Silver Nanoparticles Incorporated Within Intercalated Clay/Polymer Nanocomposite Hydrogels for Antibacterial Studies
Manjula Bandla, Babul Reddy Abbavaram, Varaprasad Kokkarachedu, Rotimi E. Sadiku Department of Chemical, Metallurgical and Materials Engineering, Tshwane University of Technology, CSIR Campus, Building 14D, Private Bag X025, Lynwood Ridge, Pretoria 0040, South Africa
In this study, we present a new process of developing silver nanoparticles (AgNPs), using intercalated clay (saponite)/poly acrylamide (PAAm) nanocomposite (NC) hydrogels, via the reduction of silver ions with NaBH4 in aqueous solutions. The –NPs obtained are very stable at room temperature due to the extended coil conformation of the PAAm chain. Size and morphology of AgNPs formed within the silicate layers depended on the surface property of clay minerals, which was confirmed by XRD and TEM measurements. The parameters concerning to the diffusion (D) and swelling (Q) of water for the PAAm/saponite NC hydrogels were estimated. In vitro antibacterial assay showed that pristine saponite could not inhibit the growth of bacteria. However, the saponite/poly PAAm NC hydrogels had strong antibacterial activity, particularly against Gram-positive bacteria. With increasing amount (and the interlayer distance) of the layered silicates in the NC hydrogels, the nanocomposites showed a strong antibacterial C 2016 Society effect. POLYM. COMPOS., 00:000–000, 2016. V of Plastics Engineers
INTRODUCTION Nowadays, nanohydrogels have attracted a great deal of attention because of their characteristic properties, such as hydrophilicity, swelling capability in water, and biocompatibility. The capability to absorb and store much water and water solutions make hydrogels as unique materials for various applications, such as food industry, biomedicine, and water purification and separation process [1–5]. Among them, acrylamide-based hydrogels are comparatively important because of their use in many sorbent applications and similar to any other hydrogels, they are usually produced by free radical polymerization in the presence of cross-linker. On the other hand, there is lack of their soft and brittle nature results weak mechanically Correspondence to: B. Manjula; e-mail:
[email protected] Contract grant sponsor: Tshwane University of Technology. DOI 10.1002/pc.23963 Published online in Wiley Online Library (wileyonlinelibrary.com). C 2016 Society of Plastics Engineers V
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properties in swollen state. To overcome this drawback, many researchers have been focused on renovate the hydrogels with high mechanical performances through incorporation of organic nanoparticle [6], interpenetrating polymer network [7–14], as well as incorporation of inorganic nanoparticles [15–22]. Among them, nanoscale dispersion of clays into polymer networks was most extensively studied due to their high mechanical strength, high chemical resistance, inherent good absorbance, and toughness. Numerous inorganic clay particles are available, that is, Na-montmorillonite, attapulgite, and laponite–synthetic hectorite have been introduced to poly acryl amide (PAAm) gel systems with or without chemical cross-linkers. Generally, the cross-linkers improved the swelling ability and mechanical performance due to their inherent multifunctional cross-linker capacity. However, the new applications of nanoparticles and nanomaterials grow up rapidly [23–25] due to their importance in human beings. Numerous techniques can be used for synthesis of metal nanoparticles, including chemical reduction or photoreduction in aqueous medium with diverse polymer surfactants [26–28], chemical reduction in soft matrices (e.g., reverse micelles) [29, 30] or in solid matrices (e.g., mesoporous silicate) [31], and chemical vapor deposition [32–35]. Among various metal nanoparticles, silver nanoparticles (AgNPs) found to be very promising in the high-sensitivity fields of biomolecular detection and diagnostics [36, 37], antimicrobials, and therapeutics [38–43] as well as catalysis [44–49] and microelectronics [50, 51]. Although, there is still a need for an ecofriendly route to synthesize AgNPs in hydrogel network. Therefore, the AgNPs within the hydrogel system would be a better choice for various microbial dieses. Three-dimensional hydrogel (nano-, micro-, and hydrogel) networks are quite appropriate for the production of AgNPs than the conventional nonaqueous or polymers, biomacromolecules, dendrimers, liquid crystals, latex particles, and so on. This may be that the hydrogels can provide free enough space between the networks in the swollen stage portion for growth and nucleation of
