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Anticancer Activity of Green Silver Nanoparticles against He-La Cervical Cancer Cell Lines
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 18 (2019) 841–847
www.materialstoday.com/proceedings
ICN3I-2017
Anticancer Activity of Green Silver Nanoparticles against He-La Cervical Cancer Cell Lines Shweta Rajawata, M.M. Malikb a, b
Department of Physics, M.A.N.I.T., Bhopal 462003, India
Abstract
In the present work, green nanotechnology was used to synthesize silver nanoparticles of different sizes to study their anti-cancer activity against He-La cervical. The synthesis method is easy, simple, environment friendly and does not require any sophisticated labs. This method utilizes principles of green chemistry. Silver nanoparticles were synthesized using electrolytic deposition technique with black tea leaf extract as capping agent. The as-synthesized nanoparticles were characterized using XRD, TEM, and UV-Visible and FTIR spectroscopy techniques. Elemental analysis of the X-ray graphs reveals that highly pure silver nanoparticles are synthesized. Activity of the Ag NPs against cell lines was observed in a dose-dependent manner using MTT assay and it was found that as-synthesized silver nanoparticles inhibited the growth of He-La cervical cancer cell lines almost up to 70-75% respectively. The IC50 value of sample against He-La cervical cancer cell lines were obtained at 30-fold dilutions of concentration of 178 ppm. © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).
Keywords: Green Technology; Silver Nanoparticles; He-La cervical cell lines; IC50 values 1. Introduction Most of the cancer drugs indiscriminately kill both cancerous and healthy cells [1], which demand the search for novel therapies. Nanotechnology is one of the most promising areas of research in material science. Silver nanoparticles have considerable potential for development as an anti-cancer agent because of their unique properties to enhance potential therapeutic efficacy. Various physical, chemical, and biological methods can be used to 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).
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S. Rajawat and M.M. Malik / Materials Today: Proceedings 18 (2019) 841–847
synthesize different types of nanoparticles. The physical methods are initially used to give a low yield [2] but most of them render highly pure nanoparticles. Chemical methods use various chemical agents to reduce metallic ions to nanoparticles. These are expensive and involve the use of toxic chemicals that pose various biological risks [3]. Therefore, the alternative methods, which are less toxic, eco-friendly and in-expansive are being explored. Silver NPs have also been synthesized by bacteria like Pseudomonas stutzeri AG259 [4], Escherichia coli [5], fungi like Fusarium oxysporum [6], Trichoderma viride and Ganoderma neojaponicum [7], Aspergillus terreus [8], yeast like Yarrowia lipolytica NCYC 789 [9], extremophilic yeasts [10], algae such as Spirulina platensis [11], Pithophora oedogonia [12], plants such as Sinapis arvensis seeds [13], Azadirachta indica leaves [14] and blackberry fruit [15]. Compared to microorganisms, the reduction of Ag+ ions with plant extracts is more rapid and produces more stable nanoparticles [15]. Morphology of the nanoparticles can be controlled using surfactants. Selection of surfactant plays a very important role. The fast-increasing number of He-La cervical patients with the passage of time is alarming. He-La cervical cancer is responsible for the higher death rate in females. Silver nanoparticles synthesized using green technologies are a boon in the field of medicine. Nano size reduces their toxicity to almost negligible level and green synthesis adds an anatomical beneficiary edge to these nanoparticles. In the present work, physical synthesis process based on principles of green chemistry, electrolytic deposition, is used for highly pure silver nanoparticles. Black tea extract is used as capping agent. The choice of black tea as surfactant/capping agent is based on its rich contents of polyphenolic compounds whose prime constituents are theaflavins and thearubigins. These polyphenols perform the role of stabilizing or capping agent’s due to their bulky and steric nature. The tea extract has a consortium effect of hindering the particle growth. Black tea leaves extract, a surfactant, contains theaflavins. Theaflavins are basically antioxidant polyphenols. Theaflavins are formed from polymerization of catechin at the fermentation or semifermentation stage during the manufacture of black tea [16]. Catechin protects the system against the oxidation of lipids and suppresses cancer growth by combining protons with the free radicals. 2. Experimental 2.1 Preparation of black tea leaves extract: Black tea leaves extract is prepared by boiling 20ml of triple de-ionized distilled water. To this 0.3433 gm of Black tea leaves are added. After 30 seconds, it is filtered using a sieve and allowed to cool down to room temperature. 