Materials Science and Engineering C 44 (2014) 234–239
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Anticancer activity of Ficus religiosa engineered copper oxide nanoparticles Renu Sankar a, Ramasamy Maheswari a, Selvaraju Karthik a, Kanchi Subramanian Shivashangari b,⁎, Vilwanathan Ravikumar a,⁎⁎ a b
Department of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu, India Regional Forensic Science Laboratory, Tiruchirapalli, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 31 October 2013 Received in revised form 30 June 2014 Accepted 8 August 2014 Available online 17 August 2014 Keywords: Ficus religiosa Green synthesis Copper oxide nanoparticles A549 cells Anticancer activity
a b s t r a c t The design, synthesis, characterization and application of biologically synthesized nanomaterials have become a vital branch of nanotechnology. There is a budding need to develop a method for environmentally benign metal nanoparticle synthesis, that do not use toxic chemicals in the synthesis protocols to avoid adverse effects in medical applications. Here, it is a report on an eco-friendly process for rapid synthesis of copper oxide nanoparticles using Ficus religiosa leaf extract as reducing and protecting agent. The synthesized copper oxide nanoparticles were confirmed by UV–vis spectrophotometer, absorbance peaks at 285 nm. The copper oxide nanoparticles were analyzed with field emission-scanning electron microscope (FE-SEM), Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS) and X-ray diffraction (XRD) spectrum. The FE-SEM and DLS analyses exposed that copper oxide nanoparticles are spherical in shape with an average particle size of 577 nm. FT-IR spectral analysis elucidates the occurrence of biomolecules required for the reduction of copper oxide ions. Zeta potential studies showed that the surface charge of the formed nanoparticles was highly negative. The XRD pattern revealed that synthesized nanoparticles are crystalline in nature. Further, biological activities of the synthesized nanoparticles were confirmed based on its stable anti-cancer effects. The apoptotic effect of copper oxide nanoparticles is mediated by the generation of reactive oxygen species (ROS) involving the disruption of mitochondrial membrane potential (Δψm) in A549 cells. The observed characteristics and results obtained in our in vitro assays suggest that the copper nanoparticles might be a potential anticancer agent. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years nanoparticles and nanomaterials could be typically used in a range of purpose, which includes targeted drug delivery, imaging, diagnosis, cosmetics and biosensor [1]. Now metal nanoparticles got extensive attraction in biological applications due to their physiochemical properties [2,3]. A number of approaches are available for the synthesis of metal nanoparticles which include physical, chemical and biological. Among these methods, biological synthesis is not only a good way to fabricate benign nanostructure materials, but also to reduce the use or generation of hazardous substances to human health and the environment. Biosynthesis of metal nanoparticles using microorganisms like bacteria, fungi and yeast are already exploited [4]. However, investigation of the plant systems as the potential nanofactories has heightened attention in the biological synthesis of nanoparticles.
⁎ Correspondence to: K.S. Shivashangari, No. 75, Second Street, Ashok Nagar, Kanchipuram-631 502, Tamilnadu, India. Tel.: +91 442 2726292. ⁎⁎ Corresponding author. Tel.: +91 431 2407071. E-mail addresses: [email protected] (K.S. Shivashangari), [email protected] (V. Ravikumar).
http://dx.doi.org/10.1016/j.msec.2014.08.030 0928-4931/© 2014 Elsevier B.V. All rights reserved.
The current research findings revealed that metal nanoparticles synthesized from plant are reliable, safe and ecofriendly when compared with physical and chemical methods. The plant mediated nanoparticle synthesis is a cost effective method and therefore a forthcoming commercial alternative for large-scale production [5,6]. Copper oxide is a semiconducting compound with a monoclinic structure. Copper oxide has concerned particular care because it is the simplest member of the family of copper compounds and exhibits a range of potentially useful physical properties [7]. As an important p-type semiconductor, copper oxide has found many varied applications such as in gas sensors, batteries, catalysis, high-temperature superconductors, solar energy conversion and field emission emitters [8]. Recently, copper oxide nanoparticles are characterized for its antimicrobial activity. Besides, copper oxide nanoparticles were found to be highly sensitive to prokaryotes and eukaryotes compared to other metal nanoparticles. It easily crosses biological barriers and reach target organ [9]. In February 2008, the US Environmental protection agency approved copper alloy based products in human use [10]. The universally available cancer chemotherapy agents are not much differentiating between cancer cells and normal cells. The development of multiple drug resistance and severe side effects also one of the major drawbacks
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refrigerated until further use. For green synthesis, 10 ml of filtrate was mixed with 90 ml of 5 mM cupric sulphate (CuSO4.5H2O) solution and incubated at room temperature. The synthesized copper oxide nanoparticles were further subjected for characterization studies. 2.2. Characterization of copper oxide nanoparticles
Fig. 1. UV–vis spectra. (a) Ficus religiosa aqueous leaf extract; (b) copper oxide nanoparticles.
