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A green approach to synthesize silver nanoparticles in starch-co-poly(acrylamide) hydrogels by Tridax procumbens leaf extract and their antibacterial activity.

1. Introduction

Hydrogels have three-dimensional polymeric networks that are fabricated from polymers stabilized through physical or chemical crosslinking. They absorb large quantities of water without losing their structural integrity [1]. Since they mimic body tissues and respond to external stimuli, they are made important and promising forms of biomaterials for various applications including tissue engineering, controlled drug release devices, biosensors, and mechanical actuators [2-4]. Due to the presence of water solubilizing groups, such as -OH, -COOH, -CON[H.sub.2], -CONH-, and - S[O.sub.3]H, these hydrogels show higher hydrophilicity. The three-dimensional network of hydrogel provides relative stability to its structure. Their swollen state results from a balance between the dispersing forces acting on hydrated chains and cohesive forces that do not prevent the penetration of water through the network [5]. Based on these properties the hydrogels have been used recently as templates for production of metallic nanoparticles. Hence the design and development of metal nanoparticles dispersed in polymer matrix have attracted potential applications in various fields like electrical, optical, or mechanical properties [6, 7] making them valuable for applications in areas like optics [8], photo imaging and patterning [9], electronic devices [10], sensors and biosensors [11-13], catalysis [14,15], and antibacterial and antimicrobial coatings [16]. Silver nanoparticles (Ag NPs) have attracted considerable interest in biological studies because of their ease of preparation, good biocompatibility, and relatively large surface area [17,18]. Ag NPs have many important applications in biomedical fields, sensors, and filters [19]. Additionally, silver is a potential antibacterial agent [20] and is thus used as a sterilizer for removing bacteria from drinking water [21, 22]. For economic and efficient use of silver, silver nanoparticle composites have been developed and tested for antimicrobial activity.

Starch is one of the natural, renewable, and biodegradable polymers produced by many plants as a source of stored energy. Starch consists of two types of molecules: amylose (normally 20-30%) and amylopectin (normally 70-80%). The relative proportion of amylose to amylopectin depends on the source of starch. It is versatile and cheap and has many uses as thickener, water binder, emulsion stabilizer, and gelling agent [23]. Starch granules are insoluble in cold water but imbibe water reversibly and swell slightly [24]. Maleic acid (MA) is a nontoxic and biodegradable hydrophilic substance which is used as a type of food additive in processing industry. The modification of starch with MA increases its hydrophilicity by converting it into ester form by the reaction between starch and MA to form starch macromonomer [25]. The chemical modification of starch with MA and tartaric acid improves its properties without sacrificing its biodegradability and biocompatibility [25-27]. Several reports are present on acrylamide (AAm) hydrogels that were developed because of their hydrophilic and inert nature that makes them suitable for applications in medicine and pharmacy [28,29]. Polyacrylamide has been used in contact lenses for a long time, and it has well been evaluated as a sustained-release wound-dressing material [30].

In continuation of our work [31] on usage of polymers in biomedical applications, the present research paper explores a simple and facile method to synthesize starch-co-poly (acrylamide) (starch-co-PAAm) hydrogels and silver nanocomposite hydrogels (SNCHs) by using a green process in which the [Ag.sup.+] ions are successfully reduced by Tridax procumbens (TD) leaf extract. Starch, MA, and AAm were chosen on the basis of their significant characteristics in biomedical fields. The most popular way used for the direct synthesis of metal ions into nanoparticles is through chemical reduction method by using several reagents such as sodium borohydride, sodium hypophosphite, and sodium citrate. Several reports are presented for the development of Ag NPs by using such reducing agents [32, 33]. But these reducing agents may have environmental problems due to their toxicity, so a considerable interest to use nontoxic materials for reduction of Ag NPs is growing rapidly these days. In order to avoid such type of toxic reagents, recently plant extracts have been used for production of Ag NPs due to their cost-effective nature and also ambient conditions for reduction [34-37]. Therefore, the development of metal nanoparticles based on natural extracts is considered the most appropriate method. Recently Tridax procumbens (TD) leaf extract was used as reducing agent for preparation of Ag NPs by direct synthesis. TD contains several flavonoids, alkyl esters, terpenes, sterols, fatty acids, and polysaccharides. Due to the presence of these constituents, TD has several potential therapeutic activities like antiviral, antioxidant, and antibiotic efficacies; wound-healing activity; insecticidal and anti-inflammatory activity [38]. Now we have used this extract for production of Ag NPs in starch-co-PAAm hydrogel networks due to its natural reduction property and high biological activity. The developed SNCHs may be biodegradable and nontoxic and were potential materials to be used for biomedical applications.