2.2 Synthesis of silver nanoparticles: Schema and picture of in-house set up for the synthesis is shown in Fig. 1 and Fig. 2. In this method, green synthesis of silver nanoparticles is done using electrolytic deposition technique. Silver nitrate (AgNO3) of 0.02N is purchased from MERCK and used, in diluted form by adding triple de-ionized distilled water, as an electrolyte. Normality of AgNO3 used in the synthesis process was 0.005N and black tea leaves was used as capping agents. For the electrolytic deposition two electrodes are used. Silver wire (99% pure) is used as anode and a carbon rod wrapped with LDPE (Low Density Poly Ethylene) material is used as cathode. LDPE material is used to collect silver nanoparticles produced in the synthesis process so that they can be easily extracted. The length of the carbon rod as well as silver wire is 4.5 cm. The diameter of the silver wire is 1.04 mm and the diameter of the carbon rod used is 4 mm. The distance between the two electrodes is 1cm. The whole assembly, with magnetic beat placed inside the beaker, is then kept on magnetic stirrer which keeps the solution in the beaker stirring continuously to avoid agglomeration. A DC power supply of rating 30volts and 3A is used. The process is carried at a room temperature (30ºC). Copper wires are used to connect the components of the circuit. Electrolysis process is started along with the addition of freshly prepared black tea extract. The concentration of the capping agent is 2.5% v/v. The black tea leaves extract is added slowly to the solution, as soon as the synthesis process starts, to avoid agglomeration. As soon as the electrolysis process starts, the color of the solution changes to light yellow within seconds which indicates the formation of the nanoparticles and then slowly the color of the solution changes to
S. Rajawat and M.M. Malik / Materials Today: Proceedings 18 (2019) 841–847
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brown as shown in Fig. 2. The color change from yellow to brown provides a piece of evidence to support the synthesis of AgNPs, and it is due to the excitation of surface plasmon vibrations, typical of AgNPs [10, 11-14]. The experiment is carried for 4 hrs.
Fig. 1 Sketch of the experimental setup in lab
3
Fig. 2 Picture of in-house experimental setup
Results and Discussions
3.1 XRD Characterization: The as-synthesized silver nanoparticles were synthesized using Rigaku Miniflex II X-ray diffractometer.
Fig. 3 XRD patterns of as-synthesized silver nanoparticles.
A characteristic XRD pattern of the silver nanoparticles synthesized by bio-base, Fig.3, with Bragg reflections at 2theta = 38.18, 64.48 and 77.63, which can be indexed to the (111), (220) and (311) planes respectively is obtained. The peaks are in accordance with JCPDS file No. 04-0783. The XRD results show Face-CenteredStructure (FCC) crystal structure of pure silver nanoparticles and a dominant peak corresponding to plane (111). The dominant plane (111) gives the low surface energy [16]. Low surface energies lead to lesser Gibbs free energy and thus lesser total energy i.e. a stable system. It is obvious from the graph that highly pure silver nanoparticles are obtained. Broadening of peaks indicate presence of silver nanoparticles. To determine and confirm the size and shape of as-synthesized silver nanoparticles, TEM characterization is done.
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3.2 TEM characterization: TEM characterization is done using JEM- 1400, Jeol, Japan. The sample (nanoparticles) is dispersed in a solution using ultrasonication for five minutes. The solution used is chemically inert, (b) should not result in agglomeration of the nanoparticles, (c) should not leave carbon residues, evaporates in a reasonable time frame (10-20 minutes) at room temperature. The grid is dried on a filter paper under an infrared lamp, followed by inspection in the TEM. TEM pictures, Fig. 4, show spherical silver nanoparticles. The size of the nanoparticles lies in the range of 2nm-25nm with an average size of 9nm. These observations confirm the results of XRD. The spherical shape of the particle can be attributed to the fact that for spherical nanoparticles the surface to volume ratio is least, thus leading to reduced total energy or state of minimum energy. A small degree of agglomeration could be seen.
Fig. 4 TEM picture of as-synthesized silver nanoparticles
4
UV-Vis spectroscopy: Silver nanoparticles were further characterized by UV- Visible spectroscopy using PC based Double Beam Spectrophotometer 2202 of Systronics was used. UV-Visible spectroscopy is based on the principle of surface plasmon resonance (SPR). Surface plasmon resonance is a physical process that occurs due to collective oscillation of the conduction electrons on interaction with EM radiations. This interaction depends on the size of the nanoparticles.
