Synthesis of silver nanoparticles

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Journal of Photochemistry & Photobiology, B: Biology 167 (2017) 282–289

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Synthesis of silver nanoparticles (Ag NPs) for anticancer activities (MCF 7 breast and A549 lung cell lines) of the crude extract of Syzygium aromaticum K Venugopal a, H A Rather a, K Rajagopal a, M P Shanthi b, K Sheriff c, M Illiyas c, R A Rather c, E Manikandan a,e,f,g,⁎, S Uvarajan e, M Bhaskar d, M Maaza f,g a

Department of Biotechnology, Vels University, Chennai, India Dept of Zoology, Nehru Memorial College, Puthanampatti-621007, Tiruchirappalli, Tamil Nadu, India c Dept of Virology, King Institute of Preventive Medicine and Research, Chennai 600032, India d Dept of Zoology, UGC SAP-DSA-I, Sri Venkateswara University, Tirupati 517502, India e Dept of Physics & Biochemistry, TUCAS, Thennangur-604408, Thiruvalluvar University, Serkadu, Vellore, India f UNESCO-UNISA AFNET in Nanosciences/Nanotechnology, College of Graduate Studies, University of South Africa, Muckleneuk Ridge, PO BOX 392, Pretoria, South Africa g Nanosciences African Network (NANO-AFNET), Materials Research Department, iThemba LABS–National Research Foundation (NRF), 1 Old Faure Road, Somerset West, PO BOX 722, Western Cape, South Africa b

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Article history: Received 15 October 2016 Accepted 5 December 2016 Available online 20 December 2016 Keywords: Silver nanoparticles Phytochemical synthesis Characterizations Anticancer activities Cellines

a b s t r a c t In the present report, silver nanoparticles were synthesized using Piper nigrum extract for in vitro cytotoxicity efficacy against MCF-7 and HEP-2 cells. The silver nanoparticles (AgNPs) were formed within 20 min and after preliminarily confirmation by UV–Visible spectroscopy (strong peak observed at ~441 nm), they were characterized by using FT-IR and HR-TEM. The TEM images show spherical shape of biosynthesized AgNPs with particle size in the range 5–40 nm while as compositional analysis were observed by EDAX. MTT assays were carried out for cytotoxicity of various concentrations of biosynthesized silver nanoparticles and Piper nigrum extract ranging from 10 to 100 μg. The biosynthesized silver nanoparticles showed a significant anticancer activity against both MCF-7 and Hep-2 cells compared to Piper nigrum extract which was dose dependent. Our study thus revealed an excellent application of greenly synthesized silver nanoparticles using Piper nigrum. The study further suggested the potential therapeutic use of these nanoparticles in cancer study. © 2016 Published by Elsevier B.V.

1. Introduction Nanoparticles, the rudiments for nanotechnology, are nowadays produced using noble metals like Ag, Pt, Au and Pd with the advancement of new materials with nanometer size including nanoparticles, nanotubes, nanowires, and so forth. In the recent times, silver nanoparticles (AgNPs) have attracted intensive research interest because of their advantageous applications in biomedical [1–3], drug delivery [4], food industries [5], agriculture [6], textile industries [7], water treatment [8], catalysis and surface-enhanced Raman scattering [9]. Diverse methods are used for the synthesis of silver nanoparticles. And the most commonly available known method is the chemical reduction of metal salt precursor using chemical reducing agents such as, citrate [10], polymer substances [11–13], borohydride, N,N-dimethyl ⁎ Corresponding author at: Department of Biotechnology, Vels University, Chennai, India. E-mail addresses: [email protected], [email protected], [email protected] (E. Manikandan). 1011-1344/© 2016 Published by Elsevier B.V.

formamide [14], sodium borohydride [15], trisodium citrate [16], sodium hydroxide [17], 2-mercaptobenzimidazole [18], sodium dodecyl sulfate [19], or other organic reagents [20–23]. The physical methods include, laser ablation method [24], sono chemical deposition [25,26], photochemical reduction [27,28], gamma ray and solar irradiation [29], UV photo reduction [19], microwave-assisted [30], electrochemical method [31–33], thermal decomposition in organic solvents [34], and molecular beam epitaxy methods [35]. Although the commercial methodologies have proven as efficient tools for synthesizing AgNPs, but their continuous use may pose a great threat to human health and the environment because of the use of toxic and hazardous reagents and generation of toxic by-products in some instances. These products tend to bind to the AgNPs surface and may adversely affect their character and performance [36]. Hence, there is a great need to find alternative methods for AgNP synthesis, which are nontoxic and eco-friendly. However, these methods suffer from disadvantages like low yield, high-energy supplies, and a need for complicated and inefficient purifications [37]. Some of the recently developed green methods utilizing

