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Fabrication of antibacterial cotton fibres loaded with silver nanoparticles via “Green Approach”
Colloids and Surfaces A: Physicochem. Eng. Aspects 367 (2010) 31–40
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Fabrication of antibacterial cotton fibres loaded with silver nanoparticles via “Green Approach” S. Ravindra, Y. Murali Mohan 1 , N. Narayana Reddy, K. Mohana Raju ∗ Synthetic Polymer Laboratory, Department of Polymer Science and Technology, Sri Krishnadevaraya University, Anantapur 515055, A.P., India
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Article history: Received 18 January 2010 Received in revised form 8 June 2010 Accepted 10 June 2010 Available online 17 June 2010 Keywords: Cotton fibre Silver nanoparticles Reduction Stability Green process Antibacterial activity Wound dressing
a b s t r a c t In the present investigation the antimicrobial efficiency of cotton fibres loaded with silver nanoparticles (AgNPs) was studied which are developed by “green process” using natural extracts, of Eucalyptus citriodora and Ficus bengalensis. The formation of AgNPs on the cotton fibres was observed by UV–vis spectrophotometer. The size of silver nanoparticles was found to have ∼20 nm. The structure and morphology of silver nanoparticles formed on the cotton fibres were confirmed by electron microscopy. The antibacterial activity of cotton fibres loaded with silver nanoparticles was evaluated against gram-negative Escherichia coli (E. coli) bacteria. The results suggest excellent antibacterial activity by the incorporation of 2% leaf extracts on cotton fibres. These fibres have also exhibited superior antibacterial activity even after several washings indicating their usage in medical and infection prevention applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Over the last few decades, considerable research effort was made to produce antibacterial coatings on the surfaces of various objects such as garments and medical devices [1,2]. The current interest is to develop efficient, non-toxic, durable and cost effective antimicrobial finishing textiles with increased applications in medical, health care, hygienic products as well as protective textile materials [3–12]. Cotton fibres are particularly suitable for manufacturing textiles for sports, non-implantable medical products, and health care/hygienic products [3]. However the ability of cotton fibres to absorb large amount of moisture makes them more prone to microbial attack under certain conditions of humidity and temperature. Cotton may acts as a nutrient, becoming suitable medium for bacterial and fungal growth [4,13]. Therefore, cotton fibres are treated with numerous chemicals to get better antimicrobial cotton textiles [14–22]. Among the various antimicrobial agents, silver nanoparticles (AgNPs) have shown strong inhibitory and antibacterial effects [23,24]. Among the various synthetic methods used for the preparation of silver nanoparticles, chemical reducing method by using a reducing agent such as sodium borohydrate, citrate or
∗ Corresponding author. Tel.: +91 8554 255655; fax: +91 8554 255655. E-mail address: [email protected] (K. Mohana Raju). 1 Present address: Cancer Biology Research Center, Sanford Research/USD, Sioux Falls, SD 57105, USA. 0927-7757/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2010.06.013
ascorbate in a silver salt solution is most common [25,26]. Synthetic reducing agents are normally associated with environmental toxicity or biological hazards. Therefore the development of silver nanoparticles based on natural extracts is considered as most appropriate method for environmental reasons. The present research is mainly focusing on the development of antimicrobial surfaces [27–29]. Of late, the incorporation of silver nanoparticles on cotton fibres has received great attention due to their high resistance to microbes. Vigneshwaran et al. [30] developed the in situ synthesis of silver nanoparticles on cotton fabrics. The terminal aldehyde group of starch not only reduces the silver nitrate into silver nanoparticles but also stabilizes them on fabric simultaneously. Perelshtein et al. [31] developed sonochemical method of coating of silver nanoparticles using ultra sound waves [31]. Duran et al. [32] incorporated silver nanoparticles on cotton fabrics having good antibacterial property by fungal process. Yu et al. [33] also reported the incorporation of silver nanoparticles onto ultrafine fibres by electro spinning. The present study involves the incorporation of silver nanoparticles on cotton fibres by “green process”. The advantages of this process are: (a) no need to have extra reducing agent(s), (b) the process can be conducted at room temperature, under normal pressure in aqueous solution, and (c) finally the developed silver nanoparticles by this process have excellent properties, including long term dispersion stability. The silver nanoparticles incorporated cotton fibres were systematically characterized and their mechanical properties were also evaluated. The antibacterial property of silver
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nanoparticles loaded cotton fibres were tested against Escherichia coli (E. coli) bacteria. 2. Materials and methods 2.1. Materials Eucalyptus citriodora (Neelagiri) and Ficus bengalensis (Marri) leaves were collected from their trees that are available in the campus of Sri Krishnadevaraya University, Anantapur, India. Silver nitrate (AgNO3 ) was purchased from Merck India Ltd. (Mumbai, India) and used as received without further purification. Cotton fibres (1 mm thickness) were purchased from SIMCO Thread mills (Salem, Chennai, India). Distilled water was used throughout this investigation. 2.2. Preparation of the leaves extract E. citriodora (Neelagiri) and F. bengalensis (Marri) leaves were collected and thoroughly washed with distilled water for 15 min at room temperature (25 ◦ C) and dried for 1 day at room temperature. The tree leaf broth solutions were prepared by taking 2, 4 and 6 g of washed leaves in an Erlenmeyer flask containing 100 ml of distilled water. The solution was heated at 100 ◦ C to extract the contents of the leaves for 30 min and filtered through 0.45 m PVDF Millex Filter units using 50 ml syringe. The extracted leaves solutions were stored at 4 ◦ C and used within a week to produce silver nanoparticles. 2.3. Synthesis of silver nanoparticles In a typical experiment 5 ml (2, 4 and 6 g) of washed leaves put in an Erlenmeyer flask containing 100 ml of distilled water and heated to 100 ◦ C for 30 min. These fresh leaves extract was added to a conical flask containing 5 ml of 1 mM aqueous AgNO3 solution (170 mg AgNO3 in 100 ml distilled water) at room temperature. The silver ions were reduced to silver nanoparticles within 2–5 min by the leaves extract. This quick conversion was observed by color change from color less to brown color. The reduction of silver nitrate into silver nanoparticles formation was monitored by using UV–vis spectrophotometer. 2.4. Impregnation of silver nanoparticles onto cotton fibres Silver nanoparticles were produced within the cotton fibres by leaf broth reduction of Ag+ ions. Pre weighed cotton fibres were immersed in the filtrate leaf broth containing silver nanopaticles and kept on a shaker at 100 rpm at room temperature for 24 h and then dried. 2.5. Characterization 2.5.1. Differential scanning calorimetry (DSC) DSC analysis of cotton fibres and silver nanoparticles incorporated cotton fibres were performed using SDT Q 600 thermal analyzer (T.A. Instruments-water LLC, Newcastle, DE, USA), from 40 to 700 ◦ C at a heating rate of 20 ◦ C per min, under a constant flow (100 ml/min) of nitrogen gas. 2.5.2. Thermogravimetric analysis (TGA) The thermal history of cotton fibre and silver nanoparticles loaded cotton fibres were evaluated on a SDT Q 600 TGA instrument (T.A. Instruments-water LLC, Newcastle, DE, USA), at a heating rate of 20 ◦ C/min under a constant nitrogen flow (100 ml/min). The samples were run from 40 to 700 ◦ C.