FIG. 1. Schematic representation of formation of AgO NC hydrogel. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
nanoparticles [52]. Recent studies on AgNPs showed that biocompatible hydrogel–silver nanocomposites are promising biomaterials for fast curing of wound burnings. As a part of our ongoing laboratory program to develop biocompatible hydrogel–silver nanocomposite materials [53, 54] for efficient antibacterial materials, we have developed silver nanoparticles within intercalated clay/polymer nanocomposite hydrogels (Fig. 1). In this study, we have taken PAAm as a model hydrogel, which is widely used for the number of applications and clay as interpenetrating inorganic particles for its excellent hydrophilic, swelling behavior, bioreactor property, and controlled release properties in addition to stabilization of AgNPs. EXPERIMENTAL Materials Acrylamide (AAm), potassium persulfate (KPS), N,N,N1,N1-tetramethyl ethylenediamine (TMEDA), silver nitrate (AgNO3), and sodium borohydride (NaBH4) were obtained from Aldrich Chemical Company (South Africa). Natural saponite clay (cationic exchange capacity (CEC) of about 75 mmol per 100 g and mean grain size was about 60–70 lm) was obtained from Xinjiang Tuogema Colloid Co., China. Double distilled water was used throughout the investigation for the preparation of all solutions. Preparation of Polyacrylamide/Saponite Nanocomposite Hydrogels (PAAm/Saponite NC Hydrogels) Nanocomposite hydrogels were prepared using initial solutions consisting of a acrylamide (AAm), a cross2 POLYMER COMPOSITES—2016
linker (clay), a solvent (water), an initiator (potassium persulfate), and an accelerator (TEMEDA). The NC hydrogels were prepared by the in situ free-radical polymerization of AAm in the presence of the water-swollen inorganic clay (saponite) without using any organic crosslinker [55]. First, a transparent aqueous solution consisting of water (3 mL), AAm (1 g), and various amounts of inorganic clay content (1–3 wt% according to monomer) were prepared. The accelerator (TEMED, 1%) and subsequently the aqueous solution of the initiator (1%) were added to the former solution with a magnetic stirrer at 200 rpm over 5 min before forming a gel. The reaction was allowed to continue overnight to obtain a hard gel. Among developed NC hydrogels, 3 wt% clay offer promising results; hence we reported in this study, 3 wt% clay having NC hydrogel. The reduction test was carried out with NaBH4 as following: preweighed silver-loaded hydrogels were immersed in 0.1% NaBH4 solution and kept it under dark condition for 8 h and kept in oven for drying at 408C.
Swelling Studies Gravimetrically weighed dry NC hydrogels were immersed in a 100 mL beaker containing double distilled water, for 48 h until the NC hydrogel reached equilibrium swelling at ambient temperature and reweighed using a PA214 analytical balance (OHAUS Corp., Pine Brook, NJ, USA) [56, 57]. Swollen NC hydrogels were treated with AgNO3 and subsequently with NaBH4 (reduction reaction) as explained in the experimental section. The swelling ratio or swelling capacity (Sg/g) of the NC DOI 10.1002/pc
FIG. 2. (a) X-ray diffraction for saponite 1 PAAm hydrogel and AgO NC hydrogel and (b) TEM image of AgO NC hydrogels. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
hydrogel developed and their corresponding nanohydrogel was calculated using Eq. 1: Swelling; Qð%Þ5
WS 2Wd 3100 Wd
(Eq. 1)
Where Ws and Wd denote the weight of the swollen hydrogel at equilibrium and the weight of the dry hydrogel, respectively. The data provided is an average value of the three individual sample readings. Fourier-Transform Infrared (FTIR) Spectroscopy FTIR spectrophotometer was used to study the transmission of light in the NC hydrogel and AgO nanoparticles patterns in the hydrogel networks. The NC hydrogels and AgO NC hydrogels were completely dried in the oven at 608C for 6 h before their FTIR experiments. Samples were examined between 500 and 4000 cm21 on a PerkinElmer UATR spectrometer (Ettlingen, Germany), using thin-film measurements.
