Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Synthesis of silver nanoparticle and its application A. Muthu Kumara Pandian a,n, C. Karthikeyan a, M. Rajasimman a, M.G. Dinesh b a b
Environmental Engineering Laboratory, Department of Chemical Engineering, Annamalai University, Annamalai Nagar, Tamil Nadu, India Department of Ayurvedic and Siddha medicine, Sriramachandra Medical College, Chennai, Tamil Nadu, India
ar t ic l e i nf o
a b s t r a c t
Article history: Received 21 November 2014 Received in revised form 25 March 2015 Accepted 31 March 2015
In this work, silver nanoparticles have been synthesized by wet chemical technique, green synthesis and microbial methods. Silver nitrate (10 3 M) was used with aqueous extract to produce silver nanoparticles. From the results it was observed that the yield of nanoparticles was high in green synthesis. The size of the silver nanoparticles was determined from Scanning Electron Microscope analysis (SEM). Fourier Transform Infrared spectroscopy (FTIR) was carried out to determine the presence of biomolecules in them. Its cytotoxic effect was studied in cancerous cell line and normal cell line. MTT assay was done to test its optimal concentration and efficacy which gives valuable information for the use of silver nanoparticles for future cancer therapy. & 2015 Elsevier Inc. All rights reserved.
Keywords: Nanotechnology Green synthesis Aillum sativum Cytotoxicity
1. Introduction
2. Materials and methods
Nanotechnology is an emerging technology that attracts researchers from various fields like physics, chemistry, electrical engineering, material sciences and life sciences especially in biomedical application and biotechnology (Catherine, 2012; Kruis et al., 1998; Pankhurst et al., 2003). The main advantage of nanotechnology is the ability to utilize the special properties that materials possess when they have nanoscale dimensions (1– 1000 nm). Further, the ability to engineer these particles on such a small scale allows them to interact in special ways with biological systems, as they are roughly the size of many native proteins (Rhyner, 2008). Nanomaterials, due to their sheer size show unique and considerably changed physical, chemical, and biological properties compared to their macroscale counterparts ( Li et al., 2001). Nanoparticles can be synthesized by chemical, green and microbial methods. Each method has its own advantages and limitations. Present study dealt with the synthesis of silver nanoparticles by chemical (Fang et al., 2005), green (Neethu Hari et al., 2013) and microbial methods (Sarkar et al., 2011). In this study we exploit the advantages of each method to find out the economical as well as eco-friendly method for the nanoparticles production and its application.
2.1. Materials
n
Corresponding author. E-mail address: [email protected] (A.M.K. Pandian).
Allium sativum commonly called as garlic was collected from local grocery shop. The plant materials were thoroughly washed with water several times. The whole plant was used for obtaining the extract. Silver nitrate was purchased from Himedia Labs, India. Silver nitrate was stored in dark colored bottles. 1 mM solution of silver nitrate was prepared using deionized water. This solution has to be stored in dark colored bottles and stored in a refrigerator. This was done to avoid photosensitive reaction of silver nitrate when exposed to light. Two microorganisms were taken for the synthesis of silver nanoparticles. They are Aspergillus niger and Aspergillus flavus. The organisms were purchased from MTCC, Chandigarh, maintained and grown in 100 ml of potato dextrose broth at 25 °C. 2.2. Procedure for synthesis of silver nanoparticles from A. sativum The plant was chopped into pieces and 6 g of this plant material was weighed. It was added to 50 ml of deionised water. This mixture was kept at room temperature for 24 h. After 24 h, solid garlic pieces were removed from the solution. The pale transparent garlic extract was collected. 50 ml of 10 3 M silver nitrate was added to the garlic extract solution. Within 2 h, light orange color change was observed, which indicates the presence of silver nanoparticles. The solution was allowed to age for 48 h to yield a deep orange/brown color. The extract was filtered using whattmann filter paper (no. 42) to remove the large aggregates. The
http://dx.doi.org/10.1016/j.ecoenv.2015.03.039 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
A.M.K. Pandian et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
extract was then lyophilized to get the silver nanoparticles.
2.3. Chemical synthesis 50 ml of 10 3 M silver nitrate was heated to boiling. To this solution 5 ml of 1% tri-sodium citrate was added drop by drop. During this process solution was mixed vigorously and heated until the color change was evident (pale brown). Then it was removed from the heating element and stirred until cooled to room temperature. The aqueous solution was lyophilized and the powdered nanoparticles were obtained and taken for further analysis.
