2. Antibacterial activity of silver nanoparticles treated cotton fabrics 2.2.1. Bacterial strains In this study, Bacillus subtilis ATCC 6633, S. aureus ATCC 29736, Methicillin-resistant S. aureus (MRSA), E. coli ATCC 8739, Klebsiella pneumoniae ATCC 2719, and Pseudomonas aeruginosa ATCC 27853 were used as test organisms. 2.2.2. Agar diffusion method (SN 195920-1992) To study the antibacterial efficacy of mycosynthesized AgNPs treated cotton fabrics, agar based diffusion method (SN 1959201992) was performed (Rajendran et al., 2011). Mycosynthesized AgNPs treated fabrics of size 10  10 mm (length  width) were used. The test pathogens were inoculated in sterile AATCC bacteriostasis broth (HiMedia) and incubated at 37
C for 24 h. The overnight grown bacterial suspension was used for preparing a lawn culture using sterile cotton swab over the sterile AATCC bacteriostasis agar. The Petri dishes were kept aside for 2 min and then AgNPs treated fabrics along with untreated fabrics were placed on AATCC bacteriostasis agar plates and were pressed gently. Following inoculation, the plates were incubated for 24 h at 37
C and then zone of inhibition (ZOI) was recorded. 2.2.3. Parallel streak method (AATCC 147-2004) A parallel streak test approved by the American Association of Textile Chemists and Colorists was used to determine the antibacterial activity of mycosynthesized AgNPs treated cotton fabrics (American Association of Textile Chemists and Colorists Technical Manual, 2010). For this study, AgNPs treated fabrics of 50  25 mm (length  width) size were used. Briefly, the test strains were inoculated in sterile nutrient broth and were allowed to grow for 18 h at 37
C. Sterile nutrient agar plates were prepared for each test strain and one loop (4 mm in size) full of overnight grown bacterial culture was inoculated on the surface of nutrient agar plate by making five parallel inoculum streaks (60 mm in length with an interval of 10 mm apart for each streak) in such a way that it should cover the central area of the Petri dish without refilling the loop of the same inoculum. Then AgNPs treated fabrics were placed transversely across five inoculum streaks made earlier on the plate. The fabrics were pressed gently using sterile spatula (flame sterilization) to ensure that AgNPs treated fabrics have an intimate contact with nutrient agar surface. Simultaneously, the plate containing untreated fabrics was also maintained as control and the plates were incubated for 24 h at 37
C. At the end of the incubation, a clear area showing no growth along the sides of AgNPs treated fabrics indicates the antibacterial activity and then ZOI was calculated. The average width of inhibition zone on either side of the AgNPs treated fabrics along the streak was calculated using the following equation,