2.5.3. Transmission electron microscopy (TEM) The size of the silver nanoparticles was determined using a Technai F12 TEM (Philips Electron Optics, Holland). TEM samples were prepared by dropping 2–3 drops of aqueous solutions of silver nanoparticles on a 200 mesh formvar-coated copper TEM grid (grid size: 97 m) (Ted Pella, Inc., Redding, CA, USA) followed by removing excess solution using a piece of fine filter paper and the samples were allowed to dry in air overnight prior to image particles. 2.5.4. Scanning electron microscopy (SEM) The surface morphology of cotton fibres and silver nanoparticles loaded cotton fibres were studied using a JEOL JSM 840A (Tokyo, Japan) scanning electron microscope (SEM) at an accelerating voltage of 15 kV. All the samples were dried in vacuum at room temperature and coated with gold before scanning. Surface morphologies were imaged at different magnifications. 2.6. Microbial activity The antibacterial activities of fibres and fibres loaded with silver nanoparticles were tested by an inhibition zone method. In this method, E. coli was taken as the model bacteria. For this study, the fibres (cotton fibre and silver nanoparticles loaded cotton fibres) were cut into small pieces (1 mm thickness and 1 cm length), put together to form a circular zone, and the antimicrobial activity was tested using modified agar diffusion assay (disc test). The plates were examined for possible clear zone formation after incubation at 37 ◦ C for 1 day. The presence of clear zone around fibres on the plates was recorded as an inhibition against the microbial species. 2.7. Mechanical properties The tensile properties of cotton fibre and silver nanoparticles loaded cotton fibres were determined using INSTRON 3369 Universal Testing Machine (Norwood, Massachusetts, USA) running at a crosshead speed of 5 mm/min. The sample fibres were cut into 1cm × 10 cm gauge length is about 5 cm. The tensile parameters, maximum stress, Young’s modulus and % elongation at break, were measured using 10 kg load cell. In each case, 3 samples were tested and the average values are reported. 3. Results and discussion In the present investigation, cotton fibres containing silver nanoparticles were developed by a “green process” without using any synthetic chemical reducing agents and stabilizers. In general, the incorporation of metal nanoparticles into polymer nanofibres can be achieved either by electrospinning of polymer solutions containing metal nanoparticles or by reducing the metal salts in the presence of electrospun polymer nanofibres. The latter method was followed in the preparation of cotton fibres containing Ag nanoparticles. The same method is adopted in the present study. Until now, different approaches have been followed for reducing silver ions to Ag nanoparticles such as UV or ␥-ray irradiation, ultrasound, prolonged reflux or by using chemicals. Often these methods are either expensive or chemically toxic that hampers their bio-compatibility. Further they raised the toxicity issues, especially the skin irritation. Therefore, the aim of the present investigation is to produce silver nanoparticles without using any toxic chemicals or protecting agents, rather by simply employing natural tree leaves extracts. The present approach involves the use of Neelagiri and Marri leaves broth. They basically consists of polysaccharides, composed of p-menthane-3,8-diol, -sitosterol, ␣-d-glucose and mesoinositol. The functional groups present in these molecules are known to reduce silver salts into silver nanoparticles and also capable of providing additional stabilization through their high molecular chains.
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Scheme 1. Schematic illustration of silver nanoparticles loaded cotton fibres preparative method consisting of 3 steps. (1) Cotton fibres immersed in silver nitrate containing extracted leaves solution, (2) The reduction process of silver nitrate into silver nanoparticles takes place by the extracted leaves solution, (3) The formed particles on fibres are stabilized by hydroxyl/carboxyl functional groups of extract solution deposited on nanoparticles and washing with water leads to leach out of few nanoparticles because of poor binding.
The fabrication of silver nanoparticles onto cotton fibres consists of three steps (Scheme 1). In detail, silver nanoparticles were prepared by adding silver nitrate solution into leaves extracts broth. An immediate reduction process takes places in the leaves extracts broth and the formed silver nanoparticles were stabilized by the polysaccharides (Step 1). The cotton fibres were immersed into the stabilized silver nanoparticles solution and were allowed to stand for 24 h under stirring (Step 2). During this step, the silver nanoparticles get deposited onto cotton fibres by interaction through the polysaccharide or any other functional groups present in the leaves extracts. The nanoparticles loaded fibres were removed from the solution. The excess or weakly bound silver nanoparticles on cotton fibres were removed by repeated washings with distilled water (Step 3). Among the three steps, step 1 is important one because this controls the particle size and their stability over a period of time. Therefore, this study initially confirms the feasibility of synthesis of silver nanoparticles in the presence of E. citriodora (Neelagiri) and F. bengalensis (Marri) leaves extracts. 3.1. Synthesis of silver nanoparticles The formation of silver nanoparticles was simply achieved by reducing aqueous silver ions in the presence of E. citriodora (Neelagiri) and F. bengalensis (Marri) leaves extracts. The formation was confirmed by UV–vis spectroscopy. It is well known that silver nanoparticles exhibit ruby red color in water, having an intense absorbance band around 400–450 nm arising due to surface plas-
mon excitation vibrations in the metal nanoparticles [32]. Fig. 1A and B shows the photographs of Neelagiri and Marri tree leaves (A and B) and their leaves extract solutions (colorless solutions, inset A and B). Fig. 1C and D illustrates the UV–vis spectra recorded for the aqueous silver nitrate solution conversion into silver nanoparticles using 2, 4, and 6% of Neelagiri and Marri leaves extracts respectively. The results confirmed that within 2 min the maximum conversion of silver ions into silver nanoparticles has taken place. Intense absorption peaks were observed in the UV–vis spectra at 412, 416 and 424 nm in the case of 2, 4, and 6% of Neelagiri extracts respectively. A clear increase in the intensity of maximum absorption peak as well as a slight shift in the peak wavelength was noticed with increase of % of Neelagiri leaves extracts. The red shift in UV–vis spectra and the broadening of surface plasmonic peak for silver nanoparticles is a possible indication that the particles are growing larger. Similarly, the Marri leaves extracts are also capable to convert silver salts solution into silver nanoparticles. But the immediately formed silver nanoparticles have exhibited surface plasmon resonance peaks at 435, 442 and 461 nm with 2, 4, and 6% of Marri leaves extracts respectively. This peak (towards red shift) clearly indicates that Marri leaves are capable of producing silver nanoparticles in bigger size compared to Neelagiri leaves. This conclusion has been arrived basing on the available literature on silver nanoparticles [8]. The conversion of silver salts into silver nanoparticles is possible by many ways. Among these, various natural and synthetic hydrophilic polymers containing hydroxyl (–OH), carboxylic
Fig. 1. Silver nanoparticles preparation using natural resources. (A) Neelagiri tree leaves, (B) Marri tree leaves. Inset images represent 2% leaves extract solution (color less solutions), (C) UV–vis spectra of nanoparticles formed using 2, 4, and 6% of Neelagiri leaves extract solutions, and (D) UV–vis spectra of nanoparticles formed using 2, 4, and 6% of Marri leaves extract solutions. In set image indicates silver nanoparticles prepared using respective leaves extract solutions.