2.4. Microbial synthesis Two microorganisms A. niger and A. flavus were utilized for the synthesis of silver nanoparticles. The organisms were grown in 100 ml of potato dextrose broth at 25° C, for 48 h. After the incubation, mycelia biomass was separated by filtration, washed with sterile distilled water to remove traces of media components, resuspended in 100 ml distilled water and incubated at 25 °C. After 24 h, the suspension was filtered through Whatman filter paper no. 42. The cell filtrate and biomass were challenged with 10 3 M silver nitrate solution and incubated at room temperature. The extract was lyophilized to obtain the silver nanoparticles.
Fig. 1. (a) UV–vis spectra of chemical, A. sativum, A. niger and A. flavus extract mediated AgNPs. (b) SEM image for A. niger mediated AgNps. (c) SEM image for A. flavus mediated AgNps. (d) SEM image for A. sativum mediated AgNps. (e) SEM image for chemical mediated AgNps. (f) FTIR spectra for silver nitrate. (g) FTIR spectra for A. niger mediated AgNPs. (h) FTIR spectra for A. flavus mediated AgNPs. (i) FTIR spectra for A. sativum mediated AgNPs. (j) FTIR spectra for chemical mediated AgNPs.
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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3. Results and discussions
absorbance was maximum at 400 nm for all the methods.
Silver nanoparticles were synthesized by chemical, plant and microbial methods. From the results it was observed that the yield of silver nanoparticles per 100 ml by chemical, A. niger, A. flavus and A. sativum were 2 g, 1.2 g, 1.5 g and 3 g, respectively. The yield is high in plant synthesis. This may be due to the higher biomolecules content present in plants which are responsible for the formation of AgNPs. Further studies were performed using the silver nanoparticles synthesized from plant extract.
3.2. Scanning electron microscopy analysis (SEM)
3.1. UV–vis spectroscopy analysis UV–vis spectroscopy analysis was carried out to confirm the presence of silver nanoparticles. The formations of the nanoparticles were confirmed by the color change observed from light green to dark brownish color. This may be due to the surface plasmon vibrations (Mie, 1908). The analysis was done by scanning the samples in the range between 200 and 800 nm and the results are depicted in Fig. 1a. From the figure it was observed that the
3
The morphology and size of the nanoparticles were determined by the Scanning Electron Microscope analysis. SEM analyses of silver nanoparticles synthesized by the three methods are shown in Fig. 1b–e. From Fig. 1b and c, it was found that the shape of A. niger and A. flavus mediated AgNPs was spherical and its average size is in the range of 100–800 nm. Fig. 1d shows that the AgNPs produced by A. sativum were spherical and their size is in the range of 100–1200 nm. Fig. 1e shows that the nanoparticles produced by chemical method were spherical and their size is in the range of 50–500 nm. The morphology and sizes of the AgNPs were correlated from the SEM images and its scale values. 3.3. Fourier transform infrared spectroscopy analysis The presence of biomolecules can be very well determined with FTIR analysis and it is been correlated with standard IR
Fig. 1. (continued)
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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fingerprint data. The broad band in the region 3100–3400 cm 1 is due to O–H stretching vibrational frequency which can be occurred due to hydroxyl group of phenol, and alcohols. The intense band at 1640 cm 1 is due to C ¼C stretching of aromatic ring which is present in terpenoids. These terpenoids play a major role in reduction of metal ions. The stretch may also due to C ¼O ketones, which is present in flavonoids. The presence of NO2 stretching was also found in the silver nitrate analysis. Peaks at 1020 and 1220 cm 1 implies C–N stretching vibration of aliphatic amides. Many medium to weak bands between 1200 and 1300 cm 1 is due to the binding of molecule to the surface of AgNPs through COOH carboxylate group. This originated from the OH bending vibrational mode from alcohol or phenol functional group or from C–O stretching and OH bending vibrations from COOH. These above bonds commonly present in protein indicate the presence of protein as a ligand for reducing silver nitrate to AgNPs and thereby increase the stability of nanoparticles. These reducing agents are usually present as large fraction in plant extract. 3.4. Cytotoxic assay (MTT) The plant extract mediated nanoparticles was tested for its cytotoxic effect using MTT (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide). The cytotoxic effects of silver nanoparticles synthesized using A. sativum extract were determined
Fig. 3. Agarose gel showing fragmented DNA.