Fig. 5 UV-Visible graph of silver nanoparticles with peak wavelength at 525nm
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Different UV-Vis absorption peaks are obtained for different size of nano particles. In the case of small nanoparticles (<30 nm), absorption generally dominates the extinction spectrum. For larger nanoparticles (>50 nm), the scattering component dominates the plasmon resonance extinction spectrum. In the present work, the size of the nanoparticles is below 50nm hence here absorption dominates. From graph in Fig 5, it is found that the peak wavelength is 525nm The concentration of the colloidal solution for the first configuration was calculated using UV-Vis graphs (Figure 6) from Beer Lambert law: A = ε c l
(1)
where A is the absorbance, ε is molar extinction coefficient, c is the concentration of the solution, and l is the dimension of the cuvette. The molar extinction coefficient for silver nanoparticles in water is calculated using Mie theory-based power law: ε= a d γ,
(2)
where a = 2.3 × 105 M-1 cm-1, γ = 3.48, and d is the diameter of the silver nanoparticle ( ≤ 38 nm) [17]. Here, A = 1.01 and l = 1 cm; therefore, c = A/ε l c = 1.01/ 2.3 x 105 x (9)3.48 c = 1.65 x 10-3 Moles/litre = 1.65 x 10 -3 x 107.8682/1000 (since 1ppm = 1mg/L) = 178.01 ~178ppm 5
FTIR characterization
Fig. 6 FTIR spectra of black tea leaves (plot1) and as-synthesized silver nanoparticles (plot 2)
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FTIR spectroscopy was done to confirm the capping of as-synthesized silver nanoparticles by the capping agent. The FTIR spectroscopy gives changes in the bonds of the capping agents when nanoparticles are being capped. In plot 1 of Fig 6, peaks at 3737 cm-1, 3323.24 cm-1 and 3242.64 cm-1 correspond to the O–H stretch. The peaks at 2232 cm-1 and 2885.14 cm-1 could be attributed to the stretching and bending modes of C–H bonds. Peak at 1637.17 cm-1 could be attributed to the C=O (found in proteins) stretch of the acid groups present in thearubigins, the peak at 1207.24 cm-1 and 1085.85 cm-1can be assigned to C–O stretch and small peaks present at 981.83 cm-1 confirms the presence of aromatic substituted rings. Peak at 1020-1091 cm-1 corresponds to C-N stretching vibrations of aliphatic amines or to alcohols or phenols representing the presence of polyphenols. Plot 2 in Fig 6 shows Shift in the CH stretch from 2232.26 cm-1 to 2237.35 cm-1 and a shift of O-H stretch from 3737.73 cm-1 to 3743.76 cm-1 indicating a weak coordination between the carbonyl group and Ag NPs and thus proves that the Ag NPs are protected by the natural compound (thearubigins) present in tea. 6
Anti-cancer studies: Anti-cancer studies were carried for the sample, colloidal silver nanoparticles with average size of 10nm. The details about cell lines is as following: Cell Line: He La Organism, Homo sapiens (human) Organ: Cervix Morphology: Epithelial Disease: Adenocarcinoma Virus Susceptibility: Human adenovirus 3 Encephalomyocarditis : virus, Human poliovirus 1, Human poliovirus 2, Human poliovirus 3 DNA Profile (STR) : Amelogenin: X CSF1PO : 9,10 D13S317 : 12,13.3 149 , D16S539 : 9,10 D5S818 : 11,12 D7S820 : 8,12 THO1 : 7 TPOX : 8,12 vWA : 16,18 Cytogenetic Analysis : Modal number = 82; range = 70 to 164. There is a small telocentric chromosome in 98% of the cells. 100% aneuploidy in 1385 cells examined. Four typical He La marker chromosomes have been reported in the literature. He La Marker Chromosomes: One copy of Ml, one copy of M2, four-five copies of M3, and two copies of M4 as revealed by G-banding patterns. M1 is a rearranged long arm and centromere of chromosome 1 and the long arm of chromosome 3. M2 is a combination of short arm of chromosome 3 and long arm of chromosome 5. M3 is an iso-chromosome of the short arm of chromosome 5. M4 consists of the long arm of chromosome 11 and an arm of chromosome 19. Isoenzymes: G6PD, A Age: 31 years adult Gender: Female, Black The cells are positive for keratin by immunoperoxidase staining. He La cells have been reported to contain human papilloma virus. 18 (HPV-18) sequences. P53 expression was reported to be low, and normal levels of pRB. (Retinoblastoma suppressor) were found. MTT assay was performed at Deshpande Laboratories, Bhopal using the standard operating procedures. MTT Assay: Briefly the compounds were dissolved in DMSO (detergent reagent) and serially diluted with complete medium to get the concentrations of range of test concentration. DMSO concentration was kept < 0.1% in all the samples. Cell lines maintained in appropriate conditions were seeded in 96 well plates and treated with different concentrations of the test samples and incubated at 37 °C, 5% CO2 for 96 hours. MTT reagent was added to the wells and incubated for 4 hours; the dark blue formazan product formed by the cells was dissolved in DMSO under a safety cabinet and read at 550nm. Percentage inhibitions were calculated and plotted, shown in Fig. 7, with the concentrations used to calculate the IC50 (50% growth inhibition concentration) values. It can be inferred from the Fig. 7 that the He-La cancer cell lines respond to the sample with nanoparticles of average size 10nm requiring 30-fold dilutions as IC50 value for as-synthesized colloidal silver solution. Growth inhibition, IC50 value, is much better than existing ones [18]