K. Venugopal et al. / Journal of Photochemistry & Photobiology, B: Biology 167 (2017) 282–289


Fig. 1. Nanoparticle synthesis using Syzygium aromaticum plant extract; (A) silver nitrate solution, (B) Syzygium aromaticum extract broth, (C) silver nanoparticle solution.

biological materials show favorable routes for their synthesis. The use of plants for the synthesis of AgNPs is in the focus of intensive research because of its eco-friendly nature. The use of plants boasts of several advantages such as the elimination of elaborate processes of maintaining cell cultures, easy scale up for large-scale synthesis and cost-effectiveness. Moreover, plant extracts may act both as reducing agents and stabilizing agents in the synthesis of nanoparticles [38]. Typically, a plantextract-mediated bio-reduction for photosynthesis of silver nanoparticles involves mixing the aqueous extract with an aqueous solution of the silver nitrate salt [39–44]. The present study directs the advantageous of silver nanoparticles from silver nitrate through a simple green route utilizing the extract of Cloves (Syzygium aromaticum) as the reducing agent. Cloves (Syzygium aromaticum), are the aromatic flower buds of a tree in the family Myrtaceae and numerous restorative uses have been most broadly connected to a toothache, and for mouth and throat aggravation. Cloves show antiseptic, antibacterial, antifungal and antiviral properties. Thus, the study proceeds with the synthesis of silver nanoparticles utilizing Syzygium aromaticum and their cytotoxicity of biosynthesized silver nanoparticles was studied against MCF-7 and A549 cancer cell lines. However, the synthesis of silver nanoparticles utilizing cloves as biosource has not yet been studied.

2. Materials and Methods

from King Institute of Preventive Medicine and Research, Chennai, India. 2.2. Preparation of Clove Extract The Syzygium aromaticum (Cloves) were collected from the local market and authenticated. The Syzygium aromaticum were finely powdered using mortar and pestle. The plant powder (20 g) was dissolved in 100 ml of millipore water and the mixture was bubbled at 80 °C for 10 min followed by filtration through Whatman Grade No. 1 filter Paper (11 μm) and the broth was stored at low temperature till further use [45,46]. 2.3. Biosynthesis of Silver Nanoparticles Syzygium aromaticum concentrate (10 ml) was added to 90 ml of 1 mM silver nitrate solution in order to achieve reduction of Ag+ ions. Various temperatures such as room temperature (RT), 40, 60, and 80 °C were maintained using a water bath to obtain the optimum synthesis. The solution was stirred at 1000 rpm for 10 min [47]. The color change was observed at various temperatures to ensure the formation of silver nanoparticles. The Syzygium aromaticum clove extract was thus employed as a reducing and stabilizing agent for 1 mM of silver nitrate [48,49].

2.1. Materials Silver nitrate (AgNO3) and MTT were purchased from Hi-Media Laboratories Pvt. Ltd. India. The MCF-7 cancer cell line was collected

Fig. 2. Silver nanoparticles synthesized at various temperature.

Fig. 3. UV–Vis spectra analysis of biosynthesized AgNPs at different temperatures.


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Fig. 4. FT-IR spectra of biosynthesized AgNPs using Syzygium aromaticum.

Fig. 6. SAED pattern of AgNPs which demonstrates that the selected-area electron diffraction (SAED) patterns exhibit concentric rings with intermittent dots, indicating that these nanoparticles are crystalline in nature.

2.4. Purification of Silver Nanoparticles 2.6. MCF-7 and A549 Cell Line Culture In order to remove the excessive silver ions and unwanted plant debris, the silver colloids were centrifuged at 10,000 rpm for 15 min and washed three times with Millipore water. A dried powder of silver nanoparticles was obtained by freeze-drying in Alpha Christ 2.0 lyophilizer for further characterization.