on SKOV3, HEPG2, PNAC1 cell lines by MTT-assay. The average total viable cells were precisely calculated and antiproliferative effects of compounds were determined by the MTT assay. Different concentrations of extract at time interval of 24 h had cytotoxicity effects in a dose-dependent manner. A dose-dependent growth inhibition was observed at concentrations ranging from 500 to 5 ng/ ml and each compound exhibited different sensitivity on cell line. Fig. 2a shows significant cytotoxic effect (85%) which was observed at 100 ng/ml concentration of AgNPs. The LD 50 value was found to be 31.25 ng/ml concentration. Fig. 2b shows the cytotoxic effect of silver nitrate on cancerous cell line and VERO cell line. It was found that 65% cell death occurs at 500 μg/ml concentrations and the LD 50 was 250 μg/ml concentration. In the nonmalignant cells (VERO cell line), a maximum cell death of 68% was observed
Fig. 2. (A) Invitro cytotoxic effect of A. sativum mediated AgNPs on HEPG2 cell line. (a) normal HEPG2 cell line. (b) High conc. 100 ng/ml. (c) LD 50 (31.25 ng/ml). (B) Invitro cytotoxic effect at increasing concentration of silver nitrate on VERO cell line. (a) Normal VERO cell line. (b) High conc. 500 mg/ml. (c) LD 50 (62.5 mg/ml).
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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at 500 mg/ml concentration and the LD 50 of 48% cell death was observed at 125 mg/ml concentration. 3.5. DNA fragmentation To confirm that A. sativum extract induced cell apoptosis, DNA fragmentation assay was performed. Fig. 3 shows the exposure of cells to A. sativum nanoparticles for 48 h, which lead to DNA fragmentation as indicated by the typical ladder pattern of DNA in the agarose gels at all concentrations tested, whereas control showed no DNA fragmentation. 3.6. Nuclear staining To further validate the cytotoxicity of A. sativum nanoparticles extract nuclear staining assays were also performed. As demonstrated by nuclear staining, toxic effects and the percentage of apoptotic bodies in malignant cells increased in a dose dependent fashion and higher doses of the extracts were found to exhibit pronounce cytotoxic and anti-proliferation effect (Fig. 4). 3.7. Apoptosis assay Furthermore, the expression levels of proteins related to apoptosis and cell cycle progression by western blotting were examined. Malignant cell lines were treated with Silver nanoparticles of A. sativum extract at different concentrations and total proteins were isolated. β-Actin was used as an internal control.
5
Fig. 5a shows A. sativum extract supplementation down regulated expression of Bcl-2 a potent suppressor of apoptosis and induces the expression of proapoptotic proteins Bax, caspase- 9 and caspase-3 in a dose dependent manner. The effect of plant extracts on the cleavage of procaspase 9 and procaspase 3 were also examined. Fig. 5b shows the western blot analysis. It demonstrated that cleavages of procaspase 9 and procaspase 3 were induced in a dose-dependent manner. The nanoparticles extract also induced the cleavage of PARP into 116 kDa and 84 kDa fragments as determined by western blot analysis. To further determine the effects of nanoparticles of A. sativum extract on cell progression through G2/M-phase, its effects on protein levels of the p53, p21, Cyclin B1, cdc25c and cdc2 genes were investigated. Flow cytometry analysis was performed to verify whether test sample induced apoptosis was related to cell cycle arrest. The flow cytometry spectra are shown in Fig. 6a. It shows regulated transition through the G2 checkpoint. As shown in Fig. 6b, the expression levels of Cyclin B, cdc2 and cdc25c decreased significantly after treating with nanoparticles extract in a dose-dependent fashion. Greater expression levels of p21 and p53 were recorded in nanoparticles extract treated HEPG2 cells. Flow cytometry analysis showed an increase in the percentage of G2/M arrest phase in malignant cell line compared to control cell line. The number of cells in the G2/M phase increased (52.34%) in A. sativum treated cells in a dose dependent manner.
Fig. 4. (a) Fluorescence microscope images of nuclear staining (control). (b) Fluorescence microscope images of nuclear staining (25 ng of A. sativum). (c) Fluorescence microscope images of nuclear staining (50 ng of A. sativum). (d) Fluorescence microscope images of nuclear staining (75 ng of A. sativum).