with the current anticancer agents [11]. So, now we are at the cutting edge stage to find the alternative effective agent to treat cancer cells. The recent research in the field of nano-cancer biology has led to develop smart engineered nanomaterial that could be used for early diagnosis and treatment purpose. A Ficus religiosa (F. religiosa) tree has a major role in an indigenous structure of medicine like Ayurveda, Siddha, Unani and Homeopathy. The different parts of the tree are commonly used to treat various human diseases such as diabetes, atherosclerosis, Alzheimer's, gastritis, cancer and AIDS [12]. The F. religiosa aqueous leaf extract has a rich source of bioactive functional compounds which includes, alkaloids, flavonoids and terpenoids [13] that could be used to reduce the metal nanoparticles from their precursor. In the present study, we attempted to synthesis of copper oxide nanoparticles using F. religiosa leaf extract and evaluation its anticancer capability in human A549 lung cancer cells. 2. Materials and methods 2.1. Synthesis of copper oxide nanoparticles The collected F. religiosa leaves were shadow dried and powdered using mixer grinder. 10 g of powder disseminated in 100 ml of deionized water followed by boiling at 60 °C for 10 min. After cooled, the extract was filtered using Whatman No. 1 filter paper and the filtrate was
The F. religiosa leaf extract mediated copper oxide nanoparticles were confirmed with UV–visible double beam spectrophotometer (UV-1601, Shimadzu, Japan). Fourier transform infrared (FT-IR) spectroscopy was achieved by spectrum RX-1 instrument in diffuse reflectance mode functioned at a resolution of 4 cm− 1. Malvern Zetazier (Nano ZS90, UK) instrument was used to find out the stability and particle size distribution of synthesized nanoparticles. The morphological and elemental analysis was attained using field emission-scanning electron microscope (FE-SEM) with EDAX (VEGA3 TESCAN, 30.0 KV) instrument. X-ray diffraction (XRD) pattern of nanoparticles was acquired using a powder X-ray diffractometer (Philips X'Pert Pro Xray diffractometer) in 2θ range from 10° to 80°. 2.3. Cell culture The A549 human lung cancer cell line was obtained from the National Center for Cell Science, Pune, India. It was cultured in Dulbecco's modified Eagle's medium (DMEM: Hi Media Laboratories Mumbai, India), supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Hi Media Laboratories Mumbai, India). 2.4. MTT assay To find out the green synthesized copper oxide nanoparticle quality of being cytotoxic to A549 cells we performed dimethyl thiazolyltetrazolium bromide (MTT) assay. Approximately 1 × 104 cells were added to each well of a 96-well culture plate and incubated for 24 h at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The A549 cells were treated with 50–500 μg/ml of green synthesized copper oxide nanoparticles and incubated for 36 h. Control cultures were treated with DMSO. After incubation time, 20 μl of MTT solution was added to each well and further incubated for 4 h. Then 200 μl of DMSO was added, the absorbance of formed crystals was read at 575 nm using Elisa reader.
Fig. 2. FTIR spectra. (a) Ficus religiosa powder; (b) copper oxide nanoparticles.
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Fig. 3. DLS measurements. (a) Particle size distribution; (b) Zeta potential measurement.
2.5. Measurement of morphological changes in A549 cells
2.6. Apoptosis study
The A549 cells were treated with green synthesized copper oxide nanoparticles (200 μg/ml) and incubated for 36 h at 37 °C in 5% CO2 atmosphere. After 36 h incubation, cell morphological changes were viewed under Nikon inverted phase contrast microscope.
The influence of copper oxide nanoparticles to induce apoptosis in lung cancer cells was confirmed using an acridine orange (AO) and ethidium bromide (EB) [1 mg/ml for both AO and EB in phosphatebuffered saline(PBS)] staining method [14]. In brief, 5 × 105 cells/well
Fig. 4. Field emission-scanning electron microscopic analysis. (a) Copper oxide nanoparticles; (b) EDAX spectrum.
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2.9. Statistical analysis The statistical analysis was done using SPSS software Version 16 (SPSS Inc., Chicago, IL, USA). The one-way ANOVA was done for the expressing significance of the present study. Statistical significance was accepted at a level of p b 0.05.
3. Results and discussion 3.1. Synthesis of copper oxide nanoparticles
Fig. 5. Copper oxide nanoparticle in vitro cytotoxicity against A549 cells.
were cultured on cover slip in 6-cell plate and incubated overnight for attachment. After attachment, the cells were treated with fresh medium containing copper oxide nanoparticles (200 μg/ml). After 36 h incubation, cover slip was removed and stained with A0/EB (10 μl) for 30 min and washed with PBS for removing excess staining dye. Cover slip was mounted on objective glass and cell images were captured using a Nikon Eclipse inverted fluorescence microscope.
2.7. Detection of intracellular reactive oxygen species (ROS) levels For quantifying the intracellular ROS, 5 × 105 cells were seeded on a coverslip in 6-well plate and incubated overnight for attachment. Next day the cells were treated with fresh medium containing copper oxide nanoparticles (200 μg/ml) and incubated for 36 h. At the end of incubation cover-slip was removed from the culture plate and stained with 40 μM of 2′,7′-dichlorofluorescein-diacetate (DCFHDA) dye and incubated for 30 min. The stained cover slip was washed with PBS solution. Using 40 × objective fluorescence microscope the cell images were captured.