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Fig 7 Dose response curve for He-La cervical cancer cell line
7
Conclusion From the present work, we conclude that highly pure silver nanoparticles can be synthesized successfully using electrolytic deposition method with black tea extract as capping agent. Silver nanoparticles capped with antioxidant polyphenols of black tea extract are more environmentally friendly and find better bio- medical applications in comparison to silver nanoparticles capped with hazardous chemicals. As-synthesized silver Nanoparticles give nearly 70% growth inhibition for cervical cancer cell lines. 30 fold dilution of 178ppm concentration of colloidal silver solution is obtained as IC50 value. Acknowledgement The author acknowledges Director, MANIT for the support in best possible ways, UGC New Delhi for funding, IUC-DAE Indore for providing XRD facilities, NIHSAD Bhopal for TEM facility, Mukul Kulshrestha, Professor, Civil engineering, MANIT for UV-Visible facility and Deshpande Labs for Anti-cancer in-vitro testing of samples of silver nanoparticles References [1] S. Kato, H. Endoh, Y. Masuhiro et al., Science, 1995, vol. 270, no. 5241, pp. 1491–1494. [2] M. A. Malik, P. O'Brien, and N. Revaprasadu, Chemistry of Materials, 2002, vol. 14, no. 5, pp. 2004–2010 [3] R. Lupu, M. Cardillo, C. Cho et al., Breast Cancer Research and Treatment, 1996, vol. 38, no. 1, pp. 57–66. [4] T. Klaus, R. Joerger, E. Olsson, C.G. Granqvist Proc. Natl. Acad. Sci. U. S. A., 96 (1999), pp. 13611-13614 [5] S. Gurunathan, K. Kalishwaralal, R. Vaidyanathan, V. Deepak, S.R.K. Pandian, J. Muniyandi, Colloids Surf. B: Biointerfaces, 74 (2009), pp. 328-335 [6] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar Colloids Surf. B: Biointerfaces, 28 (2003), pp. 313-318 [7] M. Fayaz, C.S. Tiwary, P.T. Kalaichelvan, R. Venkatesan Colloids Surf. Biointerfaces, 75 (2010), pp. 175-178 [8] L. Guangquan, H. Dan, Q. Yongqing, G. Buyuan, G. Song, C. Yan, Y. Koji, W. Li Int. J. Mol. Sci., 13 (2012), pp. 466-476 [9] A. Mugdha, S. Devashree, G. Shital, J. Swanand, B. Ashok, A. Ravi Kumar, Z. Smita AMB Express, 3 (2013), pp. 32-38 [10] A. Mourato, M. Gadanho, A.R. Lino, R. Tenreiro Bioinorg. Chem. Appl., 2011 (2011), Article 546074 (8 pages) [11] V. Sugandha, J.N. Shrivastava Int. J. Appl. Eng. Res., 9 (2014), pp. 1119-1126 [12] S. Sankar Narayan, P. Dipak, H. Nilu, S. Dipta, P. Samir Kumar Appl. Nanoscience, 5 (2015), pp. 703-709 [13] K. Mehrdad, P. Shahram, K. Mansour, H. Hadi, Z. Mehrnaz, S. Lida Biores. Bioprocess, 2 (2015), pp. 19-25 [14] S. Ahmed, M. Ahmad Saifullah, B. Swami, S. Ikram J. Radiat. Res. Appl. Sci. (2015), pp. 1-7 [15] Suk JuJang, In JunYan, Clement O Tettey, Ki MoKim, Heung MookShin,, Materials Science and Engineering: C, Volume 68, 1 November 2016, Pages 430-435 [16] Wangyu Hu, Shifang Xiao, Huiqiu Deng, Wenhua Luo and Lei Deng, http://cdn.intechopen.com/pdfs/9729/InTechThermodynamic_properties_of_nano_silver_and_alloy_particles.pdf [17] Cao, G. (2004), Imperial College Press, ISBN: 1-86094-4159, London [18] Panchanathan Manivasagan, Jayachandran Venkatesan, Kalimuthu Senthilkumar, Kannan Sivakumar, and Se-Kwon Kim, Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 287638, 9 pages,
ScienceDirect Materials Today: Proceedings 18 (2019) 841–847
www.materialstoday.com/proceedings
ICN3I-2017
Anticancer Activity of Green Silver Nanoparticles against He-La Cervical Cancer Cell Lines Shweta Rajawata, M.M. Malikb a, b
Department of Physics, M.A.N.I.T., Bhopal 462003, India
Abstract
In the present work, green nanotechnology was used to synthesize silver nanoparticles of different sizes to study their anti-cancer activity against He-La cervical. The synthesis method is easy, simple, environment friendly and does not require any sophisticated labs. This method utilizes principles of green chemistry. Silver nanoparticles were synthesized using electrolytic deposition technique with black tea leaf extract as capping agent. The as-synthesized nanoparticles were characterized using XRD, TEM, and UV-Visible and FTIR spectroscopy techniques. Elemental analysis of the X-ray graphs reveals that highly pure silver nanoparticles are synthesized. Activity of the Ag NPs against cell lines was observed in a dose-dependent manner using MTT assay and it was found that as-synthesized silver nanoparticles inhibited the growth of He-La cervical cancer cell lines almost up to 70-75% respectively. The IC50 value of sample against He-La cervical cancer cell lines were obtained at 30-fold dilutions of concentration of 178 ppm. © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).