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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Fig. 5. (a) Relative intensity of β-actin vs concentration ng/ml. (b) Western blotting showing factors which causes the cell death of cancerous cells are enhanced.
4. Conclusion This study deals with the chemical, plant and microbial extract mediated synthesis of silver nanoparticles. A. sativum, plant was
found to be more effective when compared to the other sources in terms of production as well as cytotoxic assays. From the results it was found that the silver nanoparticle synthesized from A. sativum exerted an enhanced cytotoxic effect and induced many apoptotic
Fig. 6. (a) Flow cytometry spectra. (b) Western blotting showing factors which cause the cell cycle arrest factors of cancerous cells are enhanced.
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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cells, even in the cells treated with lower concentrations. The toxicity of silver nanoparticles against rapidly dividing HEPG2 cells raises exciting opportunities for their potential use as anti-cancer agents. Since AgNPs are cytotoxic to normal cell line (VERO cells) at higher concentrations, careful usage of AgNPs at reduced concentration may be used as an efficient anticancerous agent. Apparently, the promising active principles and the possible anticancer application of these nanoparticles for human use need to be analyzed.
References Catherine, L., 2012. Gold nanoparticles for physics, chemistry and biology 29A, 15. Fang, J., Zhang, C., Mu, R., 2005. The study of deposited silver particulate films by simple method of efficient SERS. Chem. Phys. Lett. 401, 271–275. Kruis, F.E., Fissan, H., Peled, A., 1998. Synthesis of nanoparticles in the gas phase for
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electronic, optical and magnetic applications -A review. J. Aerosol Sci. 29 (5-6), 511–535. Li, L.S., Hu, J., Alivistos, A.P., 2001. Band gap variation of size- and shape-controlled colloidal CdSe quantum rods. Nano Lett. 1 (7), 349–351. Rhyner, Matthew N., 2008. Development of cancer diagnostics using nanoparticles and amphiphilic polymers, PhD Thesis. Georgia Institute of Technology, Georgia. Mie, G., 1908. Contributions to the optics of turbid media especially colloidal metal solutions. Ann. Physics 25, 377–445. Neethu Hari, Thomas, Tincy K., Jayakumaran Nair, A., 2013. Comparative Study on the Synergistic Action of Garlic Synthesized and Citrate Capped Silver Nanoparticles with β-Penem Antibiotics. International Scholarly Research Notices, Article ID 792105, 6 pages. Pankhurst, Q.A., Connolly, J., Jones, S.K., Dobson, J., 2003. Application of magnetic nanoparticles in biomedicine. J. Phys. D Appl. Phys. 36 (14), 167–181. Sarkar, J., Chattopadhyay, D., Patra, S., Singh, D.S., Sinha, S., Ghosh, M., Mukherjee, A., Acharya, K., 2011. Alternaria alternate mediated synthesis of protein capped silver nanoparticles and their genotoxic activity. Dig. J. Nanomater. Biostruct. 6 (2), 563–573.
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Synthesis of silver nanoparticle and its application A. Muthu Kumara Pandian a,n, C. Karthikeyan a, M. Rajasimman a, M.G. Dinesh b a b
Environmental Engineering Laboratory, Department of Chemical Engineering, Annamalai University, Annamalai Nagar, Tamil Nadu, India Department of Ayurvedic and Siddha medicine, Sriramachandra Medical College, Chennai, Tamil Nadu, India
ar t ic l e i nf o
a b s t r a c t
Article history: Received 21 November 2014 Received in revised form 25 March 2015 Accepted 31 March 2015
In this work, silver nanoparticles have been synthesized by wet chemical technique, green synthesis and microbial methods. Silver nitrate (10 3 M) was used with aqueous extract to produce silver nanoparticles. From the results it was observed that the yield of nanoparticles was high in green synthesis. The size of the silver nanoparticles was determined from Scanning Electron Microscope analysis (SEM). Fourier Transform Infrared spectroscopy (FTIR) was carried out to determine the presence of biomolecules in them. Its cytotoxic effect was studied in cancerous cell line and normal cell line. MTT assay was done to test its optimal concentration and efficacy which gives valuable information for the use of silver nanoparticles for future cancer therapy. & 2015 Elsevier Inc. All rights reserved.