2.8. Assessment of mitochondrial membrane potential (Δψm) The Δψm was assessed using Rhodamine-123 dye. In brief, 5 × 105 cells/well were seeded in 6-well plates and incubated overnight for attachment. After overnight attachment the cells were treated with fresh medium containing copper oxide nanoparticles (200 μg/ml). After 36 h, the cover-slip was stained with 50 μl of Rhodamine-123 dye (10 μg/ml) for 30 min, excess dye was removed by washing with PBS and cell images were captured using 40× objective under fluorescence microscope.
The copper oxide nanoparticles were efficiently synthesized from F. religiosa leaf extract with 5 mM cupric sulphate (CuSO4.5H2O) containing solution at room temperature. Within 30 min of incubation the dark brown colors change, an indicator for copper oxide nanoparticle formation. This color change wasn't observed in control (cupric sulphate) solution. The green synthesized copper oxide nanoparticles showed absorbance spectra at 285 nm in UV–visible spectroscopy (Fig. 1(a)), there was no absorption peak in the F. religiosa leaf extract (Fig. 1(b)). The UV–vis absorbance spectra result enlightens that the synthesized nanoparticles were found to be symmetrical, with spherical-polydispersed in nature.
3.2. Characterization of copper oxide nanoparticles The F. religiosa aqueous leaf extract having bioactive compounds which might play a role in the synthesis of copper oxide nanoparticles can be confirmed by FT-IR spectrum [15]. FT-IR results of F. religiosa leaf powder (Fig. 2(a)) and copper oxide nanoparticles (Fig. 2(b)) shows the spectral peaks proposing, the occurrence of bands relevant to amide N\H stretching (3402 cm− 1), amide CO stretching (1578 cm−1) and nitro N\O bending (1399 cm−1). The existence of copper oxide nanoparticle band at 618 cm−1 accepted to the vibrations of Cu\O [16]. The above result confirms that the bioactive compounds present in the leaf extract have the upper hand in the synthesis of copper oxide nanoparticles. The dynamic light scattering (DLS) analysis results reveals that the size of copper oxide nanoparticles was 577 nm (Fig. 3(a)). Zeta potential distribution exhibited a negative potential of about −25.4 mV (Fig. 3(b)). We believe that, the size and the surface charge distribution of synthesized nanoparticles anticipated the biological property. The FE-SEM morphology image of copper oxide nanoparticles was spherical in shape with evenly distributed throughout the colloidal solution (Fig. 4(a)). The high percentage of elemental copper and oxide peaks was confirmed using EDAX analysis (Fig. 4(b)). We accept as truth that, the synthesized nanoparticle shape has considerably changed their optical and electronic properties [5]. XRD analysis revealed small distinct diffraction peaks at 35.507°, 38.842°, 53.760° and 65.403°, that indexed the planes (002), (111), (020) and (022) of the
Fig. 6. Membrane integrity and morphological changes in the A549 cells after treatment of (a) control, (b) copper oxide nanoparticles.
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Fig. 7. Intracellular reactive oxygen species level in the A549 cells after treatment of (a) control, (b) copper oxide nanoparticles.
face-centered-cubic structure of copper oxide nanoparticles with a monoclinic phase (ICSD-087122) (Supplementary Fig. 1) [16].
3.3. Anticancer activity The green synthesized copper oxide nanoparticle cytotoxic potential was evaluated against human lung cancer A549 cells with different concentration (50 to 500 μg/ml). Our result exhibited that lung cancer cells respond to copper oxide nanoparticles with dose dependent concentration, revealed augmented cytotoxicity in A59 cells. The green synthesized copper oxide nanoparticles at lower concentration (50 μg/ml) showed 70% viability and the cell viability was decreased up to 6% at higher concentration (500 μg/ml). The half maximal inhibitory concentration (IC50) of copper oxide nanoparticles against A549 cells was found to be 200 μg/ml (Fig. 5). The improved cytotoxicity may be due to the presence of bioactive molecules in F. religiosa leaf extract, play as encapsulating agent in copper oxide nanoparticles [5]. The copper oxide nanoparticles induced numerous morphological alterations against A549 cells at 36 h incubation, however there is no such alterations were seen in untreated cells (Supplementary Fig. 2). The nanoparticles prompted diverse morphological alterations including cell shape alterations, cell clumping, cell communication inhibition and chromatin condensation against A549 cells which undergone cell death, whereas the untreated cells were active [11]. The F. religiosa aqueous leaf extract mediated copper oxide nanoparticles activate the apoptosis pathway through generation of reactive oxygen species, alteration of Δψm against A549 cells, leads to cell death. As shown in Fig. 6, the copper oxide nanoparticle treated A549 cells revealed loss in membrane integrity and apoptotic induction with orange fragmented nuclei when compared with control, agreement with low cell viability. The data suggested that copper oxide nanoparticles could induce cell death through apoptosis. To confirm whether the apoptosis induced by synthesized nanoparticles is mediated by reactive
oxygen species (ROS) formation, the intracellular ROS generation level was evaluated using fluorescent probe DCFH-DA [17]. The fluorescence microscopy analysis results exhibited that copper oxide nanoparticle treated cells produced increased fluorescence, indicating the generation of ROS, whereas the control cells had not been produced (Fig. 7). We believe as a truth that, the increased ROS level result in free radical attack of membrane phospholipids, leading to a loss of Δψm. The up regulated ROS level in cancer cell alters the mitochondrial functions and plays a key role in apoptosis induction [18,19]. The lipophilic cationic Rhodamine-123 is an efficient probe of vital mitochondria, it specifically accumulated in the inner mitochondrial membrane. In this assay, the mitochondrial energization induced Rhodamine-123 fluorescence decay is directly proportional to the Δψm. Loss of Δψm will result in loss of the Rhodamine-123, consequently reduced fluorescence [20]. Our results exhibited, significant reduction in Rhodamine-123 dye in copper oxide nanoparticle treated cells, leads to reduced fluorescence when compared with control cells (Fig. 8). The reduced fluorescence accompany with the loss of Δψm, the hallmark for apoptosis cascade [21]. We believe from our results that, there is affordable decisive evidence for the anticancer activity of green synthesized copper oxide nanoparticles.