Keywords: Green Technology; Silver Nanoparticles; He-La cervical cell lines; IC50 values 1. Introduction Most of the cancer drugs indiscriminately kill both cancerous and healthy cells [1], which demand the search for novel therapies. Nanotechnology is one of the most promising areas of research in material science. Silver nanoparticles have considerable potential for development as an anti-cancer agent because of their unique properties to enhance potential therapeutic efficacy. Various physical, chemical, and biological methods can be used to 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).
842
S. Rajawat and M.M. Malik / Materials Today: Proceedings 18 (2019) 841–847
synthesize different types of nanoparticles. The physical methods are initially used to give a low yield [2] but most of them render highly pure nanoparticles. Chemical methods use various chemical agents to reduce metallic ions to nanoparticles. These are expensive and involve the use of toxic chemicals that pose various biological risks [3]. Therefore, the alternative methods, which are less toxic, eco-friendly and in-expansive are being explored. Silver NPs have also been synthesized by bacteria like Pseudomonas stutzeri AG259 [4], Escherichia coli [5], fungi like Fusarium oxysporum [6], Trichoderma viride and Ganoderma neojaponicum [7], Aspergillus terreus [8], yeast like Yarrowia lipolytica NCYC 789 [9], extremophilic yeasts [10], algae such as Spirulina platensis [11], Pithophora oedogonia [12], plants such as Sinapis arvensis seeds [13], Azadirachta indica leaves [14] and blackberry fruit [15]. Compared to microorganisms, the reduction of Ag+ ions with plant extracts is more rapid and produces more stable nanoparticles [15]. Morphology of the nanoparticles can be controlled using surfactants. Selection of surfactant plays a very important role. The fast-increasing number of He-La cervical patients with the passage of time is alarming. He-La cervical cancer is responsible for the higher death rate in females. Silver nanoparticles synthesized using green technologies are a boon in the field of medicine. Nano size reduces their toxicity to almost negligible level and green synthesis adds an anatomical beneficiary edge to these nanoparticles. In the present work, physical synthesis process based on principles of green chemistry, electrolytic deposition, is used for highly pure silver nanoparticles. Black tea extract is used as capping agent. The choice of black tea as surfactant/capping agent is based on its rich contents of polyphenolic compounds whose prime constituents are theaflavins and thearubigins. These polyphenols perform the role of stabilizing or capping agent’s due to their bulky and steric nature. The tea extract has a consortium effect of hindering the particle growth. Black tea leaves extract, a surfactant, contains theaflavins. Theaflavins are basically antioxidant polyphenols. Theaflavins are formed from polymerization of catechin at the fermentation or semifermentation stage during the manufacture of black tea [16]. Catechin protects the system against the oxidation of lipids and suppresses cancer growth by combining protons with the free radicals. 2. Experimental 2.1 Preparation of black tea leaves extract: Black tea leaves extract is prepared by boiling 20ml of triple de-ionized distilled water. To this 0.3433 gm of Black tea leaves are added. After 30 seconds, it is filtered using a sieve and allowed to cool down to room temperature. 2.2 Synthesis of silver nanoparticles: Schema and picture of in-house set up for the synthesis is shown in Fig. 1 and Fig. 2. In this method, green synthesis of silver nanoparticles is done using electrolytic deposition technique. Silver nitrate (AgNO3) of 0.02N is purchased from MERCK and used, in diluted form by adding triple de-ionized distilled water, as an electrolyte. Normality of AgNO3 used in the synthesis process was 0.005N and black tea leaves was used as capping agents. For the electrolytic deposition two electrodes are used. Silver wire (99% pure) is used as anode and a carbon rod wrapped with LDPE (Low Density Poly Ethylene) material is used as cathode. LDPE