2.5. Characterization of Biosynthesized Silver Nanoparticles The preliminary characterization of silver nanoparticles was carried out using UV–Visible spectroscopy. UV–Visible spectral investigation was carried out by using NanoDrop 2000r working on a scanning range of 200–800 nm. Millipore water was used as blank. The spectra recorded were then re-plotted using Origin 8.0 version. To verify the interactions between protein-silver nanoparticles, Fourier transform infrared spectroscopy (FTIR) in the range of 4000 to 400 cm− 1 were used. The TEM images of biosynthesized AgNPs were obtained for size and shape determination using libra 200 HR-TEM (m/s Carl Zeiss, Germany) operated at an accelerating voltage 120 kV and 200 kV. The AgNPs were thoroughly sonicated for 5 min and a drop of diluted sample was placed onto the carbon-coated copper grid. The liquid fraction was allowed to evaporate at room temperature [52].

Breast (MCF-7) and Lung (A549) cancer cells were collected from King Institute of Preventive Medicine and Research, Chennai, India. It was cultured in Dulbecco's modified Eagle's medium (DMEM: Himedia Laboratories, Mumbai, India), supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin (Hi Media Laboratories Mumbai, India). Further, the MCF-7 and A549 cell lines were maintained at 5% CO2 in a CO2 incubator [53]. Cultures were routinely viewed under an inverted microscope to evaluate the quantity of confluence and the absence of bacterial and fungal contaminants was confirmed [54].

2.7. MTT Assay To determine the cytotoxic effect of silver nanoparticles and Syzygium aromaticum extract, cell viability study was done with the MTT reduction assay. MCF-7cells and A549 were seeded in a 96-well plate at the density of 5 × 103 cells/well. The cells were allowed to attach and were grown in a 96-well plate for 24 h, in 200 μl of EMEM with 10% FBS [55]. After that the media was removed and replaced with a suspension of various concentrations of silver nanoparticles 10 to100 mg/ml

Fig. 5. Topographical results of AgNPs confirming the spherical shaped particles from TEM analysis: HRTEM images of AgNPs at 5 nm.

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Fig. 7. Elemental compositional analysis of Ag NPs by TEM image with EDAX.

(minimum 4 wells were seeded with each concentration) the cells were incubated for 48 h [56]. The addition of MTT (10 ml, 5 mg/ml) was followed incubation of cells at 37 °C for another 4 h. The medium was then removed, and 200 μl of DMSO was added to each well. The optical density of the formazan product was read at 620 nm using multi well spectrophotometer [54,57]. The results were given as mean of four independent experiments. OD value was subjected to calculate the percentage of viability by using the following formula,

and fragmented nuclei were identified by their red fluorescence and the normal cells were visualized by their green fluorescence which was counted by using an upright fluorescent microscope (Nikon Eclipse, Inc., Japan) at 40× magnification with excitation filter at 510–590 nm [50].

Percentage of cell viability ¼ OD of Sample=OD of Control  100

Apoptotic cells were detected with DAPI (4′-6′-diamidino-2-phenyl indole) staining technique. The synthesized AgNPs were seeded in 6 well plates and maintained at 37 °C with 5% CO2 in a humidified CO2 incubator for 48 h. Subsequently, the cells were treated with IC50 concentrations obtained in various incubation were selected for this staining. At the indicated times, the medium was removed gently, and the cells were washed twice with phosphate buffered saline (PBS), fixed in 4% para-formaldehyde for 20 min, re-washed, and stained with DAPI at 37 °C for 20 min in the dark. Stains were then washed with methanol followed by PBS, and the plate was immediately observed in blue channel fluorescence with fluorescent microscope [51].

2.8. Apoptotic Effects by Acridine Orange (AO) and Ethidium Bromide (ETBr) Stain AO/EtBr staining technique is used to differentiate between quiescent and actively proliferating cells. It is also used to measure apoptosis. To the treated cells in cover slips, 10 μl of AO/EtBr was added and spread by placing a cover slip over it. The stained slides were incubated at room temperature for 5 min. The apoptotic cells with condensed chromatin

2.9. DAPI Staining for Nuclear Apoptosis

Fig. 8. Morphological observation of cancer cell lines under bright field microscope; (a) MCF7 Control Cells (b) MCF7 Treated Cells.


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Fig. 9. (a) A549 Control Cells (b) A549 Treated Cells.