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Fig. 2. Silver nanoparticles preparation using natural resources in dark room at room temperature. (A) UV–vis spectra of nanoparticles formed using 2, 4, and 6% Neelagiri leaves extract solutions and (B) UV–vis spectra of nanoparticles formed using 2, 4, and 6% Marri leaves extract solutions.
(–COOH), amino (–NH2 ), or thiol (–SH) groups offers reduction capacities in addition to stabilizing the formed nanoparticles [34]. Based on the above information it is thought that leaves extracts containing number of hydroxyl groups along with carbohydrate natural resources are capable of effective reduction and stabilization. Many studies have shown that there is a possibility that sun light (photoreduction) can also reduce the silver nanoparticles into silver nanoparticles [35]. But the process is slow. To confirm the formation of silver nanoparticles is not due to sun light (Photoreduction); experiments were also conducted in dark. The studies have shown (Fig. 2) that even in the dark the leaves extracts gave exactly similar absorption peak intensities in the UV–vis spectra (Fig. 2) within few minutes. 3.2. TEM analysis The formation of silver nanoparticles from silver nitrate in the presence of leaves extracts was confirmed by TEM analysis. The TEM images have shown that the formed silver nanoparticles are spherical in nature with an average particles size of ∼21 nm (Fig. 3). Mostly, the silver nanoparticles prepared by various polymer reduction and stabilization leads to particles of size above 20 nm which is consistent with our results [34].
Fig. 3. Transmission electron microscope images of silver nanoparticles generated from silver nitrate using: (A) Neelagiri and (B) Marri extracts.
and also simultaneously stabilizing the nanoparticles on the cotton fibres. The schematic representation of the formation of silver nanoparticles on cotton fibres is presented in Scheme 1. Cotton is a natural fibre consisting of cellulose with 1,4-d-glucosepyranose as its repeating units [15]. Cellulose has extensive surface area means that cotton fibre has much more surface hydroxyl groups. These surface hydroxyl groups can be used to facilitate the adsorption of silver nanoparticles quite efficiently onto cotton fibres. 3.4. DSC analysis Fig. 4 depicts the DSC thermograms for control cotton fibre and for silver nanoparticles loaded cotton fibres by using 2, 4 and 6% of Neelagiri and Marri leaves extracts. Fig. 4A and B, illustrates two peaks at 370.60 ◦ C and 492.52 ◦ C related to the glass transition and or melting temperatures of control cotton fibres. After the cotton fibres were impregnated with silver nanoparticles using different concentrations of leaves extracts the glass transition temperatures increased between 425 ◦ C and 460.66 ◦ C (Fig. 4A and B). This is an indication for efficient adsorption of silver nanoparticles on the cotton fibres. The absence of melting peaks after silver nanoparticles adsorption on cotton fibres in turn suggests improved thermal characteristics of the cotton fibre silver composites. Similar improvements in properties were found on various modified fibre [36] nanocomposites. 3.5. TGA analysis
3.3. Fabrication of silver nanoparticles loaded cotton fibres The fabrication of silver nanoparticles loaded cotton fibres was developed by in situ synthesis of silver nanoparticles on cotton fabrics. The hydroxyl groups of leaves extracts (polysaccharides) reduce the silver nitrate into silver nanoparticles on cotton fibres
Fig. 5 shows the thermograms recorded for control cotton fibres and silver nanoparticles loaded cotton fibres by using 2, 4 and 6% of Neelagiri and Marri leaves extracts. Cotton fibres shows good thermal stability until 273.42 ◦ C and maximum decomposition occurs at 397.03 ◦ C. 100% weight loss is noticed at 700 ◦ C. In the case of sil-
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Fig. 6. in vitro release patterns of silver nanoparticles from cotton fibres by varying the media such as Neelagiri and Marri leaves extracts.
Fig. 4. Differential scanning calorimetric curves of silver nanoparticles loaded cotton fibres prepared by using: (A) 2, 4, and 6% Neelagiri leaves extract solutions and (B) 2, 4, and 6% Marri leaves extract solutions.
ver nanoparticles loaded cotton fibres, an initial weight loss below 100 ◦ C was observed which may be attributed due to the loss of moisture present on the silver nanoparticles loaded cotton fibres (Fig. 5A and B). Though, the maximum decomposition temperature was lowered slightly after silver nanoparticles deposition on cotton fibres but the overall their thermal stability was extremely improved with increase of percentage (%) of extracts used. An increased concentration of leaves extracts not only helped in forming more nanoparticles and also helped in binding the formed silver nanoparticles more strongly onto the cotton fibres. This behaviour is seen in Fig. 5A and B. The cotton fibres loaded by silver nanoparticles by using 6% Neelagiri or Marri leaves extracts have shown only ∼25–30% weight loss even after 700 ◦ C. This demonstrates that the present methodology is highly successful to incorporate huge amounts of silver nanoparticles on textiles. 3.6. Release of Ag nanoparticles
Fig. 5. Thermogravimetric curves of silver nanoparticles loaded cotton fibres prepared by using: (A) 2, 4, and 6% Neelagiri leaves extract solutions and (B) 2, 4, and 6% Marri leaves extract solutions.
Recent studies have indicated that it is important to know the antibacterial activity over a period of time even after immersion in aqueous environments. To provide efficient antibacterial activity over a period of time a stable and prolonged release of silver nanoparticles/ions is necessary. Silver nanoparticles are relatively nonreactive but in aqueous media releases silver ions which are responsible for accumulation in intracellular levels causing antibacterial effects. To evaluate silver ions release from cotton fibres, silver nanoparticles impregnated cotton fibres were immersed in physiological saline solution at 37 ◦ C for different time intervals and the data is reported based on the UV–vis spectra. The release of Ag ions into saline solution is caused by diffusion process. The results obtained demonstrate that the amount of Ag ions released is different depending on the Neelagiri or Marri extracts concentration used for the preparation of nanoparticles. But, the release is not faster when compared other Ag loaded NPs on different fibres. The reason may be strong association of AgNPs on the cotton fibres by hydroxyl groups [37,38] formed by the leaves extracts. Fig. 6 shows the in vitro release patterns of silver nanoparticles from cotton fibre by varying the media such as Neelagiri and Marri leaves extract. The figure explains the effect of media on the release of silver nanoparticles which are absorbed or adsorbed on the cotton fibre. In comparison with both the leaves extract the Marri leave extract gave faster release, whereas Neelagiri has shown slower release since, better entrapment efficiency was observed for the Marri leaves extract. This is due to the presence of more hydrophilic groups in Marri than in Neelagiri. The silver nanoparticle-loaded cotton fibres, namely N2 and M2 gave 39 and 49% cumulative release upto 168 h, respectively. The release was observed over a period of 168 h. The prolonged release of silver nanoparticles sug-
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Fig. 7. Scanning electron microscope images of control cotton fibres.
