Materials Science and Engineering C 44 (2014) 209–215
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Sunflower oil mediated biomimetic synthesis and cytotoxicity of monodisperse hexagonal silver nanoparticles Sonal Thakore a,⁎, Puran Singh Rathore a, Ravirajsinh N. Jadeja b, Menaka Thounaojam b, Ranjitsinh V. Devkar b a b
Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 14 July 2014 Accepted 2 August 2014 Available online 11 August 2014 Keywords: Hexagonal nanoparticles Cytotoxicity Sunflower oil Biosynthesis Silver nanoparticles
a b s t r a c t In this work, sunflower oil was utilized for the biomimetic synthesis of silver (Ag) nanoparticles (NPs), leading to highly mono-dispersed hexagonal-shaped silver nanoparticles (NPs) at various concentrations. It was found that the biomolecules of the oil not only have the capability to reduce silver ions, due to its extended phenolic system, but also appear to recognize and affect the Ag nanocrystal growth on the (110) face, leading to hexagonal growth of the NPs of 50 nm size. Initially, some spherical AgNPs of less than 10 nm diameter were observed; however, over a longer period of time, a majority of hexagonal-shaped nanocrystals were formed. The one step synthesis can be extended for other metals. The as prepared sunflower oil capped AgNPs being completely free of toxic chemicals can be directly utilized for in vitro studies and offer a more rational approach for cellular applications. The NP solution exhibited dose-dependent cytotoxicity in human lung carcinoma cells and physiologically relevant cell model (3T3L1cells). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Over the past several years, the biological synthesis of nanoparticles (NPs) has been reported by several research groups. Compared to microorganisms [1], a plant-mediated synthesis of NPs is simple and cost-effective [2]. Nanoparticles produced by plants are more stable and the rate of synthesis is faster than that of microorganisms. There are many reviews that discussed plant components used in synthesis of metal nanoparticles [3]. The advantages of plant-derived materials based biosynthesis have created interest in researchers to investigate the possible mechanism of metal nanoparticle formation. Among various plant materials, the studies reported with oils as stabilizing or reducing agent are very few and the process is tedious too. NPs using fatty acids (oleic and lauric acid) and vegetable oils as stabilizers have been synthesized in the past [4–6]. Unlike aqueous plant extracts fatty acids of oils consist of amphiphilic molecules with polar carboxylic groups which are able to adsorb NPs and their non-polar long carbon chain prevents NP agglomeration through steric repulsion [6]. The other advantage of using edible oils is that the as synthesized nanoparticle suspension can be utilized for nanomedicinal tests as well as application. For instance the sunflower oil contains tocopherols that are related to the oxidative stability of the oil [7]. Tocopherols are also an important lipid oxidation inhibitors in food and biological
⁎ Corresponding author. Tel.: +91 265 2795552. E-mail address: [email protected] (S. Thakore).
http://dx.doi.org/10.1016/j.msec.2014.08.019 0928-4931/© 2014 Elsevier B.V. All rights reserved.
systems [8]. The medicinal value of sunflower oil can be enhanced by conjugation with nanoparticles. In spite of all this it is very necessary to develop a method that can afford control over size distribution, shape and crystallinity of nanoparticles, as it allows for tunability of the properties of the nanomaterials for various device fabrications [9]. Shape control of nanomaterials can be attained by controlling the growth of specific faces of the nanocrystals during the nucleation stage, which in turn allows the growth along a specific crystal axis over a period of time [10]. In most of the cases, the growth of nanocrystals towards a specific face is achieved by capping in the presence of organic surfactants or specific peptide sequences [11,12] or by hydrothermal and photochemical methods [13,14]. Herein, we report for the first time a simple synthesis of highly monodisperse hexagonal AgNPs in sunflower oil medium and their application as anticancer agents.
2. Materials and methods 2.1. Chemicals Silver nitrate (AgNO3), ethanol and petroleum ether were purchased from Merck Mumbai, India. All the solutions were prepared using double-distilled deionized water. The solution of AgNO3 was prepared using ethanol. Sunflower oil (Helianthus annuus L.) was obtained from a local store.