4. Conclusions In the present work, we conclude that F. religiosa aqueous leaf extract act as an agent to reduce the copper oxide nanoparticles from cupric sulphate solution. The green synthesized copper oxide nanoparticles were characterized by UV–visible spectroscopy, FT-IR, DLS, FE-SEM with EDAX and XRD. Besides, the colloidal copper oxide nanoparticles induce cytotoxicity against A549 cells through induction of apoptosis with enhanced ROS generation and altered Δψm level. We believe that in the near feature F. religiosa engineered copper oxide nanoparticles can be implemented as chemotherapy agent.
Fig. 8. Mitochondrial membrane potential level in the A549 cells after treatment of (a) control, (b) copper oxide nanoparticles.
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Acknowledgments We are grateful to the Department of Science and Technology (DST) for providing the financial assistance to Mr. Renu Sankar through the INSPIRE Fellowship scheme [Grant no. DST/INSPIRE Fellowship/2010/ 229C]. We extend our acknowledgement to the University Grant Commission (UGC) [Grant no. 40-208/2011 (SR) dated.29.06.2011] and the Science & Engineering Research Board (SERB) for their financial support [Grant no. SR/FT/LS-163/2009 dated.30.04.2012]. We also thank the Department of Science and Technology — Fund for Improvement of S & T Infrastructure in Universities and Higher Educational Institutions (DST-FIST) [Grant no. SR/FST/LSI-075/2011 dated.20.12.2011] for their infrastructure support to our department. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.08.030. References [1] V. Bansal, A. Bharde, R. Ramanathan, S.K. Bhargava, Adv. Colloid Interf. Sci. 179 (2012) 150–168. [2] J. An, D. Wang, Q. Luo, X. Yuan, Mater. Sci. Eng. C Mater. Biol. Appl. 29 (2009) 1984–1989. [3] R. Sankar, R. Dhivya, K.S. Shivashangari, V. Ravikumar, J. Mater. Sci. Mater. Med. 25 (2014) 1701–1708.
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[4] J. Wang, G. Zhang, Q. Li, H. Jiang, C. Liu, C. Amatore, X. Wang, Sci. Rep. 3 (2013) 1157. [5] R. Sankar, A. Karthik, A. Prabu, S. Karthik, K.S. Shivashangari, V. Ravikumar, Colloids Surf. B 108 (2013) 80–84. [6] G.M. Sulaiman, W.H. Mohammed, T.R. Marzoog, A.A.A. Al-Amiery, A.A.H. Kadhum, A. B. Mohamad, Asia Pac. J. Trop. Biomed. 3 (2013) 58–63. [7] J.M. Tranquada, B.J. Sternlieb, J.D. Axe, Y. Nakamura, S. Uchida, Nature 375 (1995) 561–563. [8] Ya-Nan Chang, M. Zhang, L. Xia, J. Zhang, G. Xing, Materials 5 (2012) 2850–2871. [9] F. Perreault, S.P. Melegari, C.H. da Costa, A.L. de Oliveira Franco Rossetto, R. Popovic, W.G. Matias, Sci. Total Environ. 441 (2012) 117–124. [10] J.J. Hostynek, H.I. Maibach, Dermatol. Ther. 17 (2004) 328–333. [11] M. Jeyaraj, G. Sathishkumar, G. Sivanandhan, D. MubarakAli, M. Rajesh, R. Arun, G. Kapildev, M. Manickavasagam, N. Thajuddin, K. Premkumar, A. Ganapathi, Colloids Surf. B 106 (2013) 86–92. [12] S.B. Chandrasekar, M. Bhanumathy, A.T. Pawar, T. Somasundaram, Pharmacogn. Rev. 4 (2010) 195–199. [13] J. Haneef, M. Parvathy, R.S.K. Thankayyan, H. Sithul, S. Sreeharshan, PLoS One 7 (2012) e40055. [14] S. Karthik, R. Sankar, K. Varunkumar, V. Ravikumar, Biomed. Pharmacother. 68 (2014) 327–334. [15] P. Prakash, P. Gnanaprakasam, R. Emmanuel, S. Arokiyaraj, M. Saravanan, Colloids Surf. B 108 (2013) 255–259. [16] R. Sankar, P. Manikandan, V. Malarvizhi, T. Fathima, K.S. Shivashangari, V. Ravikumar, Spectrochim. Acta A 121 (2014) 746–750. [17] D.K. Maurya, N. Nandakumar, T.P.A. Devasagayam, J. Clin. Biochem. Nutr. 48 (2011) 85–90. [18] Y.H. Ling, L. Liebes, Y. Zou, R. Perez-Soler, J. Biol. Chem. 278 (2003) 33714–33723. [19] L. Gibellini, M. Pinti, M. Nasi, S.D. Biasi, E. Roat, L. Bertoncelli, A. Cossarizza, Cancer 2 (2010) 1288–1311. [20] A. Baracca, G. Sgarbi, G. Solaini, G. Lenaz, Biochim. Biophys. Acta 1606 (2003) 137–146. [21] Y.F. Zhao, C. Zhang, Y.R. Suo, Eur. J. Pharmacol. 688 (2012) 6–13.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Anticancer activity of Ficus religiosa engineered copper oxide nanoparticles Renu Sankar a, Ramasamy Maheswari a, Selvaraju Karthik a, Kanchi Subramanian Shivashangari b,⁎, Vilwanathan Ravikumar a,⁎⁎ a b