Fig. 8. Scanning electron microscope images of silver nanoparticles loaded cotton fibres prepared by using 2, 4, and 6% Neelagiri leaves extract solutions.
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Fig. 9. Scanning electron microscope images of silver nanoparticles loaded cotton fibres prepared by using 2, 4, and 6% Marri leaves extract solutions.
gests the usefulness of the product towards wound curing for a longer period. 3.7. SEM analysis To prove visually that silver nanoparticles were formed on the cotton fibres, cotton fibres controls and silver nanoparticles impregnated fibres were scanned under scanning electron microscopy. Fig. 7 demonstrates that the SEM image of control cotton fibres exhibiting uniform neat plain spun structures. At higher magnifications cotton fibres were characterized containing smooth surfaces. Whereas, the SEM images of silver nanoparticles loaded cotton fibres using Neelagiri leaves extracts clearly
exhibited the presence of Ag nanoparticles on the overall fibres (Fig. 8). For a better view, the fibres were scanned at higher magnifications. The images demonstrated that the impregnated silver nanoparticles were smaller in size and no aggregations were found in all the fibres. Similarly, the Marri leaves extracts employed silver nanoparticles also possess impregnation on the overall fibres. But shows silver clusters made of nanocrystals of quite regular shape characterized by dimensions of the order of 100 nm (Fig. 9). This behaviour was expected because Marri leaves extracts showed complete red shift even at lower concentration in the UV–vis spectra. Overall the surface topography indicated that the fibres are extremely deposited by Ag nanoparticles by the current approach.
Fig. 10. Antibacterial activity of silver nanoparticles loaded cotton fibres prepared by using: (A) Neelagiri leaves extracts and (B) Marri leaves extracts, employing different concentrations, i.e., 2, 4, and 6%. N and M indicates Neelagiri and Marri leaves extracts.
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Fig. 11. Antibacterial activity of silver nanoparticles loaded cotton fibres prepared by using 2% Neelagiri leaves extracts, after washing with water for different time periods. (A) N1, N5, N15, N20, and N30 indicate washed for 1, 5, 15, 20, and 30 min in DI water, and (B) N3h, N5h, N10h and N24h indicate cotton fibres loaded with silver nanoparticles washed in DI water for 3, 5, 10 and 24 h.
Fig. 12. Antibacterial activity of silver nanoparticles loaded cotton fibres prepared by using 2% Marri leaves extracts, after washing with water for different time periods. (A) M5, M10, M15, and M30 indicate washed for 5, 10, 15, and 30 min in DI water, and (B) M1h, M3h, M5h, 10h and 24h indicate cotton fibres loaded with silver nanoparticles washed in DI water for 1, 3, 5, 10 and 24 h, respectively.
3.8. Microbial activity The antimicrobial efficiency of cotton fibres was tested against gram negative bacterium E. coli. Silver nanoparticles loaded cotton fibres were prepared by 2, 4 and 6% using Neelagiri and Marri leave extracts. The antimicrobial action of silver nanoparticles loaded cotton fibres, namely N2, N4, N6 (Fig. 10); and M2, M4, M6 (Fig. 10), were tested against E. coli, taking plain cotton fibre as a control. The results indicated that Ag nanoparticles loaded cotton fibres by employing 2% Neelagiri and Marri leaves extracts exhibited greater reduction of E. coli growth. This is due to the smaller nanoparticles size formation with these composition as well as faster release of AgNPs into the media. Further, these experiments also indicated that Ag loaded NPs cotton fibres obtained by using Neelagiri (2%, i.e., N2) and Marri (2% M2), leaves extracts exhibited similar microbial reduction even after washing with water for different time intervals (Minutes, hours, Figs. 11 and 12). Based on this study it is possible to use these cotton fibres as antibacterial textiles. The investigations of Morones et al. [39] revealed that the presence of smaller AgNPs can be attached readily to the cell
membrane and capable of entering inside the cell and accumulates in the bacteria. These NPs releases silver ions causing major damage to the nuclei resulting in bacterial death. Similar results were also observed in our previous studies [40] in which smaller AgNPs are effective than higher NPs. According to the Standard Antibacterial test “SNV 195920–1992”, more than 1 mm microbial zone inhibition can be considered as a good antibacterial product [41]. The AgNPs containing cotton fibres developed in the present investigation have exhibited > 1.5 mm inhibition zone in all the cases. This indicates that they are highly important from technological point of view for establishing antibacterial finishing’s. 3.9. Mechanical properties Table 1 illustrates the mechanical properties such as maximum stress, Young’s modulus and % elongation at break for cotton fibres modified with silver nanoparticles as well as cotton fibres treated with only leaves extracts. The higher elongation at break for the silver nanoparticles loaded cotton fibres was obtained
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Table 1 Mechanical properties of cotton fibres treated with leaves extracts and silver nanoparticles loaded cotton fibres. Sample code
2% Marri 4% Marri 6% Marri 2% Neelagiri 4% Neelagiri 6% Neelagiri
Stress at max. Load (MPa)
Modulus (MPa)
Elongation at break (%)
Extracts
NPs deposition
Extracts
NPs deposition
Extracts
NPs deposition
39.011 35.283 34.967 33.046 32.431 36.521
33.814 31.212 30.085 33.184 30.539 29.861
294.185 284.512 280.301 255.893 263.279 297.301
358.824 338.321 319.968 332.143 349.387 351.688
23.483 22.033 21.967 26.417 25.775 25.509
17.698 16.733 15.317 18.692 17.911 17.184
than for the control cotton fibre and leaves extracts loaded cotton fibres. This is probably due to the presence of silver nanoparticles bounded to the hydroxyl (OH) groups of cellulose chains in the cotton fibres. The higher modulus and stress at maximum load for the control cotton fibre was obtained than that of leaves extracts loaded cotton fibres and silver nanoparticles loaded cotton fibres. This study indicates that AgNPs embedded cotton fibres maintain good mechanical properties dictating their applicability as wound dressing material for chronic wounds and burns. 4. Conclusion
[13] [14]
[15]
[16]
[17] [18]
From the present investigation, it is concluded that it is possible to develop silver nanoparticles loaded cotton fibres by using natural resources in a easy manner. The SEM analysis indicates that the silver nanoparticles are well dispersed on the cotton fibres. More than that, the inclusion of silver nanoparticles into cotton fibres improved their thermal stability and elongation properties. Further the developed silver nanoparticles loaded cotton fibres have exhibited excellent antimicrobial activity against E.coli. Therefore these fibres have great potential for utilization in burn/wound dressings as well as in the fabrication of antibacterial finishings and textiles.