material is used to collect silver nanoparticles produced in the synthesis process so that they can be easily extracted. The length of the carbon rod as well as silver wire is 4.5 cm. The diameter of the silver wire is 1.04 mm and the diameter of the carbon rod used is 4 mm. The distance between the two electrodes is 1cm. The whole assembly, with magnetic beat placed inside the beaker, is then kept on magnetic stirrer which keeps the solution in the beaker stirring continuously to avoid agglomeration. A DC power supply of rating 30volts and 3A is used. The process is carried at a room temperature (30ºC). Copper wires are used to connect the components of the circuit. Electrolysis process is started along with the addition of freshly prepared black tea extract. The concentration of the capping agent is 2.5% v/v. The black tea leaves extract is added slowly to the solution, as soon as the synthesis process starts, to avoid agglomeration. As soon as the electrolysis process starts, the color of the solution changes to light yellow within seconds which indicates the formation of the nanoparticles and then slowly the color of the solution changes to
S. Rajawat and M.M. Malik / Materials Today: Proceedings 18 (2019) 841–847
843
brown as shown in Fig. 2. The color change from yellow to brown provides a piece of evidence to support the synthesis of AgNPs, and it is due to the excitation of surface plasmon vibrations, typical of AgNPs [10, 11-14]. The experiment is carried for 4 hrs.
Fig. 1 Sketch of the experimental setup in lab
3
Fig. 2 Picture of in-house experimental setup
Results and Discussions
3.1 XRD Characterization: The as-synthesized silver nanoparticles were synthesized using Rigaku Miniflex II X-ray diffractometer.
Fig. 3 XRD patterns of as-synthesized silver nanoparticles.
A characteristic XRD pattern of the silver nanoparticles synthesized by bio-base, Fig.3, with Bragg reflections at 2theta = 38.18, 64.48 and 77.63, which can be indexed to the (111), (220) and (311) planes respectively is obtained. The peaks are in accordance with JCPDS file No. 04-0783. The XRD results show Face-CenteredStructure (FCC) crystal structure of pure silver nanoparticles and a dominant peak corresponding to plane (111). The dominant plane (111) gives the low surface energy [16]. Low surface energies lead to lesser Gibbs free energy and thus lesser total energy i.e. a stable system. It is obvious from the graph that highly pure silver nanoparticles are obtained. Broadening of peaks indicate presence of silver nanoparticles. To determine and confirm the size and shape of as-synthesized silver nanoparticles, TEM characterization is done.
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3.2 TEM characterization: TEM characterization is done using JEM- 1400, Jeol, Japan. The sample (nanoparticles) is dispersed in a solution using ultrasonication for five minutes. The solution used is chemically inert, (b) should not result in agglomeration of the nanoparticles, (c) should not leave carbon residues, evaporates in a reasonable time frame (10-20 minutes) at room temperature. The grid is dried on a filter paper under an infrared lamp, followed by inspection in the TEM. TEM pictures, Fig. 4, show spherical silver nanoparticles. The size of the nanoparticles lies in the range of 2nm-25nm with an average size of 9nm. These observations confirm the results of XRD. The spherical shape of the particle can be attributed to the fact that for spherical nanoparticles the surface to volume ratio is least, thus leading to reduced total energy or state of minimum energy. A small degree of agglomeration could be seen.
Fig. 4 TEM picture of as-synthesized silver nanoparticles
4
UV-Vis spectroscopy: Silver nanoparticles were further characterized by UV- Visible spectroscopy using PC based Double Beam Spectrophotometer 2202 of Systronics was used. UV-Visible spectroscopy is based on the principle of surface plasmon resonance (SPR). Surface plasmon resonance is a physical process that occurs due to collective oscillation of the conduction electrons on interaction with EM radiations. This interaction depends on the size of the nanoparticles.