Department of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu, India Regional Forensic Science Laboratory, Tiruchirapalli, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 31 October 2013 Received in revised form 30 June 2014 Accepted 8 August 2014 Available online 17 August 2014 Keywords: Ficus religiosa Green synthesis Copper oxide nanoparticles A549 cells Anticancer activity
a b s t r a c t The design, synthesis, characterization and application of biologically synthesized nanomaterials have become a vital branch of nanotechnology. There is a budding need to develop a method for environmentally benign metal nanoparticle synthesis, that do not use toxic chemicals in the synthesis protocols to avoid adverse effects in medical applications. Here, it is a report on an eco-friendly process for rapid synthesis of copper oxide nanoparticles using Ficus religiosa leaf extract as reducing and protecting agent. The synthesized copper oxide nanoparticles were confirmed by UV–vis spectrophotometer, absorbance peaks at 285 nm. The copper oxide nanoparticles were analyzed with field emission-scanning electron microscope (FE-SEM), Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS) and X-ray diffraction (XRD) spectrum. The FE-SEM and DLS analyses exposed that copper oxide nanoparticles are spherical in shape with an average particle size of 577 nm. FT-IR spectral analysis elucidates the occurrence of biomolecules required for the reduction of copper oxide ions. Zeta potential studies showed that the surface charge of the formed nanoparticles was highly negative. The XRD pattern revealed that synthesized nanoparticles are crystalline in nature. Further, biological activities of the synthesized nanoparticles were confirmed based on its stable anti-cancer effects. The apoptotic effect of copper oxide nanoparticles is mediated by the generation of reactive oxygen species (ROS) involving the disruption of mitochondrial membrane potential (Δψm) in A549 cells. The observed characteristics and results obtained in our in vitro assays suggest that the copper nanoparticles might be a potential anticancer agent. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years nanoparticles and nanomaterials could be typically used in a range of purpose, which includes targeted drug delivery, imaging, diagnosis, cosmetics and biosensor [1]. Now metal nanoparticles got extensive attraction in biological applications due to their physiochemical properties [2,3]. A number of approaches are available for the synthesis of metal nanoparticles which include physical, chemical and biological. Among these methods, biological synthesis is not only a good way to fabricate benign nanostructure materials, but also to reduce the use or generation of hazardous substances to human health and the environment. Biosynthesis of metal nanoparticles using microorganisms like bacteria, fungi and yeast are already exploited [4]. However, investigation of the plant systems as the potential nanofactories has heightened attention in the biological synthesis of nanoparticles.
⁎ Correspondence to: K.S. Shivashangari, No. 75, Second Street, Ashok Nagar, Kanchipuram-631 502, Tamilnadu, India. Tel.: +91 442 2726292. ⁎⁎ Corresponding author. Tel.: +91 431 2407071. E-mail addresses: [email protected] (K.S. Shivashangari), [email protected] (V. Ravikumar).
http://dx.doi.org/10.1016/j.msec.2014.08.030 0928-4931/© 2014 Elsevier B.V. All rights reserved.