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2.2. Synthesis of nanoparticles
2.7. Measurement of intracellular reduced glutathione
In a typical process, 10 mL of sunflower oil and petroleum ether (1:1) was mixed with 0.6 mL of 0.01 M alcoholic AgNO3 solution with stirring in a 25 mL cap Erlenmeyer flask. It was heated in a domestic microwave (MW) oven operated at 100% power of 1350 W and frequency of 2450 MHz for about 30 s. The formation of AgNPs was marked by the characteristic pale yellow coloration of the solution. After microwave heating traces of petroleum ether and alcohol were evaporated under vacuum and the solution was kept in a refrigerator thermo-stated at 4 °C for ~48 h. The addition of petroleum ether was essential for homogeneous mixing of sunflower oil with an alcoholic AgNO3 solution.
Reduced glutathione estimation in the cell lysate was performed by the method of Moron et al. [17]. A549 cells (1 × 105 cells/well) were maintained in 6 well plates as described earlier for 24 h. At the end of the experimental period, Cell lysate was mixed with 25% of trichloroacetic acid, and centrifuged at 3000 rpm for 15 min. Supernatants were collected in microcentrifuge tubes and diluted up to 1 mL with 0.2 M sodium phosphate buffer (pH 8.0) and 2.0 mL of 0.6 mM dithionitrobenzoic acid was added and incubated at room temperature for 10 min and absorbance was read at 405 nm. 2.8. Measurement of intracellular ROS generation
2.3. Characterization of nanoparticles Characteristic optical properties of the NP solutions were recorded using a PerkinElmer Lambda 35 UV–vis spectrophotometer. FT-IR spectra of sunflower oil and vacuum dried sunflower oil capped AgNPs were recorded as KBr pellet on the Perkin Elmer RX1 model in the range of 4000–400 cm−1. Size and shape of the NPs were determined by using TEM on a Philips, Holland Technai 20 model operating at 200 kV. The sample for TEM was prepared by putting one drop of the suspension onto standard carbon-coated copper grids and then drying under an IR lamp for 30 min. The particle size distribution and zeta potential were measured using a 90 Plus DLS unit from Brookhaven (Holtsville, USA).
A549 cells (0.5 × 105 cells/well) were grown on cover slips using 12 well cell culture plates for 16 h. At the end of the incubation period, cells in cover slips were incubated with 7.5 μM CM-H2DCFDA at 37 °C for 30 min in the dark [18]. Cells were observed with a Leica DMRB fluorescence microscope. 2.9. Morphological analysis of A549 cells treated with AgNPs A549 cells (1.0 × 105 cells/well) were maintained in 6 well plates (Tarson India Pvt Ltd) for a period of 24 h in the presence of AgNPs (10–100 μg/mL) or control. At the end of experimental period, cells were fixed in 4% p-formaldehyde for 10 min, mounted in glycerin and examined under a Leica DMIL inverted microscope (40 ×) and photographed [19].
2.4. Maintenance of A549 cells 2.10. Measurement of mitochondrial membrane potential Human lung carcinoma cells (A549) obtained from National Centre for Cell Sciences, Pune, India were seeded (1 × 105 cells/25 mm T Flask) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic/anti-mycotic solution at 37 °C with 5% CO2 (Thermo scientific, forma II water jacketed CO2 incubator). Cells were subsequently sub-cultured every third day by trypsinization with trypsin phosphate versus glucose solution (TPVG). All the reagents were sterile filtered through 0.22 μ filter (Laxbro Bio-Medical Aids Pvt. Ltd) prior to use for the experiment.
The changes in mitochondrial membrane potential were measured using the fluorescent cationic dye Rhodamine 123 (rho123) as per method described in previous reports [18]. A549 cells (1.0 × 105 cells/ well) were maintained in 6 well plates (Tarson, India Pvt Ltd) for a period of 24 h as described earlier. The cells were then incubated with 1 μM rho123 for 10 min at 37 °C. The fluorescence was determined at excitation and emission wavelengths of 485 and 530 nm, respectively, using a spectrofluorometer (Jasco FP-6350) and expressed as fluorescence intensity units (FIU).