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W ¼ (T  D)/2 Where, W ¼ Width of clear zone of inhibition (mm); T ¼ Total diameter of test specimen and the zone of clearance (mm); D ¼ Diameter of the test specimen (mm). 2.3. Effect of washing on the antibacterial activity of AgNPs coated cotton fabrics The antibacterial activity of AgNPs treated cotton fabrics was quantitatively determined before and after washing using bacterial percentage reduction test (AATCC 100-2004) according to the American Association of Textile Chemists and Colorists Technical Manual (2010). In this study, AgNPs coated and uncoated swatches of size 4.8 ± 0.1 cm diameter were used. Five swatches of both AgNPs treated and untreated (sterilized) cotton fabrics were taken separately in 250 mL sterile Schott Duran flasks. Then 1 mL of overnight grown bacterial suspension was added to both the flasks, separately, using a micropipette and ensured the complete absorption of bacterial suspension. AgNPs coated and uncoated swatches were transferred to sterile AATCC bacteriostasis broth and incubated for 24 h at 37
C in rotary shaker at 120 rpm. After incubation, 1 mL of sample from both the treated and untreated swatches was taken and serially diluted using sterile distilled water up to 107 dilution. From each dilution, 0.1 mL was transferred to AATCC bacteriostasis agar and spread plate technique was performed. The plates were incubated at 37
C for 24 h and were examined for the presence of colonies. The percentage reduction of each test bacterium was calculated using the following formula, R ¼ 100 (B  A)/B Where, R ¼ Percentage reduction (%); B ¼ Total number of bacteria recovered from untreated swatches; A ¼ Total number of bacteria recovered from AgNPs treated swatches. The wash durability of mycosynthesized AgNPs treated cotton fabrics was evaluated as per IS: 687-1979 method after 5, 10, and 15 wash cycles. AgNPs treated cotton fabrics were subjected to washing process using neutral soap (5% Hiclean, HiMedia) at 40 ± 2
C for 30 min with a material to liquid ratio of 1:50. After 30 min, the fabrics were subjected to rinsing using sterile double distilled water followed by drying and this procedure was followed up to 15 wash cycles. Then the dried fabric samples after 5, 10, and 15 wash cycles were separately assessed for their antibacterial activity using AATCC 100-2004 method as described earlier. 2.4. Antifungal activity of silver nanoparticles treated cotton fabrics 2.4.1. Fungal cultures To evaluate the antifungal activity, four different fungal strains Aspergillus fumigatus, Aspergillus niger, Penicillium sp., and Rhizoctonia oryzae were obtained from the Fungal Culture Collection Centre, Centre for Advanced Studies in Botany, University of Madras, Chennai, India. These fungal pathogens were maintained in potato dextrose agar (PDA) slants at 27
C for 15e20 days and were subcultured periodically. 2.4.2. Antifungal test (AATCC 30-2004) For the evaluation of antifungal activity, AgNPs treated and untreated cotton fabrics of 2.0  2.0 cm were used. A fungal inoculum (105 spores per mL) was prepared using sterile distilled water and 1 mL was evenly spread onto the PDA plate. Then AgNPs treated and untreated swatches were made wet using 0.05% nonionic wetting agent (Triton X-100) and were placed on the PDA plates carefully. Further, 0.2 ± 0.01 mL of fungal inoculum (105

spores per mL) was distributed evenly on the fabrics using sterile pipette and the plates were incubated at 27
C for 7 days. At the end of the incubation, the antifungal activity of AgNPs treated fabrics was documented on the basis of visual assessment by determining the degree of fungal growth over the surface of each test fabric (AATCC 30-2004). This experiment was repeated thrice for reproducibility. Based on the visualization, the rating system was followed to analyze the mold growth as given below (FoksowiczFlaczyk and Walentowska, 2013). Rating system 0 e No visible growth evaluated microscopically. 1 e No visible growth evaluated with the naked eye but clearly visible microscopically. 2 e Growth visible with naked eye, covering up to 25% of tested surface. 3 e Growth visible with naked eye, covering up to 50% of tested surface. 4 e Considerable growth, covering more than 50% of tested surface. 5 e Very intense growth, covering the entire tested surface. 2.5. Sweat analysis using different pH 2.5.1. Measurement of initial silver content Cotton fabrics (weighing between 200 and 300 mg) coated with mycosynthesized AgNPs were digested using the microwave digestion method of Kulthong et al. (2010). The fabric was initially burned using furnace at 600
C for 3 h by placing it in a tightly closed crucible. The sample was then cooled at room temperature and the ash was collected and dispersed in double distilled water for acid digestion. The ashes were digested using 5 mL nitric acid (14.4 M) with a microwave irradiation cycle of 250 W for 5 min followed by 400 W for 5 min and 600 W for 5 min. The nitric acid helps in the breakdown of fabric ashes and also dissolves all the silver content in the sample. After digestion, the samples were cooled, diluted up to 50 mL using double distilled water and the silver ions were quantified using inductively coupled plasmaatomic emission spectroscopy (ICP-AES). The values were expressed in terms of silver content with respect to the fabric dry weight. 2.5.2. Measurement of silver released into artificial sweat For sweat analysis of mycosynthesized AgNPs treated cotton fabrics, four different artificial sweat formulations were prepared at different pH ranging between 4.3 and 8.0 using standard published procedures: American Association of Textile Chemists and Colorists (AATCC Test Method 15-2002), International Standard Organization (ISO 105-E04-2008E), and the British Standard (BS EN 1811-1999) (American Association of Textile Chemists and Colorists Technical Manual, 2010). In this study, the fabrics (weighing between 200 and 300 mg) treated with a known concentration of AgNPs were soaked in artificial sweat at the ratio of 1:100 (w/v). The fabrics along with different artificial sweat formulations were kept at 37
C for 24 h in a water bath. After incubation, the artificial sweat was collected and the concentration of silver ions was determined using ICP-AES following acid digestion as mentioned earlier. The experiments were performed in triplicate and the results were calculated based on the total amount of silver released into the artificial sweat with respect to the dry weight of each test fabric. 2.6. Characterization of AgNPs treated cotton fabrics The surface morphology of AgNPs treated as well as untreated (control) cotton fabrics was studied using electron microscopy.