The current research findings revealed that metal nanoparticles synthesized from plant are reliable, safe and ecofriendly when compared with physical and chemical methods. The plant mediated nanoparticle synthesis is a cost effective method and therefore a forthcoming commercial alternative for large-scale production [5,6]. Copper oxide is a semiconducting compound with a monoclinic structure. Copper oxide has concerned particular care because it is the simplest member of the family of copper compounds and exhibits a range of potentially useful physical properties [7]. As an important p-type semiconductor, copper oxide has found many varied applications such as in gas sensors, batteries, catalysis, high-temperature superconductors, solar energy conversion and field emission emitters [8]. Recently, copper oxide nanoparticles are characterized for its antimicrobial activity. Besides, copper oxide nanoparticles were found to be highly sensitive to prokaryotes and eukaryotes compared to other metal nanoparticles. It easily crosses biological barriers and reach target organ [9]. In February 2008, the US Environmental protection agency approved copper alloy based products in human use [10]. The universally available cancer chemotherapy agents are not much differentiating between cancer cells and normal cells. The development of multiple drug resistance and severe side effects also one of the major drawbacks
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refrigerated until further use. For green synthesis, 10 ml of filtrate was mixed with 90 ml of 5 mM cupric sulphate (CuSO4.5H2O) solution and incubated at room temperature. The synthesized copper oxide nanoparticles were further subjected for characterization studies. 2.2. Characterization of copper oxide nanoparticles
Fig. 1. UV–vis spectra. (a) Ficus religiosa aqueous leaf extract; (b) copper oxide nanoparticles.
with the current anticancer agents [11]. So, now we are at the cutting edge stage to find the alternative effective agent to treat cancer cells. The recent research in the field of nano-cancer biology has led to develop smart engineered nanomaterial that could be used for early diagnosis and treatment purpose. A Ficus religiosa (F. religiosa) tree has a major role in an indigenous structure of medicine like Ayurveda, Siddha, Unani and Homeopathy. The different parts of the tree are commonly used to treat various human diseases such as diabetes, atherosclerosis, Alzheimer's, gastritis, cancer and AIDS [12]. The F. religiosa aqueous leaf extract has a rich source of bioactive functional compounds which includes, alkaloids, flavonoids and terpenoids [13] that could be used to reduce the metal nanoparticles from their precursor. In the present study, we attempted to synthesis of copper oxide nanoparticles using F. religiosa leaf extract and evaluation its anticancer capability in human A549 lung cancer cells. 2. Materials and methods 2.1. Synthesis of copper oxide nanoparticles The collected F. religiosa leaves were shadow dried and powdered using mixer grinder. 10 g of powder disseminated in 100 ml of deionized water followed by boiling at 60 °C for 10 min. After cooled, the extract was filtered using Whatman No. 1 filter paper and the filtrate was
The F. religiosa leaf extract mediated copper oxide nanoparticles were confirmed with UV–visible double beam spectrophotometer (UV-1601, Shimadzu, Japan). Fourier transform infrared (FT-IR) spectroscopy was achieved by spectrum RX-1 instrument in diffuse reflectance mode functioned at a resolution of 4 cm− 1. Malvern Zetazier (Nano ZS90, UK) instrument was used to find out the stability and particle size distribution of synthesized nanoparticles. The morphological and elemental analysis was attained using field emission-scanning electron microscope (FE-SEM) with EDAX (VEGA3 TESCAN, 30.0 KV) instrument. X-ray diffraction (XRD) pattern of nanoparticles was acquired using a powder X-ray diffractometer (Philips X'Pert Pro Xray diffractometer) in 2θ range from 10° to 80°. 2.3. Cell culture The A549 human lung cancer cell line was obtained from the National Center for Cell Science, Pune, India. It was cultured in Dulbecco's modified Eagle's medium (DMEM: Hi Media Laboratories Mumbai, India), supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Hi Media Laboratories Mumbai, India). 2.4. MTT assay To find out the green synthesized copper oxide nanoparticle quality of being cytotoxic to A549 cells we performed dimethyl thiazolyltetrazolium bromide (MTT) assay. Approximately 1 × 104 cells were added to each well of a 96-well culture plate and incubated for 24 h at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The A549 cells were treated with 50–500 μg/ml of green synthesized copper oxide nanoparticles and incubated for 36 h. Control cultures were treated with DMSO. After incubation time, 20 μl of MTT solution was added to each well and further incubated for 4 h. Then 200 μl of DMSO was added, the absorbance of formed crystals was read at 575 nm using Elisa reader.
Fig. 2. FTIR spectra. (a) Ficus religiosa powder; (b) copper oxide nanoparticles.
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Fig. 3. DLS measurements. (a) Particle size distribution; (b) Zeta potential measurement.
2.5. Measurement of morphological changes in A549 cells
2.6. Apoptosis study
The A549 cells were treated with green synthesized copper oxide nanoparticles (200 μg/ml) and incubated for 36 h at 37 °C in 5% CO2 atmosphere. After 36 h incubation, cell morphological changes were viewed under Nikon inverted phase contrast microscope.