2. Experimental

2.1. Materials. AR grade soluble starch (potato), silver nitrate (AgN[O.sub.3]), potassium persulfate (KPS), maleic acid (MA), and sodium hydroxide (NaOH) were purchased from SD Fine Chemicals, Mumbai, India. Acrylamide (AAm), N,[N.sup.1]-methylenebisacrylamide (MBA), and N,N,[N.sup.1],[N.sup.11]-tetramethyl-ethylenediamine (TEMED) were purchased from Aldrich Chemical Company Inc. (Milwaukee, WI, USA). All chemicals were used without further purification. Double-distilled (DD) water is used throughout the experiment.

2.2. Preparation of Tridax procumbens (TD) Leaf Extract. Leaf extracts were prepared by a green process technique, using the standard procedure described by Prasad et al. [39]. Tridax procumbens leaves were collected from Tridax procumbens plants at our university campus in Sri Krishnadevaraya University (Anantapur, Andhra Pradesh, India), thoroughly washed with DD water to remove the dust particles, and then dried in sun to remove the residual moisture. From this 20 g leaves were chopped finely and boiled in a 250 mL glass beaker along with 200 mL of sterile DD water for 10 min in order to extract the contents of the leaves. After boiling the colour, the aqueous solution changed from watery to yellow colour. The aqueous extract was separated by filtration with 2.5 [micro]m Whatman filter paper (Sigma-Aldrich, USA) and then stored at room temperature.

2.3. Synthesis of Starch-co-PAAm Hydrogels and Their Silver Nanocomposite Hydrogels (SNCHs). Gelatinized starch solution was prepared by mixing a quantitative amount of starch powder in 10.0 mL of DD water and 1.0 mL of 0.5 M sodium hydroxide (NaOH) solution, and the mixture was heated in a water bath at 90[degrees]C for 10 min with continuous magnetic stirring. A predetermined amount of MA was then added to the gelatinized starch solution and the resulting mixture was further heated in a water bath at 80[degrees]C for 4h [25]. Subsequently, a quantitative amount of acrylamide was added and stirred for half an hour at 50[degrees] C, after that crosslinker (MBA) and initiator (KPS) were added according to the formulations mentioned in the Table 1. Finally, an aqueous solution of TEMED was added to the solution and the same temperature was maintained for another 10 min. After completion of free radical polymerization, the synthesized copolymeric hydrogel was taken out and immersed in double-distilled water at room temperature for 24 h to remove the excess of unreacted monomers and reagents present in the hydrogel network. During this period, DD water was refreshed for every 12 h in order to leach out the residue effectively. Finally, the hydrogel was dried at ambient temperature for 48 h. Similarly, other hydrogels were prepared by the above procedure according to their feed compositions as per Table 1.

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Accurately weighed dried starch-co-PAAm hydrogels were equilibrated with DD water for 48 h and immediately transferred to a beaker containing 100 mL of 5 mM AgN[O.sub.3] aqueous solution and then allowed to equilibrate for 24 h. During this process, most of the silver ions ([Ag.sup.+]) were exchanged from solution into free network spaces of copolymeric hydrogel. The hydrogel with absorbed [Ag.sup.+] ions was finally transferred to a beaker containing 50 mL Tridax procumbens leaf extract aqueous solution and kept for 24 h for reduction of [Ag.sup.+] ions into silver nanoparticles ([Ag.sup.0]). After reduction of [Ag.sup.+] ions, the hydrogel was turned into brown colour, which confirms the formation of [Ag.sup.0] nanoparticles in hydrogel matrix [31,33]. The above procedure was adopted for all other remaining hydrogels to convert them into SNCHs. The formation of hydrogels and silver nanocomposite hydrogels is shown in Scheme 1.