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Both the treated and untreated fabrics were analyzed using high resolution-scanning electron microscopy (HR-SEM, FEI Quanta FEG 200) equipped with energy dispersive X-ray spectroscopy (EDX). The samples were mounted on specimen stub with double sided adhesive tape and sputter coated with gold particles under vacuum to increase electron conduction and to improve the quality of the micrographs (El-Rafie et al., 2010). 3. Results In this present study, the antimicrobial properties of mycosynthesized AgNPs treated cotton fabrics were evaluated against various pathogens. In addition, the fabrics were also subjected to different characterization techniques to confirm the immobilization. 3.1. Antibacterial activity of silver nanoparticles treated cotton fabrics 3.1.1. Qualitative assessment by agar diffusion method (SN 1959201992) The antibacterial efficacy of mycosynthesized AgNPs treated cotton fabrics was studied using agar based diffusion method (SN 195920-1992) against both Gram positive and negative pathogens and the results were presented in Fig. 1. The AgNPs treated fabrics displayed good antibacterial activity with a clear zone of inhibition around the cotton fabrics against all the tested pathogens; however, the untreated (control) fabrics did not show any inhibition zone. 3.1.2. Qualitative assessment of antibacterial activity using parallel streak method (AATCC 147-2004) For qualitative assessment of antibacterial activity, the cotton

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Table 1 Qualitative assessment of antibacterial activity of AgNPs treated fabrics e parallel streak test (AATCC 147-2004). Bacterial strain

Zone of inhibition (diameter in mm)

S. aureus B. subtilis E. coli P. aeruginosa K. pneumoniae MRSA

16.97 18.03 13.73 15.77 14.07 8.47

± ± ± ± ± ±

0.12 0.15 0.06 0.06 0.25 0.25

Values are mean ± standard deviation of three independent experiments.

fabrics treated with and/or without mycosynthesized AgNPs were subjected to parallel streak test and the results were given in Table 1. AgNPs treated cotton fabrics showed excellent antibacterial activity against all the tested pathogens; whereas, the untreated fabrics did not show any activity. The maximum ZOI was observed against B. subtilis (18.03 mm) followed by S. aureus (16.97 mm), and P. aeruginosa (15.77 mm). However, the least activity was recorded against MRSA with a ZOI of 8.47 mm. 3.2. Effect of washing on the antibacterial activity of AgNPs coated cotton fabrics The antibacterial activity of AgNPs coated cotton fabrics was carried out before and after several wash cycles and the results were presented in Fig. 2 and Table 2. AgNPs coated cotton fabrics depicted excellent laundering durability with pronounced antibacterial activity even after 15 wash cycles. Before washing, more than 99.9% reduction of bacterial colonies was recorded against all the tested pathogens including MRSA. However, this percentage was found to be decreased with increasing wash cycles from 5 to 15.