[19] [20] [21]
[22] [23] [24] [25]
Acknowledgement KMR thanks the Defence Research & Development Organization (DRDO) and Ministry of Defence, Govt. of India, New Delhi and SR thanks the UGC-SAP, New Delhi for the partial financial support.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Fabrication of antibacterial cotton fibres loaded with silver nanoparticles via “Green Approach” S. Ravindra, Y. Murali Mohan 1 , N. Narayana Reddy, K. Mohana Raju ∗ Synthetic Polymer Laboratory, Department of Polymer Science and Technology, Sri Krishnadevaraya University, Anantapur 515055, A.P., India
a r t i c l e
i n f o
Article history: Received 18 January 2010 Received in revised form 8 June 2010 Accepted 10 June 2010 Available online 17 June 2010 Keywords: Cotton fibre Silver nanoparticles Reduction Stability Green process Antibacterial activity Wound dressing
a b s t r a c t In the present investigation the antimicrobial efficiency of cotton fibres loaded with silver nanoparticles (AgNPs) was studied which are developed by “green process” using natural extracts, of Eucalyptus citriodora and Ficus bengalensis. The formation of AgNPs on the cotton fibres was observed by UV–vis spectrophotometer. The size of silver nanoparticles was found to have ∼20 nm. The structure and morphology of silver nanoparticles formed on the cotton fibres were confirmed by electron microscopy. The antibacterial activity of cotton fibres loaded with silver nanoparticles was evaluated against gram-negative Escherichia coli (E. coli) bacteria. The results suggest excellent antibacterial activity by the incorporation of 2% leaf extracts on cotton fibres. These fibres have also exhibited superior antibacterial activity even after several washings indicating their usage in medical and infection prevention applications. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Over the last few decades, considerable research effort was made to produce antibacterial coatings on the surfaces of various objects such as garments and medical devices [1,2]. The current interest is to develop efficient, non-toxic, durable and cost effective antimicrobial finishing textiles with increased applications in medical, health care, hygienic products as well as protective textile materials [3–12]. Cotton fibres are particularly suitable for manufacturing textiles for sports, non-implantable medical products, and health care/hygienic products [3]. However the ability of cotton fibres to absorb large amount of moisture makes them more prone to microbial attack under certain conditions of humidity and temperature. Cotton may acts as a nutrient, becoming suitable medium for bacterial and fungal growth [4,13]. Therefore, cotton fibres are treated with numerous chemicals to get better antimicrobial cotton textiles [14–22]. Among the various antimicrobial agents, silver nanoparticles (AgNPs) have shown strong inhibitory and antibacterial effects [23,24]. Among the various synthetic methods used for the preparation of silver nanoparticles, chemical reducing method by using a reducing agent such as sodium borohydrate, citrate or
∗ Corresponding author. Tel.: +91 8554 255655; fax: +91 8554 255655. E-mail address: [email protected] (K. Mohana Raju). 1 Present address: Cancer Biology Research Center, Sanford Research/USD, Sioux Falls, SD 57105, USA. 0927-7757/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2010.06.013
ascorbate in a silver salt solution is most common [25,26]. Synthetic reducing agents are normally associated with environmental toxicity or biological hazards. Therefore the development of silver nanoparticles based on natural extracts is considered as most appropriate method for environmental reasons. The present research is mainly focusing on the development of antimicrobial surfaces [27–29]. Of late, the incorporation of silver nanoparticles on cotton fibres has received great attention due to their high resistance to microbes. Vigneshwaran et al. [30] developed the in situ synthesis of silver nanoparticles on cotton fabrics. The terminal aldehyde group of starch not only reduces the silver nitrate into silver nanoparticles but also stabilizes them on fabric simultaneously. Perelshtein et al. [31] developed sonochemical method of coating of silver nanoparticles using ultra sound waves [31]. Duran et al. [32] incorporated silver nanoparticles on cotton fabrics having good antibacterial property by fungal process. Yu et al. [33] also reported the incorporation of silver nanoparticles onto ultrafine fibres by electro spinning. The present study involves the incorporation of silver nanoparticles on cotton fibres by “green process”. The advantages of this process are: (a) no need to have extra reducing agent(s), (b) the process can be conducted at room temperature, under normal pressure in aqueous solution, and (c) finally the developed silver nanoparticles by this process have excellent properties, including long term dispersion stability. The silver nanoparticles incorporated cotton fibres were systematically characterized and their mechanical properties were also evaluated. The antibacterial property of silver
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nanoparticles loaded cotton fibres were tested against Escherichia coli (E. coli) bacteria. 2. Materials and methods 2.1. Materials Eucalyptus citriodora (Neelagiri) and Ficus bengalensis (Marri) leaves were collected from their trees that are available in the campus of Sri Krishnadevaraya University, Anantapur, India. Silver nitrate (AgNO3 ) was purchased from Merck India Ltd. (Mumbai, India) and used as received without further purification. Cotton fibres (1 mm thickness) were purchased from SIMCO Thread mills (Salem, Chennai, India). Distilled water was used throughout this investigation. 2.2. Preparation of the leaves extract E. citriodora (Neelagiri) and F. bengalensis (Marri) leaves were collected and thoroughly washed with distilled water for 15 min at room temperature (25 ◦ C) and dried for 1 day at room temperature. The tree leaf broth solutions were prepared by taking 2, 4 and 6 g of washed leaves in an Erlenmeyer flask containing 100 ml of distilled water. The solution was heated at 100 ◦ C to extract the contents of the leaves for 30 min and filtered through 0.45 m PVDF Millex Filter units using 50 ml syringe. The extracted leaves solutions were stored at 4 ◦ C and used within a week to produce silver nanoparticles. 2.3. Synthesis of silver nanoparticles In a typical experiment 5 ml (2, 4 and 6 g) of washed leaves put in an Erlenmeyer flask containing 100 ml of distilled water and heated to 100 ◦ C for 30 min. These fresh leaves extract was added to a conical flask containing 5 ml of 1 mM aqueous AgNO3 solution (170 mg AgNO3 in 100 ml distilled water) at room temperature. The silver ions were reduced to silver nanoparticles within 2–5 min by the leaves extract. This quick conversion was observed by color change from color less to brown color. The reduction of silver nitrate into silver nanoparticles formation was monitored by using UV–vis spectrophotometer. 2.4. Impregnation of silver nanoparticles onto cotton fibres Silver nanoparticles were produced within the cotton fibres by leaf broth reduction of Ag+ ions. Pre weighed cotton fibres were immersed in the filtrate leaf broth containing silver nanopaticles and kept on a shaker at 100 rpm at room temperature for 24 h and then dried. 2.5. Characterization 2.5.1. Differential scanning calorimetry (DSC) DSC analysis of cotton fibres and silver nanoparticles incorporated cotton fibres were performed using SDT Q 600 thermal analyzer (T.A. Instruments-water LLC, Newcastle, DE, USA), from 40 to 700 ◦ C at a heating rate of 20 ◦ C per min, under a constant flow (100 ml/min) of nitrogen gas. 2.5.2. Thermogravimetric analysis (TGA) The thermal history of cotton fibre and silver nanoparticles loaded cotton fibres were evaluated on a SDT Q 600 TGA instrument (T.A. Instruments-water LLC, Newcastle, DE, USA), at a heating rate of 20 ◦ C/min under a constant nitrogen flow (100 ml/min). The samples were run from 40 to 700 ◦ C.