2.10. Statistical Analysis

3.4. Transmission Electron Microscopic (TEM) Analysis

The grouped data were statistically evaluated using Graph Pad Prism 6 software and Origin 6.0 version. Values are presented as the mean ± SD of the four replicates of each experiment.

Fig. 5(I) shows the TEM images of AgNPs at different magnifications. The images reveal that the AgNPs are predominantly spherical in shape and are not in physical contact with each other as reported previously [52]. The AgNPs varied in particle size in the range from 5 to 50 nm. Lower magnification image (HRTEM) reveals the nanoparticles are embedded in a dense matrix which may be the organic stabilizing components of Syzygium aromaticum extract Fig. 5. The SAED results demonstrated that the AgNPs were crystalline in nature Fig. 6. This clearly demonstrated that aqueous extract of clove was all that much fit for reducing silver particles in the medium and changing over them

3. Results and Discussion 3.1. Formation of Silver Nanoparticles (AgNPs) Silver nitrate solution (Fig. 1A) is colorless and extract of Cloves is dark red in color (Fig. 1B). After adding Syzygium aromaticum extract to Silver nitrate solution, the solution became grayish red in color (Fig. 1C). The color change confirms that the silver nitrate was reduced and transformed into silver nanoparticles. 3.2. UV–Vis Spectra Analysis UV–Vis spectroscopy is one of the essential systems used to determine the primary existence of metal nanoparticles in a liquid medium. The color change demonstrating the presence of Ag nanoparticles was further characterized by UV–Vis spectrophotometer and observed by taking readings at a distinctive temperature (Fig. 2). The absorption peaks show the UV–Vis spectra of silver nanoparticle formation at different temperature using Syzygium aromaticum nanoparticle extract (i) Room temperature (RT), (ii) 40 °C, (iii) 60 °C, (iv) 80 °C in aqueous medium shown in Fig. 2. The peaks shows due to surface plasma resonance of silver colloid for diverse temperature were seen in the range of 420 to 470 nm spectral lines were obseved and the UV visible spectra showed in Fig. 3. The intense SPR bands were observed around ~470 nm at 80 °C compared other samples. Our results confirm the observations of Shalini Chauhan et al. [53] (Fig. 4). 3.3. FTIR Analysis The FT-IR transmission spectra of silver nanoparticles from Syzygium aromaticum are represented in Fig. 5. FTIR spectra of AgNPs demonstrated the peaks at 3444, 1722, 1621, 1388, 1055, and 587 cm−1. The band at 3444 cm− 1 corresponds to “polymeric” OH stretching mode. The 1722 cm−1 peak corresponds to normal aldehyde group. The absorption at 1621 cm−1 represents amide and Open chain imino (\\C_N\\). 1388 cm−1 relates methyl C\\H says./sym bend. The 1388 cm−1 peak relates to trimethyl or “tert-butyl” (multiplet). The 1055 cm−1 peak corresponds to the C\\C stretch and the aliphatic fluoro compounds C\\F stretch. The peaks at 587 cm−1 correspond the aliphatic do compounds, C\\I stretch, alcohol and OH out-of-plane bending. This confirmation proposes that the protein particles could perform the capacity of the arrangement and adjustment of AgNPs in the aqueous medium [45,59].

Fig. 10. Cytotoxicity evaluation of Syzygium aromaticum extract and biosynthesized AgNPs at various concentrations against cancer (MCF7) and (A549) cell line. The figure demonstrates the Inhibitory Concentration (IC50) value of the Phyto mediated AgNPs against MCF7 and A549 cells and that of Syzygium aromaticum extract.

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Fig. 11. Apoptotic effects (a) fluorescence microscopy study of AO/EtBr stain respective (a) control cells appear in live cells in green color (b) orange color apoptotic cells and necrotic cells appearing in red color.

into all around the well-dispersed silver nanoparticle. SAED showed a diffraction pattern of AgNPs directed toward (111), (200), (220), and (311) crystalline planes which correspond to the face-centered cubic (FCC) structure of elemental silver thus confirming the crystalline nature of AgNPs. The SAED patterns analyzed here were also shown in the study conducted previously by Mohan and co-workers [48]. The identified elements are indexed in the EDAX spectrum as follows C, Cu, Ag and their corresponding Kα, Kβ X-ray energy lines shown in Fig. 7. The preeminent peaks of silver (Ag) concentrations from the extracted Syzygium aromaticum, C and Cu from the sample gird.