a heating rate of 108C/min under a constant nitrogen flow (100 mL/min). Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS) Scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) analysis of plain hydrogel and AgO nanohydrogels were coated with iridium and performed using a JEOL FESEM JSM-7600F (Tokyo, Japan) operated at an accelerating voltage of 15 kV. All samples were examined on a field-emission scanning electron microscope. X-Ray Diffraction (XRD) To determine the formation of AgO nanoparticles in the NC hydrogel network, X-ray diffraction (XRD) was employed. The measurements were carried out for dried and finely grounded samples on a Bruker D8 advanced refractometer X-ray diffraction (XRD) diffractometer (Cu Ka radiation, k 5 0.1546 nm) at 40 kV and 50 mA.
UV–Vis Spectroscopy UV–vis spectra of AgO NC hydrogels were recorded on a Cary 60 Model UV–vis spectrophotometer (The Elico co., Hyderabad, India) in 300–500 nm range. For this study, 100 mg of AgO NC hydrogels were dispersed in 10 mL of distilled water and allowed to stand for 24 h to extract, as much AgO nanoparticles as possible into aqueous phase and these solutions were used to record their UV–vis absorption spectra.
Transmission Electron Microscopy (TEM)
Thermogravimetric Analysis
Antibacterial Studies
Thermal analysis (TGA) of the samples was carried out using PerkinElmer TGA 7 instrument (T.A. Instruments-water LLC, Newcastle, DE 19720, USA), at
The antibacterial activity of the AgO nanohydrogels were investigated by disc method, using the standard procedure described elsewhere [58, 59]. Nutrient agar
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Transmission electron microscope (TEM) (JEM1200EX, JEOL, Tokyo, Japan) was used for morphological observation of the AgO nanoparticles. TEM sample was prepared by dispersing two to three drops of finely grinded AgO NC hydrogel (1 mg/1 mL) solution on a 3 mm copper grid and dried at ambient temperature after removing excess solution using filter paper.
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FIG. 3. SEM micrographs of (a) saponite 1 PAAm hydrogel, (b) silver-loaded hydrogel and AgO NC hydrogel with their EDS spectroscopy.
medium was prepared by mixing peptone (5.0 g), beef extract (3.0 g), and sodium chloride (NaCl) (5.0 g) in 1000 mL distilled water and the pH was adjusted to 7.0. Finally, agar (15.0 g) was added to the solution. The agar medium was sterilized in an autoclave at a pressure of 6.8 kg (15 lbs) for 30 min. This medium was transferred into sterilized Petri dishes in a laminar air flow chamber (Microfilt Laminar Flow Ultra Clean Air Unit, Mumbai India). After solidification of the media, bacteria (Staphylococcus and Escherichia coli) (50 mL) culture was spread on the solid surface of the media. Over the inoculated Petri dish, one drop of gel solutions (20 mg/10 mL distilled water) was added using a 10 mL tip and the plates were incubated for 48 h at 378C.