2.5. Cytotoxicity assay
2.11. Statistical analysis
Mitochondrial MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay, as an index of cytotoxicity and antiproliferative, was performed as mentioned earlier [15]. For the cell viability assay, A549 cells (5.0 × 103 cells/well) were maintained in 96 well plates (Tarson India Pvt Ltd) for a period of 24 h. In the presence of sunflower oil as well as oil containing AgNPs (10–100 μg/mL). At the end of the incubation period, 10 μL of MTT (5 mg/mL) was added to the wells and the plates were incubated for 4 h at 37 °C. At the end of 4 h, culture media were discarded and all the wells were washed with phosphate buffer saline (HiMedia Pvt Ltd, Mumbai, India). This was followed by the addition of 150 μL of dimethyl sulphoxide (DMSO) and incubated for 30 min with constant shaking. Absorbance was read at 540 nm in ELX800 Universal Microplate Reader (Bio-Tek instruments, Inc., Winooski, VT).
Data was analyzed for statistical significance using one way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test and the results were expressed as mean ± S.E.M. using GraphPad Prism version 3.0 for Windows, Graph Pad Software, San Diego California USA. 3. Result and discussion 3.1. Composition of sunflower oil Sunflower oil has a perfect chemical composition for reduction as well as stabilization of AgNPs. Typical constituents of sunflower oil are mainly triglycerides of palmitic acid (4–9%), stearic acid (1–7%), oleic acid (14–40%) and linoleic acid (48–74%). The oil also contains lecithin, tocopherols, carotenoids, wax derivatives and other phenolics like 4hydroxybenzaldehyde and oryzanol [20,21].
2.6. Lipid peroxidation assay
3.2. Optical properties of sunflower oil capped AgNPs
A549 cells (1 × 105 cells/well) were maintained in 6 well plates as described earlier for 24 h. Subsequently, cells were collected from the plate with a cell scraper (Tarson India Pvt Ltd) into a 2 mL centrifuge tube. Lipid peroxidation was assayed in the cell suspension using TBA–TCA–HCL reagent [16].
Digital photograph shows change in oil color due to formation of AgNPs (Fig. 1). The characteristic absorption peak in UV–vis absorption spectra (Fig. 2) due to the surface-plasmon resonance (SPR) of Ag colloids was observed at 428 nm. This further supports the formation of the AgNPs.
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An intense band is observed at 3420 cm−1, due to the associated \OH (stretching) group present in phenolic compounds as well as fatty acids. A significant band at 1748 cm−1 is due to carbonyl groups of triglyceride, tocopherols and other phenolics. The band at 2929 cm−1 is due to asymmetric and symmetric stretch of C\H groups of alkanes, which are abundantly found in the sunflower oil and 3008 cm− 1 for C_C (stretching) alkene. The IR spectra of the oil obtained after formation of AgNPs show the presence of peak at 3420 cm−1 shifted to 3473 cm−1 accompanied by a decrease in intensity of peak (Fig. 4B). Also, a shift in the peaks at 1020 and 1180 cm−1 to 1091 and 1111 cm−1 respectively indicates that the phenolic \OH groups participated in the bio-reduction of AgNPs. Other bands which shifted during reduction were 1748 cm−1 to 1719 cm−1 due to changes in C_O band and 1460 cm−1 to 1417 cm− 1 due to changes in C_C band. 3.5. DLS and zeta potential of sunflower oil capped AgNPs Fig. 1. Digital photograph showing formation of AgNPs.