Keywords: Nanotechnology Green synthesis Aillum sativum Cytotoxicity
1. Introduction
2. Materials and methods
Nanotechnology is an emerging technology that attracts researchers from various fields like physics, chemistry, electrical engineering, material sciences and life sciences especially in biomedical application and biotechnology (Catherine, 2012; Kruis et al., 1998; Pankhurst et al., 2003). The main advantage of nanotechnology is the ability to utilize the special properties that materials possess when they have nanoscale dimensions (1– 1000 nm). Further, the ability to engineer these particles on such a small scale allows them to interact in special ways with biological systems, as they are roughly the size of many native proteins (Rhyner, 2008). Nanomaterials, due to their sheer size show unique and considerably changed physical, chemical, and biological properties compared to their macroscale counterparts ( Li et al., 2001). Nanoparticles can be synthesized by chemical, green and microbial methods. Each method has its own advantages and limitations. Present study dealt with the synthesis of silver nanoparticles by chemical (Fang et al., 2005), green (Neethu Hari et al., 2013) and microbial methods (Sarkar et al., 2011). In this study we exploit the advantages of each method to find out the economical as well as eco-friendly method for the nanoparticles production and its application.
2.1. Materials
n
Corresponding author. E-mail address: [email protected] (A.M.K. Pandian).
Allium sativum commonly called as garlic was collected from local grocery shop. The plant materials were thoroughly washed with water several times. The whole plant was used for obtaining the extract. Silver nitrate was purchased from Himedia Labs, India. Silver nitrate was stored in dark colored bottles. 1 mM solution of silver nitrate was prepared using deionized water. This solution has to be stored in dark colored bottles and stored in a refrigerator. This was done to avoid photosensitive reaction of silver nitrate when exposed to light. Two microorganisms were taken for the synthesis of silver nanoparticles. They are Aspergillus niger and Aspergillus flavus. The organisms were purchased from MTCC, Chandigarh, maintained and grown in 100 ml of potato dextrose broth at 25 °C. 2.2. Procedure for synthesis of silver nanoparticles from A. sativum The plant was chopped into pieces and 6 g of this plant material was weighed. It was added to 50 ml of deionised water. This mixture was kept at room temperature for 24 h. After 24 h, solid garlic pieces were removed from the solution. The pale transparent garlic extract was collected. 50 ml of 10 3 M silver nitrate was added to the garlic extract solution. Within 2 h, light orange color change was observed, which indicates the presence of silver nanoparticles. The solution was allowed to age for 48 h to yield a deep orange/brown color. The extract was filtered using whattmann filter paper (no. 42) to remove the large aggregates. The
http://dx.doi.org/10.1016/j.ecoenv.2015.03.039 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
A.M.K. Pandian et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
extract was then lyophilized to get the silver nanoparticles.
2.3. Chemical synthesis 50 ml of 10 3 M silver nitrate was heated to boiling. To this solution 5 ml of 1% tri-sodium citrate was added drop by drop. During this process solution was mixed vigorously and heated until the color change was evident (pale brown). Then it was removed from the heating element and stirred until cooled to room temperature. The aqueous solution was lyophilized and the powdered nanoparticles were obtained and taken for further analysis.
2.4. Microbial synthesis Two microorganisms A. niger and A. flavus were utilized for the synthesis of silver nanoparticles. The organisms were grown in 100 ml of potato dextrose broth at 25° C, for 48 h. After the incubation, mycelia biomass was separated by filtration, washed with sterile distilled water to remove traces of media components, resuspended in 100 ml distilled water and incubated at 25 °C. After 24 h, the suspension was filtered through Whatman filter paper no. 42. The cell filtrate and biomass were challenged with 10 3 M silver nitrate solution and incubated at room temperature. The extract was lyophilized to obtain the silver nanoparticles.
Fig. 1. (a) UV–vis spectra of chemical, A. sativum, A. niger and A. flavus extract mediated AgNPs. (b) SEM image for A. niger mediated AgNps. (c) SEM image for A. flavus mediated AgNps. (d) SEM image for A. sativum mediated AgNps. (e) SEM image for chemical mediated AgNps. (f) FTIR spectra for silver nitrate. (g) FTIR spectra for A. niger mediated AgNPs. (h) FTIR spectra for A. flavus mediated AgNPs. (i) FTIR spectra for A. sativum mediated AgNPs. (j) FTIR spectra for chemical mediated AgNPs.