Fig. 1. Antibacterial activity of AgNPs coated cotton fabrics against human pathogenic bacteria by SN 195920-1992 method. (i) Uncoated (control) fabrics; (ii) cotton fabrics loaded with AgNPs. (a) Bacillus subtilis, (b) Staphylococcus aureus, (c) Methicillin-resistant Staphylococcus aureus (MRSA), (d) Escherichia coli, (e) Klebsiella pneumoniae, (f) Pseudomonas aeruginosa.

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Fig. 2. Laundering durability of AgNPs coated cotton fabrics after several wash cycles and their potent antibacterial activity against different bacterial pathogens by AATCC 100-2004 method. (i) AgNPs loaded fabric after 5 wash cycles; (ii) AgNPs loaded fabric after 10 wash cycles; (iii) AgNPs loaded fabric after 15 wash cycles. (a) Bacillus subtilis, (b) Staphylococcus aureus, (c) Methicillin-resistant Staphylococcus aureus (MRSA), (d) Escherichia coli, (e) Klebsiella pneumoniae, (f) Pseudomonas aeruginosa.

Table 2 Effect of washing on the antibacterial activity of AgNPs loaded cotton fabrics. Bacteria strains

Antibacterial activity (reduction percentage, %) Wash cycles Before washing

S. aureus B. subtilis E. coli P. aeruginosa K. pneumoniae MRSA

3.4. Measurement of silver nanoparticles released into artificial sweat

99.99 99.95 99.98 99.96 99.97 99.95

± ± ± ± ± ±

0.004 0.001 0.002 0.001 0.002 0.001

5 98.94 98.34 98.54 98.84 98.88 97.95

10 ± ± ± ± ± ±

0.05 0.09 0.06 0.10 0.05 0.07

97.06 97.43 97.71 97.45 97.39 96.25

15 ± ± ± ± ± ±

0.06 0.04 0.05 0.12 0.06 0.10

95.44 95.16 95.61 93.38 95.10 94.73

± ± ± ± ± ±

0.04 0.05 0.11 0.05 0.05 0.10

Values are mean ± standard deviation of three independent experiments.

As it can be seen from Table 2, more than 93% reduction of bacterial colony number was recorded against all the tested pathogens even after 15 washings and the maximum bacterial reduction percentage was observed against E. coli with 95.61% followed by S. aureus (95.44%), and B. subtilis (95.16%).

3.3. Antifungal activity of silver nanoparticles treated cotton fabrics Similar to the antibacterial activity, AgNPs treated cotton fabrics were also subjected to antifungal activity. In this study, mold growth was visually observed in all the plates treated with normal (control) cotton fabrics after 7 days of incubation. On the other hand, AgNPs treated fabrics showed good antifungal activity against all the test pathogens. Among the tested fungi, Penicillium sp., A. niger, and R. oryzae were rated as ‘2’ since the visible growth was observed with naked eye (Fig. 3). In case of A. fumigatus, there was no visible growth observed with naked eye; however, few mycelia were seen under light microscopy. Hence, it was rated as ‘1’.

The extent of release of silver ions from AgNPs treated fabrics into artificial sweat was determined using four standard formulations (AATCC, ISO, and EN) with pH values of 4.3, 5.5, 6.5, and 8.0. A gradual increase in the release of silver ions was observed at lower pH (4.3 & 5.5) compared to higher pH (8.0). As it can be seen from Table 3, the maximum release of silver ions (31.71 ± 4.33 mg/kg) was observed at pH 4.3 (AATCC formulation) followed by pH 5.5 (ISO formulation), and pH 6.5 (EN formulation). However, the least amount of release (12.27 ± 2.21 mg/kg) was recorded at pH 8.0 (ISO formulation). The untreated cotton fabrics showed no detectable silver in all the tested samples. 3.5. Characterization of silver nanoparticles coated cotton fabrics The surface morphology of AgNPs coated and uncoated cotton fabrics was studied by HR-SEM equipped with EDX. The SEM image of untreated cotton fabric showed smooth surface at higher magnification without any physical disturbance (Fig. 4a); whereas, AgNPs treated fabric showed rough surface (Fig. 4b). The SEM micrograph of treated fabric also revealed AgNPs on the surface of the cotton fabric (Fig. 4b). In addition, the EDX spectrum of treated cotton fabric also showed peaks at silver region, thus, confirming the existence of AgNPs on the fabric surface (Fig. 4c). 4. Discussion More recently, in our laboratory, stable AgNPs synthesized from A. terreus mycelial free filtrate demonstrated pronounced antimicrobial activity against a wide range of Gram-positive and negative