3.5. Cytotoxicity Analysis of MCF-7 and A549 Cell Line The cytotoxicity of the silver nanoparticle and Syzygium aromaticum extract was studied against the MCF-7 and A549 cell line by MTT assay. Figs. 8 and 9 shows the cytotoxicity of AgNPs and Syzygium aromaticum against MCF-7 and A549 disease cell lines. The cytotoxicity impact on cell growth was examined at different concentration (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg) shown in Fig.10. The percentage of cancer cell growth inhibition was found to be high with the increasing the concentrations of AgNPs. The Inhibitory Concentration (IC50) of the phytomediated AgNPs was recorded at 60 μg·ml−1 against MCF7 and 50 μg·ml−1 A549 cells. The IC50 for Syzygium aromaticum was recorded at 70 μg·ml−1 against MCF7 and 70 μg·ml−1 for A549 cells. The cell viability decreased with increasing concentrations of phytomediated AgNPs compared with Syzygium aromaticum Fig. 10. This study demonstrates that the dosage needed was less for the cancer cell line. In fact, silver nanoparticles may stimulate reactive oxygen species and effect in damage cellular components which lead to cell death [52].

3.6. Apoptotic Effects by AO/EtBr and DAPI Staining In the present study apoptotic potential of synthesized AgNPs from Syzygium aromaticum were confirmed by morphological evaluation of the MCF7 treated cells by Acridine orange (AO) Ethidium Bromide (EtBr) staining and DAPI Fig. 11 & Fig 12. From the observations made from AO/EtBr staining it was clear that the mechanism of cell death induced by the nanoparticles is through apoptosis as evident by the characteristic nuclear changes such as chromatin condensation and nuclear fragmentation in the cells. The results of DAPI staining revealed that the treatment with the synthesized nanoparticles increased the number of apoptotic cells in breast cancer cell line MCF-7 when compared to the control (no drug is added) [50–52].

4. Conclusion In the present study, different temperatures were used for silver nanoparticle biosynthesized by using Syzygium aromaticum clove extract. By fluctuating the temperature of the clove extract (Room temperature) RT to 80 °C the plasmon resonance band raised slowly. Most extreme plasmon resonance band was achieved at 80 °C, which demonstrates that the reduction of the silver particle was directly proportional to the concentration of clove extract. There was no development of the plasmon resonance band when RT of the extract was used, which showed that there was an insufficient reducing agent in the extract to reduce the silver particle. The greatest Plasmon resonance was acquired at 60 °C previously, improved the procedure parameters (different temperature of extract) for incorporating AgNPs by utilizing aqueous extracts of Syzygium aromaticum. UV–Vis spectroscopy and FT-IR

Fig. 12. (a) DAPI nuclear stain of control cells (b) DAPI stain of AgNPs treated cells exhibited condensed form of nuclear materials in apoptotic cells.