The influence of copper oxide nanoparticles to induce apoptosis in lung cancer cells was confirmed using an acridine orange (AO) and ethidium bromide (EB) [1 mg/ml for both AO and EB in phosphatebuffered saline(PBS)] staining method [14]. In brief, 5 × 105 cells/well
Fig. 4. Field emission-scanning electron microscopic analysis. (a) Copper oxide nanoparticles; (b) EDAX spectrum.
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2.9. Statistical analysis The statistical analysis was done using SPSS software Version 16 (SPSS Inc., Chicago, IL, USA). The one-way ANOVA was done for the expressing significance of the present study. Statistical significance was accepted at a level of p b 0.05.
3. Results and discussion 3.1. Synthesis of copper oxide nanoparticles
Fig. 5. Copper oxide nanoparticle in vitro cytotoxicity against A549 cells.
were cultured on cover slip in 6-cell plate and incubated overnight for attachment. After attachment, the cells were treated with fresh medium containing copper oxide nanoparticles (200 μg/ml). After 36 h incubation, cover slip was removed and stained with A0/EB (10 μl) for 30 min and washed with PBS for removing excess staining dye. Cover slip was mounted on objective glass and cell images were captured using a Nikon Eclipse inverted fluorescence microscope.
2.7. Detection of intracellular reactive oxygen species (ROS) levels For quantifying the intracellular ROS, 5 × 105 cells were seeded on a coverslip in 6-well plate and incubated overnight for attachment. Next day the cells were treated with fresh medium containing copper oxide nanoparticles (200 μg/ml) and incubated for 36 h. At the end of incubation cover-slip was removed from the culture plate and stained with 40 μM of 2′,7′-dichlorofluorescein-diacetate (DCFHDA) dye and incubated for 30 min. The stained cover slip was washed with PBS solution. Using 40 × objective fluorescence microscope the cell images were captured.
2.8. Assessment of mitochondrial membrane potential (Δψm) The Δψm was assessed using Rhodamine-123 dye. In brief, 5 × 105 cells/well were seeded in 6-well plates and incubated overnight for attachment. After overnight attachment the cells were treated with fresh medium containing copper oxide nanoparticles (200 μg/ml). After 36 h, the cover-slip was stained with 50 μl of Rhodamine-123 dye (10 μg/ml) for 30 min, excess dye was removed by washing with PBS and cell images were captured using 40× objective under fluorescence microscope.
The copper oxide nanoparticles were efficiently synthesized from F. religiosa leaf extract with 5 mM cupric sulphate (CuSO4.5H2O) containing solution at room temperature. Within 30 min of incubation the dark brown colors change, an indicator for copper oxide nanoparticle formation. This color change wasn't observed in control (cupric sulphate) solution. The green synthesized copper oxide nanoparticles showed absorbance spectra at 285 nm in UV–visible spectroscopy (Fig. 1(a)), there was no absorption peak in the F. religiosa leaf extract (Fig. 1(b)). The UV–vis absorbance spectra result enlightens that the synthesized nanoparticles were found to be symmetrical, with spherical-polydispersed in nature.
3.2. Characterization of copper oxide nanoparticles The F. religiosa aqueous leaf extract having bioactive compounds which might play a role in the synthesis of copper oxide nanoparticles can be confirmed by FT-IR spectrum [15]. FT-IR results of F. religiosa leaf powder (Fig. 2(a)) and copper oxide nanoparticles (Fig. 2(b)) shows the spectral peaks proposing, the occurrence of bands relevant to amide N\H stretching (3402 cm− 1), amide CO stretching (1578 cm−1) and nitro N\O bending (1399 cm−1). The existence of copper oxide nanoparticle band at 618 cm−1 accepted to the vibrations of Cu\O [16]. The above result confirms that the bioactive compounds present in the leaf extract have the upper hand in the synthesis of copper oxide nanoparticles. The dynamic light scattering (DLS) analysis results reveals that the size of copper oxide nanoparticles was 577 nm (Fig. 3(a)). Zeta potential distribution exhibited a negative potential of about −25.4 mV (Fig. 3(b)). We believe that, the size and the surface charge distribution of synthesized nanoparticles anticipated the biological property. The FE-SEM morphology image of copper oxide nanoparticles was spherical in shape with evenly distributed throughout the colloidal solution (Fig. 4(a)). The high percentage of elemental copper and oxide peaks was confirmed using EDAX analysis (Fig. 4(b)). We accept as truth that, the synthesized nanoparticle shape has considerably changed their optical and electronic properties [5]. XRD analysis revealed small distinct diffraction peaks at 35.507°, 38.842°, 53.760° and 65.403°, that indexed the planes (002), (111), (020) and (022) of the
Fig. 6. Membrane integrity and morphological changes in the A549 cells after treatment of (a) control, (b) copper oxide nanoparticles.
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Fig. 7. Intracellular reactive oxygen species level in the A549 cells after treatment of (a) control, (b) copper oxide nanoparticles.
face-centered-cubic structure of copper oxide nanoparticles with a monoclinic phase (ICSD-087122) (Supplementary Fig. 1) [16].