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Fig. 3. Antifungal activity of AgNPs coated cotton fabrics against different fungal pathogens by AATCC 30-2004 method. (i) AgNPs loaded fabric; (ii) control (unloaded fabric). (a) Penicillium sp., (b) Aspergillus fumigatus, (c) Aspergillus niger, (d) Rhizoctonia oryzae.

Table 3 Determination of release of silver ions from AgNPs treated cotton fabrics into artificial sweat. Sample

Initial silver content (mg/kg)

Test Control

65.05 ± 3.77 N.D.

Silver ions released into artificial sweat (mg/kg) AATCC pH 4.3

ISO pH 5.5

ISO pH 8.0

EN pH 6.5

31.71 ± 4.33 N.D.

26.34 ± 4.41 N.D.

12.27 ± 2.21 N.D.

14.97 ± 1.41 N.D.

Experiments were conducted for 24 h. Values are mean ± standard deviation of three independent experiments. N.D. ¼ not detected.

bacteria and yeast pathogens (Balakumaran et al., 2016). In this current study, we aimed to test whether these AgNPs could impart antimicrobial activity to cotton fabrics or not. About 100 ppm of AgNPs was coated in each cotton fabric using pad-dry-cure method without any binder or cross linker and the fabrics were assessed for their antibacterial as well as antifungal activities using standard published protocols mentioned in the AATCC technical manual. In agar based diffusion method (SN 195920-1992), AgNPs coated cotton fabrics exhibited remarkable antibacterial activity against six different human pathogens. Similarly, Pollini et al. (2009) have also shown antibacterial activity for silver coated cotton and polyester against E. coli. In parallel streak test (AATCC 147-2004), AgNPs coated cotton fabrics showed considerable antibacterial activity against all the test pathogens and the ZOI was ranged between 8.47 and 18.03 mm. Similarly, herbal products loaded cotton fabrics have also shown good antibacterial activity with a ZOI ranging from 4.9 to 12.5 mm (Sathianarayanan et al., 2010). Thus, it is evident that the present result shows better antibacterial activity

as ZOI was found to be higher than those reported by Sathianarayanan et al. (2010). The possible mechanism for antibacterial activity of AgNPs coated cotton fabrics was probably due to the formation of chemical bond between the silver and the functional groups of the textile substrates and the physical adsorption of AgNPs on the fabric surface (Perelshtein et al., 2008). Effect of washing on the antibacterial activity of AgNPs coated cotton fabrics was analyzed before and after several wash cycles. In this study, AgNPs treated cotton fabrics showed outstanding laundering durability with antibacterial activity even after 15 wash cycles. More than 99.9% reduction of bacterial colonies was observed before washing process; however, this percentage was decreased with the increase in wash cycles from 5 to 15. After 15 washings, above 93% reduction of bacterial colonies was recorded against all the test pathogens. Much similar to our results, after 20 wash cycles, El-Rafie et al. (2014) have observed higher than 85% reduction of bacterial colonies irrespective of the concentration of AgNPs used or silver content of the fabrics. Although the

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Fig. 4. High resolution-scanning electron micrographs of unloaded cotton fabric showing smooth surface (a & b); AgNPs loaded cotton fabrics showing slight change in the surface from smooth to rough (c & d) and the arrows indicate AgNPs on the surface of the AgNPs coated cotton fabric; EDX spectrum of AgNPs loaded cotton fabric showing strong peaks at silver region (e).