2.4. Swelling Studies. Fully dried copolymeric hydrogels were weighed and equilibrated in DD water at 37[degrees]C for three days. The equilibrium swelling capacity (Q) of the hydrogel was calculated by the following equation:

Q = [W.sub.s]/[W.sub.d], (1)

where [W.sub.s] and [W.sub.d] are the weight of swollen hydrogel and weight of the dry hydrogel, respectively. The data provided is an average value of 3 individual sample readings.

2.5. Characterization Methods. Fourier transform infrared (FT-IR) spectra of hydrogel and SNCH are recorded on Perkin Elmer (model impact 410, Wisconsin, MI, USA) spectrophotometer. The samples were crushed with potassium bromide (KBr) to make pellets under hydraulic pressure of 600 kg and scanned between 4000 and 400 [cm.sup.-1]. UV-visible (UV-Vis) spectra of starch-co-PAAm hydrogel and SNCHs were recorded using UV-Vis spectrophotometer (Perkin Elmer). For this, the grinded samples (10 mg/mL) were stored for 10 days to leach out Ag NPs into water (media) and then the measurements were carried out. X-ray diffraction (X-RD) measurements were carried out using Rikagu diffractometer (Cu radiation, [lambda] = 0.1546 nm) running at 40 kV and 40 mA. TGA curves of hydrogel and SNCH are recorded using TA instrument's sequential thermal analyser (Model-SDT Q600, USA). The samples were heated from room temperature to 700[degrees]C at a heating rate of 10[degrees]C per min. Scanning electron microscopy (SEM) images were taken with a high resolution SEM instrument (MIRA LMH, H.S.) at an accelerating voltage of 15 kV. SEM specimens were prepared by the dry hydrogel and SNCHs, coated with a thin layer of palladium and gold alloy, and then studied for morphological variations. The size and shape of [Ag.sup.0] nanoparticles were investigated by transmission electron microscopy (TEM) (FEI Tecnai [G.sup.2] S-Twin 200 kV) at an acceleration voltage of 15 kV. For TEM measurements, the samples were prepared by dropping 5-10 [micro]L of finely grinded SNCH dispersion on a carbon-coated copper grid and dried at room temperature after removing excess moisture using filter paper.

2.6. Antibacterial Activity of Silver Nanocomposite Hydrogels. Antimicrobial activity studies of starch-co-PAAm hydrogel and its SNCHs were carried out by disc diffusion method [40, 41] on bacterial strains, namely, Escherichia coli and Bacillus. Nutrient agar medium was prepared by mixing peptone (5.0 g), beef extract (3.0 g), and sodium chloride (NaCl) (5.0 g) in 1,000 mL distilled water and the pH was adjusted to 7.0. Finally, agar (15.0 g) was added to this solution and this medium was sterilized in an autoclave at a pressure of 15 lbs for 30 min at 121[degrees]C. This medium was transferred into sterilized Petri dishes in a laminar air flow chamber. After solidification of the media, Escherichia coli and Bacillus culture (50 [micro]L) was spread on the solid surface of the media. Paper discs (6 mm diameter) were soaked inside the test compounds (20 mg/20 mL) overnight before loading them on culture plates. The plates were incubated at 37[degrees] C for 24 h. The inhibition zone appeared around the disc was measured and recorded as the antibacterial effect of Ag NPs.

3. Results and Discussion

3.1. Swelling Studies. Water uptake by hydrogels was monitored for an extended period of time till equilibrium was achieved. Swelling characteristics of starch-co-PAAm hydrogels and SNCH systems at 37[degrees]C were determined using (1) and are plotted as a function of composition in Figure 1. The hydrogels swelled rapidly in water and attained equilibrium within 12 h. Figure 1 shows the influence of various amounts of starch, MA, acrylamide, and MBA on swelling characteristics of the copolymeric hydrogels and their SNCHs. The order of swelling capacity values of starch-co-PAAm hydrogels and SNCHs is copolymeric SNCHs > pure hydrogels. This pattern of swelling is reasonable because once the [Ag.sup.+] ions loaded hydrogels were treated with TD leaf extract, the reduction of [Ag.sup.+] ions into [Ag.sup.0] nanoparticles takes place which may enhance the overall porosity of the gel and hence promote higher water molecules uptake capacity. The other reason can be that the particles have different sizes and different surface charges in the gel networks that cause absolute expansion of the network.