Fig. 5 UV-Visible graph of silver nanoparticles with peak wavelength at 525nm
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Different UV-Vis absorption peaks are obtained for different size of nano particles. In the case of small nanoparticles (<30 nm), absorption generally dominates the extinction spectrum. For larger nanoparticles (>50 nm), the scattering component dominates the plasmon resonance extinction spectrum. In the present work, the size of the nanoparticles is below 50nm hence here absorption dominates. From graph in Fig 5, it is found that the peak wavelength is 525nm The concentration of the colloidal solution for the first configuration was calculated using UV-Vis graphs (Figure 6) from Beer Lambert law: A = ε c l
(1)
where A is the absorbance, ε is molar extinction coefficient, c is the concentration of the solution, and l is the dimension of the cuvette. The molar extinction coefficient for silver nanoparticles in water is calculated using Mie theory-based power law: ε= a d γ,
(2)
where a = 2.3 × 105 M-1 cm-1, γ = 3.48, and d is the diameter of the silver nanoparticle ( ≤ 38 nm) [17]. Here, A = 1.01 and l = 1 cm; therefore, c = A/ε l c = 1.01/ 2.3 x 105 x (9)3.48 c = 1.65 x 10-3 Moles/litre = 1.65 x 10 -3 x 107.8682/1000 (since 1ppm = 1mg/L) = 178.01 ~178ppm 5
FTIR characterization
Fig. 6 FTIR spectra of black tea leaves (plot1) and as-synthesized silver nanoparticles (plot 2)
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S. Rajawat and M.M. Malik / Materials Today: Proceedings 18 (2019) 841–847
FTIR spectroscopy was done to confirm the capping of as-synthesized silver nanoparticles by the capping agent. The FTIR spectroscopy gives changes in the bonds of the capping agents when nanoparticles are being capped. In plot 1 of Fig 6, peaks at 3737 cm-1, 3323.24 cm-1 and 3242.64 cm-1 correspond to the O–H stretch. The peaks at 2232 cm-1 and 2885.14 cm-1 could be attributed to the stretching and bending modes of C–H bonds. Peak at 1637.17 cm-1 could be attributed to the C=O (found in proteins) stretch of the acid groups present in thearubigins, the peak at 1207.24 cm-1 and 1085.85 cm-1can be assigned to C–O stretch and small peaks present at 981.83 cm-1 confirms the presence of aromatic substituted rings. Peak at 1020-1091 cm-1 corresponds to C-N stretching vibrations of aliphatic amines or to alcohols or phenols representing the presence of polyphenols. Plot 2 in Fig 6 shows Shift in the CH stretch from 2232.26 cm-1 to 2237.35 cm-1 and a shift of O-H stretch from 3737.73 cm-1 to 3743.76 cm-1 indicating a weak coordination between the carbonyl group and Ag NPs and thus proves that the Ag NPs are protected by the natural compound (thearubigins) present in tea. 6
Anti-cancer studies: Anti-cancer studies were carried for the sample, colloidal silver nanoparticles with average size of 10nm. The details about cell lines is as following: Cell Line: He La Organism, Homo sapiens (human) Organ: Cervix Morphology: Epithelial Disease: Adenocarcinoma Virus Susceptibility: Human adenovirus 3 Encephalomyocarditis : virus, Human poliovirus 1, Human poliovirus 2, Human poliovirus 3 DNA Profile (STR) : Amelogenin: X CSF1PO : 9,10 D13S317 : 12,13.3 149 , D16S539 : 9,10 D5S818 : 11,12 D7S820 : 8,12 THO1 : 7 TPOX : 8,12 vWA : 16,18 Cytogenetic Analysis : Modal number = 82; range = 70 to 164. There is a small telocentric chromosome in 98% of the cells. 100% aneuploidy in 1385 cells examined. Four typical He La marker chromosomes have been reported in the literature. He La Marker Chromosomes: One copy of Ml, one copy of M2, four-five copies of M3, and two copies of M4 as revealed by G-banding patterns. M1 is a rearranged long arm and centromere of chromosome 1 and the long arm of chromosome 3. M2 is a combination of short arm of chromosome 3 and long arm of chromosome 5. M3 is an iso-chromosome of the short arm of chromosome 5. M4 consists of the long arm of chromosome 11 and an arm of chromosome 19. Isoenzymes: G6PD, A Age: 31 years adult Gender: Female, Black The cells are positive for keratin by immunoperoxidase staining. He La cells have been reported to contain human papilloma virus. 