antibacterial activity of AgNPs treated cotton fabrics decreased slightly after 15 washings, however, the fabrics exhibited excellent durability. This observation was in good agreement with the studies of El-Rafie et al. (2014). However, in their earlier study, El-Rafie et al. (2012) have observed nearly 50% loss of antibacterial activity for AgNPs coated cotton fabrics after 20 wash cycles and suggested the use of binder or cross linker to improve the durability of the fabrics. In our study, even without any binder, merely 5e7% loss was recorded after 15 washings and this result was found to be consistent with the studies of Rajendran et al. (2013), in which Ocimum sanctum encapsulated nanoparticle treated fabrics showed 100% antibacterial activity against P. aeruginosa, Bacillus cereus, and S. aureus after 20 wash cycles. However, 2e5% loss of antibacterial activity was observed after 30 washes (Rajendran et al., 2013). As shown by FTIR study, AgNPs were stabilized by the extracellular proteins of A. terreus during the synthesis (Balakumaran et al., 2016). Hence, it is here speculated that the proteins capped around the surface of AgNPs may serve as binding agents and thereby enhance the durability of the fabrics. The higher antibacterial activity and laundering durability of AgNPs coated cotton fabrics was probably due to the smaller particle size, uniform coating, and controlled release of nanoparticles (Rajendran et al., 2013). In this current study, AgNPs coated fabrics were also tested for their antifungal activity. After 7 days of incubation period, visible mold growth was observed in the plates treated with normal (control) cotton fabrics. As expected, AgNPs coated cotton fabrics exhibited good antifungal activity against all the pathogens. Similarly, Vankar and Shukla (2012) have also evaluated antifungal activity for AgNPs dyed silk as well as cotton fabrics against F. oxysporum and Alternaria brassicicola. Contrary to our present results, the higher antifungal activity was observed in AgNPs dyed

silk fabrics rather than the cotton (Vankar and Shukla, 2012). ICP-AES measurements of sweat solutions after 24 h contact with AgNPs coated cotton fabrics clearly revealed the release of silver ions. The release of silver ions was steadily increased at lower pH (4.3 & 5.5) than the higher pH (8.0). The maximum release of silver ions (31.71 ± 4.33 mg/kg) was observed at pH 4.3 (AATCC formulation) while the minimum release (12.27 ± 2.21 mg/kg) was recorded at pH 8.0 (ISO formulation). Similarly, Lazic et al. (2012) have also assessed the stability of AgNPs coated cotton fabrics by measuring the release of silver ions into artificial sweat. Contrary to our present results, the alkaline sweat (pH 8.0) released 39% more silver ions than the acidic sweat (pH 5.5). In another study, Kulthong et al. (2010) have observed 15e322 mg/kg of silver release from the laboratory-prepared fabrics; whereas, in our study, about 12e31 mg/kg of silver ions were released. Thus, our study shows much less amount of silver release than those reported by Kulthong et al. (2010). It is obvious from these studies that the release of silver ions from AgNPs coated fabrics was relied much on the initial amount of silver, fabric quality, pH, and the artificial sweat formulation (Kulthong et al., 2010). It is also likely that artificial sweat may assist the transfer of AgNPs coated fabric to the skin surface. In line with our results, von Goetz et al. (2013) have recently demonstrated that the release of nanoparticles from engineered nanoparticles (ENP)-treated products was less (11e45 mg/g/L). Owing to their much smaller percutaneous uptake rates, silver ions have much less internal exposure on the skin; therefore, they have far less toxic potential (von Goetz et al., 2013). The surface characteristics of cotton fabrics coated with AgNPs were analyzed using HR-SEM and EDX. The HR-SEM image of uncoated cotton fabric showed smooth surface; but, AgNPs coated fabric showed rough surface. The SEM micrograph of AgNPs coated fabric revealed the presence of AgNPs on the surface of the cotton