3.3. Anticancer activity The green synthesized copper oxide nanoparticle cytotoxic potential was evaluated against human lung cancer A549 cells with different concentration (50 to 500 μg/ml). Our result exhibited that lung cancer cells respond to copper oxide nanoparticles with dose dependent concentration, revealed augmented cytotoxicity in A59 cells. The green synthesized copper oxide nanoparticles at lower concentration (50 μg/ml) showed 70% viability and the cell viability was decreased up to 6% at higher concentration (500 μg/ml). The half maximal inhibitory concentration (IC50) of copper oxide nanoparticles against A549 cells was found to be 200 μg/ml (Fig. 5). The improved cytotoxicity may be due to the presence of bioactive molecules in F. religiosa leaf extract, play as encapsulating agent in copper oxide nanoparticles [5]. The copper oxide nanoparticles induced numerous morphological alterations against A549 cells at 36 h incubation, however there is no such alterations were seen in untreated cells (Supplementary Fig. 2). The nanoparticles prompted diverse morphological alterations including cell shape alterations, cell clumping, cell communication inhibition and chromatin condensation against A549 cells which undergone cell death, whereas the untreated cells were active [11]. The F. religiosa aqueous leaf extract mediated copper oxide nanoparticles activate the apoptosis pathway through generation of reactive oxygen species, alteration of Δψm against A549 cells, leads to cell death. As shown in Fig. 6, the copper oxide nanoparticle treated A549 cells revealed loss in membrane integrity and apoptotic induction with orange fragmented nuclei when compared with control, agreement with low cell viability. The data suggested that copper oxide nanoparticles could induce cell death through apoptosis. To confirm whether the apoptosis induced by synthesized nanoparticles is mediated by reactive
oxygen species (ROS) formation, the intracellular ROS generation level was evaluated using fluorescent probe DCFH-DA [17]. The fluorescence microscopy analysis results exhibited that copper oxide nanoparticle treated cells produced increased fluorescence, indicating the generation of ROS, whereas the control cells had not been produced (Fig. 7). We believe as a truth that, the increased ROS level result in free radical attack of membrane phospholipids, leading to a loss of Δψm. The up regulated ROS level in cancer cell alters the mitochondrial functions and plays a key role in apoptosis induction [18,19]. The lipophilic cationic Rhodamine-123 is an efficient probe of vital mitochondria, it specifically accumulated in the inner mitochondrial membrane. In this assay, the mitochondrial energization induced Rhodamine-123 fluorescence decay is directly proportional to the Δψm. Loss of Δψm will result in loss of the Rhodamine-123, consequently reduced fluorescence [20]. Our results exhibited, significant reduction in Rhodamine-123 dye in copper oxide nanoparticle treated cells, leads to reduced fluorescence when compared with control cells (Fig. 8). The reduced fluorescence accompany with the loss of Δψm, the hallmark for apoptosis cascade [21]. We believe from our results that, there is affordable decisive evidence for the anticancer activity of green synthesized copper oxide nanoparticles.
4. Conclusions In the present work, we conclude that F. religiosa aqueous leaf extract act as an agent to reduce the copper oxide nanoparticles from cupric sulphate solution. The green synthesized copper oxide nanoparticles were characterized by UV–visible spectroscopy, FT-IR, DLS, FE-SEM with EDAX and XRD. Besides, the colloidal copper oxide nanoparticles induce cytotoxicity against A549 cells through induction of apoptosis with enhanced ROS generation and altered Δψm level. We believe that in the near feature F. religiosa engineered copper oxide nanoparticles can be implemented as chemotherapy agent.
Fig. 8. Mitochondrial membrane potential level in the A549 cells after treatment of (a) control, (b) copper oxide nanoparticles.
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Acknowledgments We are grateful to the Department of Science and Technology (DST) for providing the financial assistance to Mr. Renu Sankar through the INSPIRE Fellowship scheme [Grant no. DST/INSPIRE Fellowship/2010/ 229C]. We extend our acknowledgement to the University Grant Commission (UGC) [Grant no. 40-208/2011 (SR) dated.29.06.2011] and the Science & Engineering Research Board (SERB) for their financial support [Grant no. SR/FT/LS-163/2009 dated.30.04.2012]. We also thank the Department of Science and Technology — Fund for Improvement of S & T Infrastructure in Universities and Higher Educational Institutions (DST-FIST) [Grant no. SR/FST/LSI-075/2011 dated.20.12.2011] for their infrastructure support to our department. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.08.030. References [1] V. Bansal, A. Bharde, R. Ramanathan, S.K. Bhargava, Adv. Colloid Interf. Sci. 179 (2012) 150–168. [2] J. An, D. Wang, Q. Luo, X. Yuan, Mater. Sci. Eng. C Mater. Biol. Appl. 29 (2009) 1984–1989. [3] R. Sankar, R. Dhivya, K.S. Shivashangari, V. Ravikumar, J. Mater. Sci. Mater. Med. 25 (2014) 1701–1708.
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