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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3. Results and discussions
absorbance was maximum at 400 nm for all the methods.
Silver nanoparticles were synthesized by chemical, plant and microbial methods. From the results it was observed that the yield of silver nanoparticles per 100 ml by chemical, A. niger, A. flavus and A. sativum were 2 g, 1.2 g, 1.5 g and 3 g, respectively. The yield is high in plant synthesis. This may be due to the higher biomolecules content present in plants which are responsible for the formation of AgNPs. Further studies were performed using the silver nanoparticles synthesized from plant extract.
3.2. Scanning electron microscopy analysis (SEM)
3.1. UV–vis spectroscopy analysis UV–vis spectroscopy analysis was carried out to confirm the presence of silver nanoparticles. The formations of the nanoparticles were confirmed by the color change observed from light green to dark brownish color. This may be due to the surface plasmon vibrations (Mie, 1908). The analysis was done by scanning the samples in the range between 200 and 800 nm and the results are depicted in Fig. 1a. From the figure it was observed that the
3
The morphology and size of the nanoparticles were determined by the Scanning Electron Microscope analysis. SEM analyses of silver nanoparticles synthesized by the three methods are shown in Fig. 1b–e. From Fig. 1b and c, it was found that the shape of A. niger and A. flavus mediated AgNPs was spherical and its average size is in the range of 100–800 nm. Fig. 1d shows that the AgNPs produced by A. sativum were spherical and their size is in the range of 100–1200 nm. Fig. 1e shows that the nanoparticles produced by chemical method were spherical and their size is in the range of 50–500 nm. The morphology and sizes of the AgNPs were correlated from the SEM images and its scale values. 3.3. Fourier transform infrared spectroscopy analysis The presence of biomolecules can be very well determined with FTIR analysis and it is been correlated with standard IR
Fig. 1. (continued)
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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4
fingerprint data. The broad band in the region 3100–3400 cm 1 is due to O–H stretching vibrational frequency which can be occurred due to hydroxyl group of phenol, and alcohols. The intense band at 1640 cm 1 is due to C ¼C stretching of aromatic ring which is present in terpenoids. These terpenoids play a major role in reduction of metal ions. The stretch may also due to C ¼O ketones, which is present in flavonoids. The presence of NO2 stretching was also found in the silver nitrate analysis. Peaks at 1020 and 1220 cm 1 implies C–N stretching vibration of aliphatic amides. Many medium to weak bands between 1200 and 1300 cm 1 is due to the binding of molecule to the surface of AgNPs through COOH carboxylate group. This originated from the OH bending vibrational mode from alcohol or phenol functional group or from C–O stretching and OH bending vibrations from COOH. These above bonds commonly present in protein indicate the presence of protein as a ligand for reducing silver nitrate to AgNPs and thereby increase the stability of nanoparticles. These reducing agents are usually present as large fraction in plant extract. 3.4. Cytotoxic assay (MTT) The plant extract mediated nanoparticles was tested for its cytotoxic effect using MTT (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide). The cytotoxic effects of silver nanoparticles synthesized using A. sativum extract were determined
Fig. 3. Agarose gel showing fragmented DNA.
on SKOV3, HEPG2, PNAC1 cell lines by MTT-assay. The average total viable cells were precisely calculated and antiproliferative effects of compounds were determined by the MTT assay. Different concentrations of extract at time interval of 24 h had cytotoxicity effects in a dose-dependent manner. A dose-dependent growth inhibition was observed at concentrations ranging from 500 to 5 ng/ ml and each compound exhibited different sensitivity on cell line. Fig. 2a shows significant cytotoxic effect (85%) which was observed at 100 ng/ml concentration of AgNPs. The LD 50 value was found to be 31.25 ng/ml concentration. Fig. 2b shows the cytotoxic effect of silver nitrate on cancerous cell line and VERO cell line. It was found that 65% cell death occurs at 500 μg/ml concentrations and the LD 50 was 250 μg/ml concentration. In the nonmalignant cells (VERO cell line), a maximum cell death of 68% was observed
Fig. 2. (A) Invitro cytotoxic effect of A. sativum mediated AgNPs on HEPG2 cell line. (a) normal HEPG2 cell line. (b) High conc. 100 ng/ml. (c) LD 50 (31.25 ng/ml). (B) Invitro cytotoxic effect at increasing concentration of silver nitrate on VERO cell line. (a) Normal VERO cell line. (b) High conc. 500 mg/ml. (c) LD 50 (62.5 mg/ml).