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fabric. Similar to our findings, El-Rafie et al. (2014) have observed well dispersed AgNPs in 100 ppm AgNPs coated cotton fabrics than the 50 ppm coated ones. The EDX spectrum of AgNPs coated fabric showed strong peaks at silver region, confirming the successful immobilization of AgNPs into cotton fabrics. Similarly, El-Rafie et al. (2014) have also verified the presence of AgNPs in AgNPs coated cotton fabrics. 5. Conclusions In this present study, antimicrobial cotton fabrics were prepared by simple pad-dry-cure method using stable AgNPs synthesized from A. terreus. AgNPs treated fabrics showed excellent laundering durability besides pronounced antibacterial activity even after 15 wash cycles. In addition, effective antifungal activity was also recorded against all the test pathogens. The release of silver ions from AgNPs coated fabrics into artificial sweat was relied much on the initial amount of silver, fabric quality, and the sweat formulation including its pH. HR-SEM and EDX analyses showed the presence of AgNPs on the AgNPs coated cotton fabrics, thus, confirming the successful immobilization of AgNPs into cotton fabrics. Taken together, these findings clearly show that stable AgNPs synthesized from biological entities could be explored as promising candidates to impart antimicrobial activity to the cotton fabrics. This application can also be extended to produce antiseptic dressing or bandage for medical purposes in the near future. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors thank the Ministry of Human Resource Development (MHRD), Government of India, New Delhi for supporting this work under Special Grants through National Centre for Nanoscience and Nanotechnology (NCNSNT), University of Madras, Chennai. We are grateful to the Director, CAS in Botany, School of Life Sciences, University of Madras for providing adequate laboratory facilities. The Head, SAIF, IIT-Madras is gratefully acknowledged for HR-SEM and EDX analyses. Also, we thank the Head, SAIF, IIT-Bombay for ICP-AES analysis. References Ali, S.W., Rajendran, S., Joshi, M., 2011. Synthesis and characterization of chitosan and silver loaded chitosan nanoparticles for bioactive polyester. Carbohydr. Polym. 83 (2), 438e446. http://dx.doi.org/10.1016/j.carbpol.2010.08.004. American Association of Textile Chemists and Colorists (AATCC) Technical Manual, 2010, p. 85. Balakumaran, M.D., 2015. Effect of Biologically Synthesized Silver/gold Nano Particles, Their Anti-bacterial, Anti-fungal, and Anti-cancer Properties and Their Applications in Nano Medicine (Ph. D. thesis). University of Madras, Chennai, India. Balakumaran, M.D., Ramachandran, R., Balashanmugam, P., Mukeshkumar, D.J., Kalaichelvan, P.T., 2016. Mycosynthesis of silver and gold nanoparticles: optimization, characterization and antimicrobial activity against human pathogens. Microbiol. Res. 182, 8e20. http://dx.doi.org/10.1016/j.micres.2015.09.009. Chaloupka, K., Malam, Y., Seifalian, A.M., 2010. Nanosilver as a new generation of nanoproduct in biomedical applications. Trends Biotechnol. 28 (11), 580e588. http://dx.doi.org/10.1016/j.tibtech.2010.07.006. Dastjerdi, R., Montazer, M., 2010. A review on the application of inorganic nanostructured materials in the modification of textiles: focus on anti-microbial properties. Colloids Surf. B Biointerfaces 79 (1), 5e18. http://dx.doi.org/ 10.1016/j.colsurfb.2010.03.029. Dur an, N., Marcato, P.D., De Souza, G.I.H., Alves, O.L., Esposito, E., 2007. Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment. J. Biomed. Nanotechnol. 3 (2), 203e208. El-Rafie, M.H., Mohamed, A.A., Shaheen, T.I., Hebeish, A., 2010. Antimicrobial effect of silver nanoparticles produced by fungal process on cotton fabrics. Carbohydr. Polym. 80 (3), 779e782. http://dx.doi.org/10.1016/j.carbpol.2009.12.028.

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