It is observed from Figures 1(a), 1(b), and 1(c) as the amount of starch, MA, and AAm increases, the swelling of hydrogels increased. This is due to the increase in hydrophilic groups (-OH, -COOH, -CON[H.sub.2]) and increase in hydrodynamic free volume to accommodate more of the solvent molecules. As the amount of MBA increased (Figure 1(d)) the swelling has been decreased due to the high crosslinking density, which tightens the polymeric chains thereby making the hydrogels rigid.

3.2. FT-IR Spectral Studies. In Figure 2, the IR spectrum of starch-co-PAAm hydrogel shows the characteristic absorption peaks at 1118 [cm.sup.-1] and the absorption bands in the range of 2900-3400 [cm.sup.-1] which are attributed to -C-O and -OH stretching vibrations of starch, respectively. The characteristic absorption peak at 1680 [cm.sup.-1] results from -C=O stretching vibration of the - CON[H.sub.2] group, and a broad absorption band at 3219 [cm.sup.-1] is related to the -NH asymmetric and -OH symmetric stretching vibrational group; band at 2812 [cm.sup.-1] is attributed to stretching vibrations of - C[H.sup.3] units. The absorption band at 1710 [cm.sup.-1] is due to asymmetrical stretching for -COOH group of maleic acid. The substitution of MA was also confirmed by the absorption band observed at 1230 [cm.sup.-1] which indicates the -C-O-O-stretching vibrations of ester. These peaks have shifted to 1110 [cm.sup.-1],1214 [cm.sup.-1],1669 [cm.sup.-1], and 2804 [cm.sup.-1], and 3202 [cm.sup.-1] in SNCH indicates that [Ag.sup.0] nanoparticles are successfully incorporated in SNCH matrices.

3.3. UV-Visible Spectroscopy. The formation of [Ag.sup.0] nanoparticles in the copolymeric hydrogel networks can be expected in our current strategy because the [Ag.sup.+]-loaded starch-co-PAAm hydrogel was readily reduced by TD leaf extract, which immediately turned into brown colour. This change in colour shows that the [Ag.sup.0] nanoparticles are entrapped inside the networks through strong localization and stabilization established by the polymer matrix. The existence of [Ag.sup.0] nanoparticles in the gel networks was estimated by UV-visible spectroscopy analysis. Figure 3 illustrates the absorption peaks of SNCHs in 325-500 nm ranges that are assigned to Ag NPs which arose from the surface plasmon resonance (SPR).

A significant improvement in the absorption peak at 420 nm was observed for [Ag.sup.0]-loaded hydrogels due to the surface plasmon resonance band [42]. The UV-visible absorption band is not symmetrical for all the formulations, because the formation of nanoparticles and their size depends on the network structure of hydrogels and their functionality. This absorption peak was not observed in the case of starch-co-PAAm hydrogel. No additional absorption peaks around 325-500 nm indicates that SNCH network is protecting the nanoparticles from the aggregation or cluster formation. This analysis confirms the formation of highly dispersed [Ag.sup.0] nanoparticles in the copolymeric hydrogels.

3.4. X-Ray Diffraction. The crystallinity of the hydrogel and SNCH was confirmed by the analysis of X-ray diffraction (X-RD) pattern (Figure 4). The dry powders of hydrogel and SNCH were used for X-RD analysis. In the diffractogram, four diffraction peaks were obtained at an angle, 2[theta] = 38.39, 49.25, 64.10, and 77.87 which could be attributed to the Brags reflections of (111), (200), (220), and (311) planes of face centred cubic (FCC) structure of Ag NPs. A broad diffraction peak 2[theta] in both samples can be seen at 20[degrees] due to the amorphous nature of starch-co-PAAm hydrogel and SNCH. The (200), (220), and (311) peaks were less intense compared to (111) peak. So, in this case, we might conclude that the nanoassemblies were mainly composed of (111) lattice planes. Similar type of observations was found previously in reported work [31, 33].