RESULTS AND DISCUSSION X-Ray Diffraction The X-ray diffraction was suitable technique to know the formation of the AgO nanoparticles in the hydrogel networks. Figure 2(a) shows the XRD pattern of saponite 1 PAAm hydrogel and AgO nanoparticles-loaded nanohydrogel, via chemical process at room temperature. The diffraction peaks are consistent with the standard patterns of crystal silver (JCPDS file No. 04-0783). The diffraction peaks at 2h 5 37.92, 44.13, 64.29, and 76.838 and these peaks indexed to the reflections of (111), (200), (220), (311) planes, indicate face centered-cubic silver, respectively [5, 60, 61]. Similar common peaks was observed in both blank hydrogel and AgO nanohydrogel, 4 POLYMER COMPOSITES—2016
which exhibits strong and broad diffraction peaks at 2h 5 24.29. Transmission Electron Microscopy (TEM) TEM analysis also confirmed the formation of spherical AgO nanoparticles in the clay hydrogels network. Their TEM image is shown in Fig. 2(b). The average size of the AgO nanoparticles was found to be about 20 nm. It is evident that AgO nanoparticles were highly stabilized by using clay in the hydrogel network. These observations are due to mainly the strong interaction between the AgO nanoparticles and clay hydrogel; this is further supported to FTIR analysis (Fig. 4). Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS) The surface morphology of PAAm-clay hydrogel and AgO nanohydrogels were investigated with SEM. Figure 3a–c shows the SEM images of the of saponite 1 PAAm hydrogel silver-loaded hydrogel and AgO NC hydrogels. Figure 3a indicates a clear rough surface with clay particles morphology for the saponite 1 PAAm hydrogel, when compared to silver-loaded hydrogel shown in Fig. 3b, whereas AgO nanoparticles incorporated hydrogel exhibited smaller nanoparticles distributed throughout the hydrogel matrix (Fig. 3c). It is worth mentioning that no individual AgO nanoparticles were observed outside the saponite 1 PAAm hydrogels, indicating a strong interaction between the saponite 1 PAAm hydrogel and the AgO nanoparticles. To confirm the presence of AgO, DOI 10.1002/pc
FIG. 4. (a) FTIR spectra of saponite 1 PAAm hydrogel, saponite, silver-loaded hydrogel and AgO NC hydrogel and (b) TGA of saponite 1 PAAm hydrogel and AgO NC hydrogel. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
nanoparticles in the saponite 1 PAAm hydrogel network by EDS analysis were carried out. EDS image of the saponite 1 PAAm did not show the characteristic peak of AgO nanoparticles, while the EDS spectrum of AgO nanoparticles-loaded hydrogel showed clearly the peak of AgO nanoparticles. Hence, the existence of AgO nanoparticles in the hydrogel was confirmed by EDS spectra. The use of clay in the hydrogel to stabilized the AgO nanoparticles in the hydrogel networks.
at 1651 cm21 corresponds to the C@O moiety of the AAm unit, the peak at 1448 cm21 corresponds to the CAN stretching of the AAm unit and the peak at 1028 cm21 corresponds to the SiAO stretching of clay [62] and also observed at 815, 652, 592, and 430 cm21 showing the presence of quartz related to m (SiAOASi) and d (SiAO) bands also support the presence of quartz [63]. Thermogravimetric Analysis
Fourier-Transform Infrared Spectroscopy The formation of the AgO NC hydrogels developed in this study was characterized by FTIR analysis. FTIR spectroscopy of saponite, saponite 1 PAAm hydrogel, silver-loaded hydrogel, and AgO NC hydrogels are depicted in Fig. 4(a). As seen in the figure, the characteristic absorptions of both PAAm (2941, 2855, 2753, 1651, 1506, and 1448 cm21) and clay (1028 and 603 cm21) were shown in the spectra. In the FTIR spectra, the peak
Thermogravimetric analysis was used to study the formation of AgO nanoparticles in the hydrogel network. The primary thermogram of the saponite 1 PAAm hydrogel and AgO nanohydrogels are shown in Fig. 4(b). The saponite 1 PAAm hydrogel was two decomposition curves at below 9008C and 32% degradation of the hydrogel chains occurs at below 10008C. However, in case of AgO nanohydrogels, only one decomposition curve is observed below 9008C and only 27% weight loss occurred