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analysis confirmed the formation of the silver nanoparticles. The size of AgNPs was confirmed by TEM analysis, which illustrates that AgNPs were spherical in shape with size ranging from 5 to 40 nm. Synthesized AgNPs appeared polydispersed and well scattered. The synthesized silver nanoparticles and Syzygium aromaticum extracts were compared and showed promising cytotoxicity activity against MCF-7 and A549 cell line. From the study, it can be concluded that the silver nanoparticles synthesized using plant, possess high cytotoxicity activity against cell lines which suggests the potential therapeutic use of these nanoparticles. Acknowledgement The author (K.V.) would like to thank Dr. L Karthik, Senior Scientist from Marine Biotechnology Laboratory, SJTU & State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, China for his valuable suggestion and technical corrections for this manuscript. We are grateful to Dr. G.M. Bhalerao, Scientist-D (Electron Microscope Unit) for HR-TEM measurements, UGC-DAE Consortium for Scientific Research, Kalpakkam node. We also thank Dr. P. Gunasekaran, G. Fathima and Dr. K. Kaveri, Dept of Virology, King institute of preventive medicine & research, for cancer studies. References [1] K. Chaloupka, Y. Malam, A.M. Seifalian, Nanosilver as a new generation of nanoproduct in biomedical applications, Trends Biotechnol. 28 (2010) 580–588. [2] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobials, Biotechnol. Adv. 27 (2009) 76–83. [3] Y.A. Krutyakov, A.A. Kudrinskiy, A.Y. Olenin, G.V. Lisichkin, Synthesis and properties of silver nanoparticles: advances and prospects, Russ. Chem. Rev. 77 (233) (2008). [4] T.W. Prow, J.E. Grice, L.L. Lin, R. Faye, M. Butler, W. Becker, E.M.T. Wurm, C. Yoong, T.A. Robertson, H.P. Soyer, M.S. Roberts, Nanoparticles and microparticles for skin drug delivery, Adv. Drug Deliv. Rev. 63 (2011) 470–491. [5] Q. Chaudhry, L. Castle, Food applications of nanotechnologies: an overview of opportunities and challenges for developing countries, Trends Food Sci. Technol. 22 (2011) 595. [6] R. Nair, S.H. Varghese, B.G. Nair, T. Maekawa, Y. Yoshida, D. Sakthi Kumar, Nanoparticulate material delivery to plants, Plant Sci. 179 (2010) 154–163. [7] F.M. Kelly, J.H. Johnston, Colored and functional silver nanoparticle wool fiber composites, ACS Appl. Mater. Interfaces 3 (2011) 1083–1092. [8] T.A. Dankovich, D.G. Gray, Bactericidal paper impregnated with silver nanoparticles for point-of-use water treatment, Environ. Sci. Technol. 45 (2011) 1992–1998. [9] V.K. Sharma, R.A. Yngard, Y. Lin, Silver nanoparticles: green synthesis and their microbial activities, Adv. Colloid Interf. Sci. 145 (2009) 83–96. [10] L. Rivas, S. Sanchez-cartos, J.V. Garcia-Ramos, G. Marcillo, Growth of silver colloidal particles obtained by citrate reduction to increase the Raman enhancement factor, Langmuir 17 (2001) 574–577. [11] D. Maity, M.K. Bain, B. Bhowmick, J. Sarkar, S. Saha, K. Acharya, In situ synthesis, characterization, and antimicrobial activity of silver nanoparticles using water soluble polymer, J. Appl. Polym. Sci. 122 (4) (2011) 2189–2196. [12] K. Ohno, K. Koh, Y. Tsujii, T. Fukada, Fabrication of ordered arrays of gold nanoparticles coated with high-density polymer brushes, Angew. Chem. Int. Ed. 42 (2003) 2751–2754. [13] Z. Zhang, R.C. Patel, R. Kothari, C.P. Johnson, S.E. Friberg, P.A. Aikens, Stable silver clusters and nanoparticles prepared in polyacrylate and inverse micellar solutions, J. Phys. Chem. B 104 (2000) 1176–1182. [14] Y. Gauri, C. Thieuleux, A. Mehadi, C. Reye, R.J.P. Corriu, S. Gomez-Gallardo, In situ formation of gold nanoparticles within thiol functionalized HMS-C16 and SBA-15 type materials via an organometallic two-step approach, Chem. Mater. 15 (2003) 2017–2024. [15] M.R.D. Moura, L.H.C. Mattoso, V. Zucolotto, Development of cellulose based bactericidal nanocomposites containing silver nanoparticles and their use as active food packaging, J. Food Eng. 109 (2012) 520–524. [16] A. Hebeish, M. Hashem, M.M. Abd El-Hady, S. Sharaf, Development of CMC hydrogels loaded with silver nano-particles for medical applications, Carbohydr. Polym. 92 (2013) 407–413. [17] A. Bankar, B. Joshi, A.R. Kumar, S. Zinjarde, Banana peel extract mediated novel route for the synthesis of silver nanoparticles, Colloids Surf. A Physicochem. Eng. Asp. 368 (2010) 58–63. [18] R.M. Stiger, S. Gorer, B. Craft, P.M. Penner, Investigations of electrochemical silver nanocrystal growth on hydrogen-terminated silicon (1 0 0), Langmuir 15 (3) (1999) 790–798. [19] A.J. Kora, J. Arunachalam, Assessment of antibacterial activity of silver nanoparticles on Pseudomonas aeruginosa and its mechanism of action, World J. Microbiol. Biotechnol. 27 (2011) 1209–1216. [20] K.S. Mayya, B. Schoeler, F. Caruso, Preparation and organization of nanoscale polyelectrolyte-coated gold nanoparticles, Adv. Funct. Mater. 13 (2003) 183–188.

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