18 (HPV-18) sequences. P53 expression was reported to be low, and normal levels of pRB. (Retinoblastoma suppressor) were found. MTT assay was performed at Deshpande Laboratories, Bhopal using the standard operating procedures. MTT Assay: Briefly the compounds were dissolved in DMSO (detergent reagent) and serially diluted with complete medium to get the concentrations of range of test concentration. DMSO concentration was kept < 0.1% in all the samples. Cell lines maintained in appropriate conditions were seeded in 96 well plates and treated with different concentrations of the test samples and incubated at 37 °C, 5% CO2 for 96 hours. MTT reagent was added to the wells and incubated for 4 hours; the dark blue formazan product formed by the cells was dissolved in DMSO under a safety cabinet and read at 550nm. Percentage inhibitions were calculated and plotted, shown in Fig. 7, with the concentrations used to calculate the IC50 (50% growth inhibition concentration) values. It can be inferred from the Fig. 7 that the He-La cancer cell lines respond to the sample with nanoparticles of average size 10nm requiring 30-fold dilutions as IC50 value for as-synthesized colloidal silver solution. Growth inhibition, IC50 value, is much better than existing ones [18]
S. Rajawat and M.M. Malik / Materials Today: Proceedings 18 (2019) 841–847
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Fig 7 Dose response curve for He-La cervical cancer cell line
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Conclusion From the present work, we conclude that highly pure silver nanoparticles can be synthesized successfully using electrolytic deposition method with black tea extract as capping agent. Silver nanoparticles capped with antioxidant polyphenols of black tea extract are more environmentally friendly and find better bio- medical applications in comparison to silver nanoparticles capped with hazardous chemicals. As-synthesized silver Nanoparticles give nearly 70% growth inhibition for cervical cancer cell lines. 30 fold dilution of 178ppm concentration of colloidal silver solution is obtained as IC50 value. Acknowledgement The author acknowledges Director, MANIT for the support in best possible ways, UGC New Delhi for funding, IUC-DAE Indore for providing XRD facilities, NIHSAD Bhopal for TEM facility, Mukul Kulshrestha, Professor, Civil engineering, MANIT for UV-Visible facility and Deshpande Labs for Anti-cancer in-vitro testing of samples of silver nanoparticles References [1] S. Kato, H. Endoh, Y. Masuhiro et al., Science, 1995, vol. 270, no. 5241, pp. 1491–1494. [2] M. A. Malik, P. O'Brien, and N. Revaprasadu, Chemistry of Materials, 2002, vol. 14, no. 5, pp. 2004–2010 [3] R. Lupu, M. Cardillo, C. Cho et al., Breast Cancer Research and Treatment, 1996, vol. 38, no. 1, pp. 57–66. [4] T. Klaus, R. Joerger, E. Olsson, C.G. Granqvist Proc. Natl. Acad. Sci. U. S. A., 96 (1999), pp. 13611-13614 [5] S. Gurunathan, K. Kalishwaralal, R. Vaidyanathan, V. Deepak, S.R.K. Pandian, J. Muniyandi, Colloids Surf. B: Biointerfaces, 74 (2009), pp. 328-335 [6] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar Colloids Surf. B: Biointerfaces, 28 (2003), pp. 313-318 [7] M. Fayaz, C.S. Tiwary, P.T. Kalaichelvan, R. Venkatesan Colloids Surf. Biointerfaces, 75 (2010), pp. 175-178 [8] L. Guangquan, H. Dan, Q. Yongqing, G. Buyuan, G. Song, C. Yan, Y. Koji, W. Li Int. J. Mol. Sci., 13 (2012), pp. 466-476 [9] A. Mugdha, S. Devashree, G. Shital, J. Swanand, B. Ashok, A. Ravi Kumar, Z. Smita AMB Express, 3 (2013), pp. 32-38 [10] A. Mourato, M. Gadanho, A.R. Lino, R. Tenreiro Bioinorg. Chem. Appl., 2011 (2011), Article 546074 (8 pages) [11] V. Sugandha, J.N. Shrivastava Int. J. Appl. Eng. Res., 9 (2014), pp. 1119-1126 [12] S. Sankar Narayan, P. Dipak, H. Nilu, S. Dipta, P. Samir Kumar Appl. Nanoscience, 5 (2015), pp. 703-709 [13] K. Mehrdad, P. Shahram, K. Mansour, H. Hadi, Z. Mehrnaz, S. Lida Biores. Bioprocess, 2 (2015), pp. 19-25 [14] S. Ahmed, M. Ahmad Saifullah, B. Swami, S. Ikram J. Radiat. Res. Appl. Sci. (2015), pp. 1-7 [15] Suk JuJang, In JunYan, Clement O Tettey, Ki MoKim, Heung MookShin,, Materials Science and Engineering: C, Volume 68, 1 November 2016, Pages 430-435 [16] Wangyu Hu, Shifang Xiao, Huiqiu Deng, Wenhua Luo and Lei Deng, http://cdn.intechopen.com/pdfs/9729/InTechThermodynamic_properties_of_nano_silver_and_alloy_particles.pdf [17] Cao, G. (2004), Imperial College Press, ISBN: 1-86094-4159, London [18] Panchanathan Manivasagan, Jayachandran Venkatesan, Kalimuthu Senthilkumar, Kannan Sivakumar, and Se-Kwon Kim, Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 287638, 9 pages,