3.5. SEM Studies. The morphology of starch-co-PAAm hydrogel and SNCH was investigated with SEM. Figure 5 shows the SEM micrographs of the starch-co-PAAm and SNCHs. Figure 5(a) shows a clear surface feature for the pure hydrogel, whereas Ag NPs-loaded starch-co-PAAm hydrogel (Figure 5(b)) exhibits smaller nanoparticles distributed throughout the hydrogel networks. It is worth mentioning that no individual silver particles were observed outside the SNCH, indicating a strong interaction between the polymer matrix and the silver particles.

3.6. TEM Studies. Shape and size distribution of the synthesized Ag NPs were characterized by transmission electron microscopic (TEM) analysis. Figures 6(a) and 6(b) are the low magnification images and Figure 6(c) is the high magnification image obtained by transmission electron microscopy at an acceleration voltage of 15 kV. These images indicate that the particles are well defined, spherical in shape with a narrow size distribution, and highly dispersed with an average diameter of 7 nm in size (Figure 6(d)). Moreover, the electron diffraction pattern of [Ag.sup.0] nanoparticles is clearly visible in Figure 6(e) as three diffraction rings and from that pattern it is evident that the nanoparticles are crystalline and have a face centered cubic (FCC) structure.

3.7. Thermogravimetrical Analysis. The starch-co-PAAm hydrogel and its SNCHs are characterized by thermogravi-metrical analysis to determine the percentage of weight loss of pure hydrogel as well as [Ag.sup.0] nanoparticles loaded hydrogel, as shown in Figure 7. The starch-co-PAAm hydrogel has followed two decomposition steps below 400[degrees]C and 82% weight loss occurred below 700[degrees]C. However, two decomposition states and only 60% weight loss were observed in the case of SNCH below 700[degrees]C. The difference in decomposition between the hydrogel and SNCH is found to be 22%, and it confirms the presence of [Ag.sup.0] nanoparticles in the SNCH network. From these results it is concluded that SNCH showed a higher thermal stability when compared with the starch-co-PAAm hydrogel due to the undecomposed remaining weight of [Ag.sup.0] nanoparticles present in the copolymeric networks.

3.8. Antibacterial Studies. The antimicrobial activity of different molar weight ratios of SNCHs was examined against gram-negative and gram-positive bacteria. All SNCHs proved effective against the tested microorganisms Escherichia coli and Bacillus, but growth inhibitory effects varied from one another as shown in Figures 8(a) and 8(b). The results indicate that starch-co-PAAm-3, starch-co-PAAm-4, starch-co-PAAm-5, starch-co-PAAm-8, and starch-co-PAAm-9 SNCHs exhibited greater reduction of Escherichia coli and Bacillus growth, while the pure hydrogels (c) were generally inefficient as shown in Table 2. The inhibition zone follows the order starch-co-PAAm-3 > starch-co-PAAm-5 > starch-co-PAAm-8 > starch-co-PAAm-4 > starch-co-PAAm- 9. These results are expected due to the small sizes of [Ag.sup.0] nanoparticles and the same order is expected for swelling ratio. Ag NPs adhered to the cell wall of bacteria and penetrated through the cell membrane; they kill microorganisms instantly by blocking their respiratory enzyme systems and having no negative effect on human cells.

4. Conclusions

In conclusion, we have demonstrated a facile and simple green methodology for preparation of starch-co-PAAm silver nanocomposite hydrogels by free radical polymerization and thereby reducing [Ag.sup.+] into [Ag.sup.0] nanoparticles using Tridax procumbens leaf extract. The main aim of this study was to develop a new antimicrobial/wound-dressing agent. These antimicrobial agents are nontoxic materials which can be used systemically as an alternative to conventional systemic antibiotic, antiviral, and antifungal therapies that do not incur the development of resistance by the target pathogens. The synthesis method adopted here is very simple and can be easily implemented for any kind of scientific as well as industrial application due to its cost-effective nature. A number of SNCHs were formulated with high dispersion rates of [Ag.sup.0] nanoparticles throughout the hydrogel networks by varying the concentrations of carbohydrate polymer, monomers, and crosslinker. The synthesized nanocomposites are confirmed by using various spectral, thermal, and electron microscopy methods. These SNCHs having [Ag.sup.0] nanoparticles concentrations as low as 1 mg/mL showed excellent antibacterial activity against gram-positive and gram-negative bactericide. This resulted into inhibition of bacterial cell growth and multiplication. So these nanocomposites could be used as successful antibacterial agents such as wound-dressing materials.