FIG. 5. (a) UV–visible spectra of saponite 1 PAAm hydrogel, silver-loaded hydrogel and AgO NC hydrogel and (b) swelling behavior of hydrogels. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
DOI 10.1002/pc
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FIG. 6. Antimicrobial activity of bionanocomposite films against (a) Salmonella and (b) Staphylococcus aureus. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
even at 10008C. It is expected that the AgO nanohydrogels showed a higher thermal stability than the saponite 1 PAAm hydrogel. The difference in decomposition between the saponite 1 PAAm hydrogel and their AgO NC hydrogel was found to be 15.2% and it confirms the presence of AgO nanoparticles (weight loss) in the hydrogel [64] and further conformed from Fig. 4(a). In the present invention, silver nanoparticles play a role in degradation of hydrogel, which restricts the percentage of weight loss. UV–Visible Spectroscopy To understand the formation of the AgO nanoparticles, UV–visible spectroscopy technique was used. Figure 5(a) shows the absorption characteristics of saponite 1 PAAm hydrogel, silver-loaded hydrogel, and their corresponding AgO NC hydrogel. The characteristic plasmon resonance excitation peak of AgO nanoparticles was observed at kmax 351 nm. However, there is no intensity peak was observed in the case of saponite 1 PAAm hydrogel at 351 nm. This is clearly indicated that AgO nanoparticles are stabilized in saponite 1 PAAm hydrogel system. Swelling Studies The swelling ability of NC hydrogel plays a significant role in their wound healing capacity, antibacterial activity, and generally in their biomedical applications, due to their high water clenching capacity [65]. They can further absorb a significant amount of the wound exudates by swelling, which helps in fast curing of the wound. Figure 5(b) shows the swelling capacity as a function of time of the NC hydrogel developed in this study. The 3 wt% clay hydrogel showed the highest swelling capacity than other hydrogels. This is may be due to the intermolecular interaction between water molecules in clay galleries and the lone pair electrons of ANH2 group present in polyacrylamide. 6 POLYMER COMPOSITES—2016
Antibacterial Activity Nowadays, inorganic nanohydrogels have been actively investigated for antibacterial applications [59, 66]. AgO nanoparticles are ecofriendly and nontoxic antibacterial material; however, its main disadvantage is poor binding characteristic and stability, which restricted their application. However, polymer-stabilized nanoparticles [67] and nanoparticles-embedded hydrogel networks [59] are outstanding approaches for their preparations. Hence, antibacterial abilities of saponite 1 PAAm hydrogel and AgO nanoparticles contained hydrogels were studied. The efficiency of antibacterial activity was evaluated by calculating their ability to inhibit zone of Staphylococcus aureus (as Gram-positive bacteria) and Salmonella (as Gramnegative bacteria) growth over agar culture dishes. After 48 h of incubation at 378C, there was the inactivation of the bacterial zones and no bacterial colonies were observed for saponite 1 PAAm hydrogel and silver-loaded hydrogel (not shown here), whereas AgO NC hydrogel shown a promising results as shown in the Petri dishes (Fig. 6). These results are quite expected and seemed to be followed according to the nanoparticles content in the hydrogel matrixes. It was evident from the TGA analysis that the percentage of nanoparticles content had been found higher for 3 wt% AgO NC hydrogel. Hence, inhibition zones were observed higher (17 mm) for 3 wt% AgO NC hydrogel than 1 wt% AgO NC hydrogel (10 mm) [68]. Further, according to the various literatures and by standard antibacterial test “SNV 195920–1992,” the developed hydrogels in this study showing >1 mm microbial zone inhibition can be considered as better antibacterial agents [69, 70]. Hence, the inorganic AgO nanohydrogels developed from this study can be considered as good antibacterial agents, effective in killing the bacteria. CONCLUSIONS In conclusion, AgNPs have been successfully prepared in hydrogels template. The natural clay displayed excellent characteristics in anchoring and stabilizing the DOI 10.1002/pc
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