2.5.3. Transmission electron microscopy (TEM) The size of the silver nanoparticles was determined using a Technai F12 TEM (Philips Electron Optics, Holland). TEM samples were prepared by dropping 2–3 drops of aqueous solutions of silver nanoparticles on a 200 mesh formvar-coated copper TEM grid (grid size: 97 m) (Ted Pella, Inc., Redding, CA, USA) followed by removing excess solution using a piece of fine filter paper and the samples were allowed to dry in air overnight prior to image particles. 2.5.4. Scanning electron microscopy (SEM) The surface morphology of cotton fibres and silver nanoparticles loaded cotton fibres were studied using a JEOL JSM 840A (Tokyo, Japan) scanning electron microscope (SEM) at an accelerating voltage of 15 kV. All the samples were dried in vacuum at room temperature and coated with gold before scanning. Surface morphologies were imaged at different magnifications. 2.6. Microbial activity The antibacterial activities of fibres and fibres loaded with silver nanoparticles were tested by an inhibition zone method. In this method, E. coli was taken as the model bacteria. For this study, the fibres (cotton fibre and silver nanoparticles loaded cotton fibres) were cut into small pieces (1 mm thickness and 1 cm length), put together to form a circular zone, and the antimicrobial activity was tested using modified agar diffusion assay (disc test). The plates were examined for possible clear zone formation after incubation at 37 ◦ C for 1 day. The presence of clear zone around fibres on the plates was recorded as an inhibition against the microbial species. 2.7. Mechanical properties The tensile properties of cotton fibre and silver nanoparticles loaded cotton fibres were determined using INSTRON 3369 Universal Testing Machine (Norwood, Massachusetts, USA) running at a crosshead speed of 5 mm/min. The sample fibres were cut into 1cm × 10 cm gauge length is about 5 cm. The tensile parameters, maximum stress, Young’s modulus and % elongation at break, were measured using 10 kg load cell. In each case, 3 samples were tested and the average values are reported. 3. Results and discussion In the present investigation, cotton fibres containing silver nanoparticles were developed by a “green process” without using any synthetic chemical reducing agents and stabilizers. In general, the incorporation of metal nanoparticles into polymer nanofibres can be achieved either by electrospinning of polymer solutions containing metal nanoparticles or by reducing the metal salts in the presence of electrospun polymer nanofibres. The latter method was followed in the preparation of cotton fibres containing Ag nanoparticles. The same method is adopted in the present study. Until now, different approaches have been followed for reducing silver ions to Ag nanoparticles such as UV or ␥-ray irradiation, ultrasound, prolonged reflux or by using chemicals. Often these methods are either expensive or chemically toxic that hampers their bio-compatibility. Further they raised the toxicity issues, especially the skin irritation. Therefore, the aim of the present investigation is to produce silver nanoparticles without using any toxic chemicals or protecting agents, rather by simply employing natural tree leaves extracts. The present approach involves the use of Neelagiri and Marri leaves broth. They basically consists of polysaccharides, composed of p-menthane-3,8-diol, -sitosterol, ␣-d-glucose and mesoinositol. The functional groups present in these molecules are known to reduce silver salts into silver nanoparticles and also capable of providing additional stabilization through their high molecular chains.
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Scheme 1. Schematic illustration of silver nanoparticles loaded cotton fibres preparative method consisting of 3 steps. (1) Cotton fibres immersed in silver nitrate containing extracted leaves solution, (2) The reduction process of silver nitrate into silver nanoparticles takes place by the extracted leaves solution, (3) The formed particles on fibres are stabilized by hydroxyl/carboxyl functional groups of extract solution deposited on nanoparticles and washing with water leads to leach out of few nanoparticles because of poor binding.
The fabrication of silver nanoparticles onto cotton fibres consists of three steps (Scheme 1). In detail, silver nanoparticles were prepared by adding silver nitrate solution into leaves extracts broth. An immediate reduction process takes places in the leaves extracts broth and the formed silver nanoparticles were stabilized by the polysaccharides (Step 1). The cotton fibres were immersed into the stabilized silver nanoparticles solution and were allowed to stand for 24 h under stirring (Step 2). During this step, the silver nanoparticles get deposited onto cotton fibres by interaction through the polysaccharide or any other functional groups present in the leaves extracts. The nanoparticles loaded fibres were removed from the solution. The excess or weakly bound silver nanoparticles on cotton fibres were removed by repeated washings with distilled water (Step 3). Among the three steps, step 1 is important one because this controls the particle size and their stability over a period of time. Therefore, this study initially confirms the feasibility of synthesis of silver nanoparticles in the presence of E. citriodora (Neelagiri) and F. bengalensis (Marri) leaves extracts. 3.1. Synthesis of silver nanoparticles The formation of silver nanoparticles was simply achieved by reducing aqueous silver ions in the presence of E. citriodora (Neelagiri) and F. bengalensis (Marri) leaves extracts. The formation was confirmed by UV–vis spectroscopy. It is well known that silver nanoparticles exhibit ruby red color in water, having an intense absorbance band around 400–450 nm arising due to surface plas-
mon excitation vibrations in the metal nanoparticles [32]. Fig. 1A and B shows the photographs of Neelagiri and Marri tree leaves (A and B) and their leaves extract solutions (colorless solutions, inset A and B). Fig. 1C and D illustrates the UV–vis spectra recorded for the aqueous silver nitrate solution conversion into silver nanoparticles using 2, 4, and 6% of Neelagiri and Marri leaves extracts respectively. The results confirmed that within 2 min the maximum conversion of silver ions into silver nanoparticles has taken place. Intense absorption peaks were observed in the UV–vis spectra at 412, 416 and 424 nm in the case of 2, 4, and 6% of Neelagiri extracts respectively. A clear increase in the intensity of maximum absorption peak as well as a slight shift in the peak wavelength was noticed with increase of % of Neelagiri leaves extracts. The red shift in UV–vis spectra and the broadening of surface plasmonic peak for silver nanoparticles is a possible indication that the particles are growing larger. Similarly, the Marri leaves extracts are also capable to convert silver salts solution into silver nanoparticles. But the immediately formed silver nanoparticles have exhibited surface plasmon resonance peaks at 435, 442 and 461 nm with 2, 4, and 6% of Marri leaves extracts respectively. This peak (towards red shift) clearly indicates that Marri leaves are capable of producing silver nanoparticles in bigger size compared to Neelagiri leaves. This conclusion has been arrived basing on the available literature on silver nanoparticles [8]. The conversion of silver salts into silver nanoparticles is possible by many ways. Among these, various natural and synthetic hydrophilic polymers containing hydroxyl (–OH), carboxylic
Fig. 1. Silver nanoparticles preparation using natural resources. (A) Neelagiri tree leaves, (B) Marri tree leaves. Inset images represent 2% leaves extract solution (color less solutions), (C) UV–vis spectra of nanoparticles formed using 2, 4, and 6% of Neelagiri leaves extract solutions, and (D) UV–vis spectra of nanoparticles formed using 2, 4, and 6% of Marri leaves extract solutions. In set image indicates silver nanoparticles prepared using respective leaves extract solutions.