http://dx.doi.org/10.1155/2013/539636

Acknowledgments

The authors thank University Grants Commission (UGC), New Delhi (India), for providing financial support to one of the authors (Siraj Shaik) under UGC-RFSMS (Letter no. F.7-290/2009 (BSR)/10-01/2009). The authors are thankful to Professor T. P. Radhakrishnan, School of Chemistry, University of Hyderabad, Hyderabad, Andhra Pradesh, for helping them in obtaining TEM images.

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Siraj Shaik, (1) Madhusudana Rao Kummara, (2) Sudhakar Poluru, (1) Chandrababu Allu, (1) Jaffer Mohiddin Gooty, (3) Chowdoji Rao Kashayi, (4) and Marata Chinna Subbarao Subha (1)

(1) Department of Chemistry, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh 515 003, India

(2) Department of Polymer Science and Engineering, Pusan National University, Busan 609-735, Republic of Korea

(3) Department of Microbiology, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh 515 003, India

(4) Department of Polymer Science & Tech, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh 515 003, India

Correspondence should be addressed to Marata Chinna Subbarao Subha; mcssubha3@gmail.com

Received 31 August 2013; Accepted 13 November 2013

Academic Editor: Roland J. Pieters

TABLE 1: Various formulation parameters used in the
preparation of starch-co-PAA hydrogels.

Hydrogel code                           Chemicals used

                   Starch    Maleic     Acrylamide    MBA     KPS
                    (g)     acid (mM)      (mM)      (mM)    (mM)

Starch-co-PAAm-1    0.50      14.91       14.06      0.648   1.849
Starch-co-PAAm-2    1.00      14.91       14.06      0.648   1.849
Starch-co-PAAm-3    1.50      14.91       14.06      0.648   1.849
Starch-co-PAAm-4    0.50      7.03        14.06      0.648   1.849
Starch-co-PAAm-5    0.50      21.10       14.06      0.648   1.849
Starch-co-PAAm-6    0.50      14.91        7.03      0.648   1.849
Starch-co-PAAm-7    0.50      14.91       21.10      0.648   1.849
Starch-co-PAAm-8    0.50      14.91       14.06      0.129   1.849
Starch-co-PAAm-9    0.50      14.91       14.06      0.324   1.849

Hydrogel code
                   TMEDA
                   (mM)

Starch-co-PAAm-1   0.860
Starch-co-PAAm-2   0.860
Starch-co-PAAm-3   0.860
Starch-co-PAAm-4   0.860
Starch-co-PAAm-5   0.860
Starch-co-PAAm-6   0.860
Starch-co-PAAm-7   0.860
Starch-co-PAAm-8   0.860
Starch-co-PAAm-9   0.860

TABLE 2: Antibacterial activities of the synthesized hydrogels
on gram-positive and gram-negative organisms.

Name of the           Inhibition zone in diameter (mm)
bacteria             for silver nanocomposite hydrogels
(pathogens)
                Pure     Starch-co-   Starch-co-   Starch-co-
              hydrogel     PAAm-3       PAAm-4       PAAm-5
                (c)

E. coli          0          4.5          3.0          4.0
Bacillus         0          5.0          3.5          4.5

Name of the           Inhibition zone in diameter (mm)
bacteria             for silver nanocomposite hydrogels
(pathogens)
                         Starch-co-   Starch-co-
                           PAAm-8       PAAm-9

E. coli                     3.5          2.5
Bacillus                    4.0          3.5
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Title Annotation:Research Article
Author:Shaik, Siraj; Kummara, Madhusudana Rao; Poluru, Sudhakar; Allu, Chandrababu; Gooty, Jaffer Mohiddin;
Publication:International Journal of Carbohydrate Chemistry
Article Type:Report
Date:Jan 1, 2014
Words:5279
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