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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at 500 mg/ml concentration and the LD 50 of 48% cell death was observed at 125 mg/ml concentration. 3.5. DNA fragmentation To confirm that A. sativum extract induced cell apoptosis, DNA fragmentation assay was performed. Fig. 3 shows the exposure of cells to A. sativum nanoparticles for 48 h, which lead to DNA fragmentation as indicated by the typical ladder pattern of DNA in the agarose gels at all concentrations tested, whereas control showed no DNA fragmentation. 3.6. Nuclear staining To further validate the cytotoxicity of A. sativum nanoparticles extract nuclear staining assays were also performed. As demonstrated by nuclear staining, toxic effects and the percentage of apoptotic bodies in malignant cells increased in a dose dependent fashion and higher doses of the extracts were found to exhibit pronounce cytotoxic and anti-proliferation effect (Fig. 4). 3.7. Apoptosis assay Furthermore, the expression levels of proteins related to apoptosis and cell cycle progression by western blotting were examined. Malignant cell lines were treated with Silver nanoparticles of A. sativum extract at different concentrations and total proteins were isolated. β-Actin was used as an internal control.
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Fig. 5a shows A. sativum extract supplementation down regulated expression of Bcl-2 a potent suppressor of apoptosis and induces the expression of proapoptotic proteins Bax, caspase- 9 and caspase-3 in a dose dependent manner. The effect of plant extracts on the cleavage of procaspase 9 and procaspase 3 were also examined. Fig. 5b shows the western blot analysis. It demonstrated that cleavages of procaspase 9 and procaspase 3 were induced in a dose-dependent manner. The nanoparticles extract also induced the cleavage of PARP into 116 kDa and 84 kDa fragments as determined by western blot analysis. To further determine the effects of nanoparticles of A. sativum extract on cell progression through G2/M-phase, its effects on protein levels of the p53, p21, Cyclin B1, cdc25c and cdc2 genes were investigated. Flow cytometry analysis was performed to verify whether test sample induced apoptosis was related to cell cycle arrest. The flow cytometry spectra are shown in Fig. 6a. It shows regulated transition through the G2 checkpoint. As shown in Fig. 6b, the expression levels of Cyclin B, cdc2 and cdc25c decreased significantly after treating with nanoparticles extract in a dose-dependent fashion. Greater expression levels of p21 and p53 were recorded in nanoparticles extract treated HEPG2 cells. Flow cytometry analysis showed an increase in the percentage of G2/M arrest phase in malignant cell line compared to control cell line. The number of cells in the G2/M phase increased (52.34%) in A. sativum treated cells in a dose dependent manner.
Fig. 4. (a) Fluorescence microscope images of nuclear staining (control). (b) Fluorescence microscope images of nuclear staining (25 ng of A. sativum). (c) Fluorescence microscope images of nuclear staining (50 ng of A. sativum). (d) Fluorescence microscope images of nuclear staining (75 ng of A. sativum).
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
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Fig. 5. (a) Relative intensity of β-actin vs concentration ng/ml. (b) Western blotting showing factors which causes the cell death of cancerous cells are enhanced.
4. Conclusion This study deals with the chemical, plant and microbial extract mediated synthesis of silver nanoparticles. A. sativum, plant was
found to be more effective when compared to the other sources in terms of production as well as cytotoxic assays. From the results it was found that the silver nanoparticle synthesized from A. sativum exerted an enhanced cytotoxic effect and induced many apoptotic
Fig. 6. (a) Flow cytometry spectra. (b) Western blotting showing factors which cause the cell cycle arrest factors of cancerous cells are enhanced.
Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
A.M.K. Pandian et al. / Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
cells, even in the cells treated with lower concentrations. The toxicity of silver nanoparticles against rapidly dividing HEPG2 cells raises exciting opportunities for their potential use as anti-cancer agents. Since AgNPs are cytotoxic to normal cell line (VERO cells) at higher concentrations, careful usage of AgNPs at reduced concentration may be used as an efficient anticancerous agent. Apparently, the promising active principles and the possible anticancer application of these nanoparticles for human use need to be analyzed.
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Please cite this article as: Pandian, A.M.K., et al., Synthesis of silver nanoparticle and its application. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.039i