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Fig. 2. Silver nanoparticles preparation using natural resources in dark room at room temperature. (A) UV–vis spectra of nanoparticles formed using 2, 4, and 6% Neelagiri leaves extract solutions and (B) UV–vis spectra of nanoparticles formed using 2, 4, and 6% Marri leaves extract solutions.
(–COOH), amino (–NH2 ), or thiol (–SH) groups offers reduction capacities in addition to stabilizing the formed nanoparticles [34]. Based on the above information it is thought that leaves extracts containing number of hydroxyl groups along with carbohydrate natural resources are capable of effective reduction and stabilization. Many studies have shown that there is a possibility that sun light (photoreduction) can also reduce the silver nanoparticles into silver nanoparticles [35]. But the process is slow. To confirm the formation of silver nanoparticles is not due to sun light (Photoreduction); experiments were also conducted in dark. The studies have shown (Fig. 2) that even in the dark the leaves extracts gave exactly similar absorption peak intensities in the UV–vis spectra (Fig. 2) within few minutes. 3.2. TEM analysis The formation of silver nanoparticles from silver nitrate in the presence of leaves extracts was confirmed by TEM analysis. The TEM images have shown that the formed silver nanoparticles are spherical in nature with an average particles size of ∼21 nm (Fig. 3). Mostly, the silver nanoparticles prepared by various polymer reduction and stabilization leads to particles of size above 20 nm which is consistent with our results [34].
Fig. 3. Transmission electron microscope images of silver nanoparticles generated from silver nitrate using: (A) Neelagiri and (B) Marri extracts.
and also simultaneously stabilizing the nanoparticles on the cotton fibres. The schematic representation of the formation of silver nanoparticles on cotton fibres is presented in Scheme 1. Cotton is a natural fibre consisting of cellulose with 1,4-d-glucosepyranose as its repeating units [15]. Cellulose has extensive surface area means that cotton fibre has much more surface hydroxyl groups. These surface hydroxyl groups can be used to facilitate the adsorption of silver nanoparticles quite efficiently onto cotton fibres. 3.4. DSC analysis Fig. 4 depicts the DSC thermograms for control cotton fibre and for silver nanoparticles loaded cotton fibres by using 2, 4 and 6% of Neelagiri and Marri leaves extracts. Fig. 4A and B, illustrates two peaks at 370.60 ◦ C and 492.52 ◦ C related to the glass transition and or melting temperatures of control cotton fibres. After the cotton fibres were impregnated with silver nanoparticles using different concentrations of leaves extracts the glass transition temperatures increased between 425 ◦ C and 460.66 ◦ C (Fig. 4A and B). This is an indication for efficient adsorption of silver nanoparticles on the cotton fibres. The absence of melting peaks after silver nanoparticles adsorption on cotton fibres in turn suggests improved thermal characteristics of the cotton fibre silver composites. Similar improvements in properties were found on various modified fibre [36] nanocomposites. 3.5. TGA analysis
3.3. Fabrication of silver nanoparticles loaded cotton fibres The fabrication of silver nanoparticles loaded cotton fibres was developed by in situ synthesis of silver nanoparticles on cotton fabrics. The hydroxyl groups of leaves extracts (polysaccharides) reduce the silver nitrate into silver nanoparticles on cotton fibres
Fig. 5 shows the thermograms recorded for control cotton fibres and silver nanoparticles loaded cotton fibres by using 2, 4 and 6% of Neelagiri and Marri leaves extracts. Cotton fibres shows good thermal stability until 273.42 ◦ C and maximum decomposition occurs at 397.03 ◦ C. 100% weight loss is noticed at 700 ◦ C. In the case of sil-
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Fig. 6. in vitro release patterns of silver nanoparticles from cotton fibres by varying the media such as Neelagiri and Marri leaves extracts.
Fig. 4. Differential scanning calorimetric curves of silver nanoparticles loaded cotton fibres prepared by using: (A) 2, 4, and 6% Neelagiri leaves extract solutions and (B) 2, 4, and 6% Marri leaves extract solutions.
ver nanoparticles loaded cotton fibres, an initial weight loss below 100 ◦ C was observed which may be attributed due to the loss of moisture present on the silver nanoparticles loaded cotton fibres (Fig. 5A and B). Though, the maximum decomposition temperature was lowered slightly after silver nanoparticles deposition on cotton fibres but the overall their thermal stability was extremely improved with increase of percentage (%) of extracts used. An increased concentration of leaves extracts not only helped in forming more nanoparticles and also helped in binding the formed silver nanoparticles more strongly onto the cotton fibres. This behaviour is seen in Fig. 5A and B. The cotton fibres loaded by silver nanoparticles by using 6% Neelagiri or Marri leaves extracts have shown only ∼25–30% weight loss even after 700 ◦ C. This demonstrates that the present methodology is highly successful to incorporate huge amounts of silver nanoparticles on textiles. 3.6. Release of Ag nanoparticles
Fig. 5. Thermogravimetric curves of silver nanoparticles loaded cotton fibres prepared by using: (A) 2, 4, and 6% Neelagiri leaves extract solutions and (B) 2, 4, and 6% Marri leaves extract solutions.
Recent studies have indicated that it is important to know the antibacterial activity over a period of time even after immersion in aqueous environments. To provide efficient antibacterial activity over a period of time a stable and prolonged release of silver nanoparticles/ions is necessary. Silver nanoparticles are relatively nonreactive but in aqueous media releases silver ions which are responsible for accumulation in intracellular levels causing antibacterial effects. To evaluate silver ions release from cotton fibres, silver nanoparticles impregnated cotton fibres were immersed in physiological saline solution at 37 ◦ C for different time intervals and the data is reported based on the UV–vis spectra. The release of Ag ions into saline solution is caused by diffusion process. The results obtained demonstrate that the amount of Ag ions released is different depending on the Neelagiri or Marri extracts concentration used for the preparation of nanoparticles. But, the release is not faster when compared other Ag loaded NPs on different fibres. The reason may be strong association of AgNPs on the cotton fibres by hydroxyl groups [37,38] formed by the leaves extracts. Fig. 6 shows the in vitro release patterns of silver nanoparticles from cotton fibre by varying the media such as Neelagiri and Marri leaves extract. The figure explains the effect of media on the release of silver nanoparticles which are absorbed or adsorbed on the cotton fibre. In comparison with both the leaves extract the Marri leave extract gave faster release, whereas Neelagiri has shown slower release since, better entrapment efficiency was observed for the Marri leaves extract. This is due to the presence of more hydrophilic groups in Marri than in Neelagiri. The silver nanoparticle-loaded cotton fibres, namely N2 and M2 gave 39 and 49% cumulative release upto 168 h, respectively. The release was observed over a period of 168 h. The prolonged release of silver nanoparticles sug-
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Fig. 7. Scanning electron microscope images of control cotton fibres.
Fig. 8. Scanning electron microscope images of silver nanoparticles loaded cotton fibres prepared by using 2, 4, and 6% Neelagiri leaves extract solutions.
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Fig. 9. Scanning electron microscope images of silver nanoparticles loaded cotton fibres prepared by using 2, 4, and 6% Marri leaves extract solutions.
gests the usefulness of the product towards wound curing for a longer period. 3.7. SEM analysis To prove visually that silver nanoparticles were formed on the cotton fibres, cotton fibres controls and silver nanoparticles impregnated fibres were scanned under scanning electron microscopy. Fig. 7 demonstrates that the SEM image of control cotton fibres exhibiting uniform neat plain spun structures. At higher magnifications cotton fibres were characterized containing smooth surfaces. Whereas, the SEM images of silver nanoparticles loaded cotton fibres using Neelagiri leaves extracts clearly
exhibited the presence of Ag nanoparticles on the overall fibres (Fig. 8). For a better view, the fibres were scanned at higher magnifications. The images demonstrated that the impregnated silver nanoparticles were smaller in size and no aggregations were found in all the fibres. Similarly, the Marri leaves extracts employed silver nanoparticles also possess impregnation on the overall fibres. But shows silver clusters made of nanocrystals of quite regular shape characterized by dimensions of the order of 100 nm (Fig. 9). This behaviour was expected because Marri leaves extracts showed complete red shift even at lower concentration in the UV–vis spectra. Overall the surface topography indicated that the fibres are extremely deposited by Ag nanoparticles by the current approach.
Fig. 10. Antibacterial activity of silver nanoparticles loaded cotton fibres prepared by using: (A) Neelagiri leaves extracts and (B) Marri leaves extracts, employing different concentrations, i.e., 2, 4, and 6%. N and M indicates Neelagiri and Marri leaves extracts.
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Fig. 11. Antibacterial activity of silver nanoparticles loaded cotton fibres prepared by using 2% Neelagiri leaves extracts, after washing with water for different time periods. (A) N1, N5, N15, N20, and N30 indicate washed for 1, 5, 15, 20, and 30 min in DI water, and (B) N3h, N5h, N10h and N24h indicate cotton fibres loaded with silver nanoparticles washed in DI water for 3, 5, 10 and 24 h.
Fig. 12. Antibacterial activity of silver nanoparticles loaded cotton fibres prepared by using 2% Marri leaves extracts, after washing with water for different time periods. (A) M5, M10, M15, and M30 indicate washed for 5, 10, 15, and 30 min in DI water, and (B) M1h, M3h, M5h, 10h and 24h indicate cotton fibres loaded with silver nanoparticles washed in DI water for 1, 3, 5, 10 and 24 h, respectively.
3.8. Microbial activity The antimicrobial efficiency of cotton fibres was tested against gram negative bacterium E. coli. Silver nanoparticles loaded cotton fibres were prepared by 2, 4 and 6% using Neelagiri and Marri leave extracts. The antimicrobial action of silver nanoparticles loaded cotton fibres, namely N2, N4, N6 (Fig. 10); and M2, M4, M6 (Fig. 10), were tested against E. coli, taking plain cotton fibre as a control. The results indicated that Ag nanoparticles loaded cotton fibres by employing 2% Neelagiri and Marri leaves extracts exhibited greater reduction of E. coli growth. This is due to the smaller nanoparticles size formation with these composition as well as faster release of AgNPs into the media. Further, these experiments also indicated that Ag loaded NPs cotton fibres obtained by using Neelagiri (2%, i.e., N2) and Marri (2% M2), leaves extracts exhibited similar microbial reduction even after washing with water for different time intervals (Minutes, hours, Figs. 11 and 12). Based on this study it is possible to use these cotton fibres as antibacterial textiles. The investigations of Morones et al. [39] revealed that the presence of smaller AgNPs can be attached readily to the cell
membrane and capable of entering inside the cell and accumulates in the bacteria. These NPs releases silver ions causing major damage to the nuclei resulting in bacterial death. Similar results were also observed in our previous studies [40] in which smaller AgNPs are effective than higher NPs. According to the Standard Antibacterial test “SNV 195920–1992”, more than 1 mm microbial zone inhibition can be considered as a good antibacterial product [41]. The AgNPs containing cotton fibres developed in the present investigation have exhibited > 1.5 mm inhibition zone in all the cases. This indicates that they are highly important from technological point of view for establishing antibacterial finishing’s. 3.9. Mechanical properties Table 1 illustrates the mechanical properties such as maximum stress, Young’s modulus and % elongation at break for cotton fibres modified with silver nanoparticles as well as cotton fibres treated with only leaves extracts. The higher elongation at break for the silver nanoparticles loaded cotton fibres was obtained
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Table 1 Mechanical properties of cotton fibres treated with leaves extracts and silver nanoparticles loaded cotton fibres. Sample code
2% Marri 4% Marri 6% Marri 2% Neelagiri 4% Neelagiri 6% Neelagiri
Stress at max. Load (MPa)
Modulus (MPa)
Elongation at break (%)
Extracts
NPs deposition
Extracts
NPs deposition
Extracts
NPs deposition
39.011 35.283 34.967 33.046 32.431 36.521
33.814 31.212 30.085 33.184 30.539 29.861
294.185 284.512 280.301 255.893 263.279 297.301
358.824 338.321 319.968 332.143 349.387 351.688
23.483 22.033 21.967 26.417 25.775 25.509
17.698 16.733 15.317 18.692 17.911 17.184
than for the control cotton fibre and leaves extracts loaded cotton fibres. This is probably due to the presence of silver nanoparticles bounded to the hydroxyl (OH) groups of cellulose chains in the cotton fibres. The higher modulus and stress at maximum load for the control cotton fibre was obtained than that of leaves extracts loaded cotton fibres and silver nanoparticles loaded cotton fibres. This study indicates that AgNPs embedded cotton fibres maintain good mechanical properties dictating their applicability as wound dressing material for chronic wounds and burns. 4. Conclusion
[13] [14]
[15]
[16]
[17] [18]
From the present investigation, it is concluded that it is possible to develop silver nanoparticles loaded cotton fibres by using natural resources in a easy manner. The SEM analysis indicates that the silver nanoparticles are well dispersed on the cotton fibres. More than that, the inclusion of silver nanoparticles into cotton fibres improved their thermal stability and elongation properties. Further the developed silver nanoparticles loaded cotton fibres have exhibited excellent antimicrobial activity against E.coli. Therefore these fibres have great potential for utilization in burn/wound dressings as well as in the fabrication of antibacterial finishings and textiles.
[19] [20] [21]
[22] [23] [24] [25]
Acknowledgement KMR thanks the Defence Research & Development Organization (DRDO) and Ministry of Defence, Govt. of India, New Delhi and SR thanks the UGC-SAP, New Delhi for the partial financial support.
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