3.3. Transmission electron microscopy study of sunflower oil capped AgNPs The TEM image in Fig. 3A of sunflower oil capped AgNPs before cold storage at 4 °C reveals the presence of spherical or semi-hexagonal particles of size less than or around 10 nm, whereas, Fig. 3B shows the TEM image of sunflower oil capped AgNPs after cold storage wherein hexagonal nanoparticles along with some spherical particles of smaller diameter are clearly visible. A high magnification TEM image in Fig. 3C suggests that the size of the hexagonal AgNPs is about 40–50 nm, while the spherical particles have a diameter of around 10 nm. It is possible that during cold storage the spherical particles gradually grow into larger hexagonal particles. Inset of Fig. 3B gives an electron diffraction (ED) pattern of the single hexagonal silver nanoparticle. Analyzing these diffraction patterns suggested that AgNPs have an fcc crystal structure. This indicates that the hexagonal silver NP is a single crystal grown along the [110] direction and enclosed by the [200] and [220] planes at the top and side surfaces, respectively.
3.4. FT-IR spectroscopy study of sunflower oil capped AgNPs The presence of triglyceride and various phenolic compounds in the oil is verified by using FT-IR spectroscopy. As clearly visible in the IR spectra of sunflower oil (Fig. 4A), the spectra exhibited a band at 1020 and 1180 cm−1 which is a depiction of 1° and 2° alcoholic groups and phenolic derivatives. The bands visible at 622 to 789 cm−1 signify the presence of R\CH group and aromatic C\H bending. The bands at 1381 cm−1 due to ether linkage and 1460 cm− 1 are due to aromatic C_C stretching. The band at 1630 cm−1 is for C_C bending of alkene.
The DLS (supporting information) of sunflower oil capped AgNPs before cold storage at 4 °C showed a single narrow distribution with average particle size in the range of 1–21 nm, whereas, after cold storage, the average particle size of NPs was 22–72 nm (supporting information). The measurement of zeta potential reveals that the oil as well as oil capped NPs showed negative zeta potential values (supporting information). 3.6. Probable mechanism of formation of sunflower oil capped AgNPs It has been reported that polyphenols and phenolic derivatives exhibit chelating properties towards transition and redox metals [25] and in some cases this aspect has been shown to play an important role in the formation of metal nanoparticles [26]. Sunflower seed oil is a good source of phenolics mainly α-tocopherol (Vitamin-E). It appears that during MW heating the tocopherols may develop an affinity towards silver ions and consequently may form silver nanoparticles. αTocopherol being an important constituent having reducing properties may be responsible for synthesis of AgNPs. It is reported in literature that during heating of oil α-tocopherol is degraded sharply to αtocopherylquinone [27]. It is possible that during microwave heating the conversion from α-tocopherol to α-tocopherylquinone might have occurred. This generally happens in two successive univalent steps; the initial step is reversible and consists of the production of a phenoxyl radical which forms an unstable oxidation–reduction system with the phenol. In a second step the phenoxyl radical gives αtocopherylquinone irreversibly and an electron which facilitate the reduction of the Ag ion as shown in scheme-S1 (Supplementary data) [28]. Initially NPs having a spherical shape or semi-hexagonal shape were formed, but over a longer period of time (After 48 h.), most of the NPs adapted hexagonal shape. Although the exact mechanism is not known we hypothesize that free fatty acids and phenolic compounds attach to the silver nuclei, leading to lower surface energy of the (110) face during cold storage. Thus the components of sunflower oil don't only have the capability to reduce silver ions, but also appear to recognize and affect the Ag nanocrystal growth on the (110) face, leading to the formation of hexagon-shaped Ag nanocrystals. 3.7. Morphological evaluation of AgNPs treated with A549 cells
Fig. 2. UV–vis absorption spectra of sunflower oil capped AgNPs.
Morphological evaluation of cultured cells following toxic insult is a very useful parameter to study cell death [29]. As shown in Fig. 5, there was a dose dependent alteration in cell shape and morphology of A549 cells treated with AgNPs. Majority of the cells appeared to be rounded and shrunken with higher doses of AgNPs compared to the control cells which depicted a characteristic spindle to oblong cellular morphology (Fig. 5).
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Fig. 3. TEM images of sunflower oil capped AgNPs (A) before cold storage, (B) after cold storage (inset showing the electron diffraction pattern) and (C) high resolution image after cold storage.
3.8. Cytotoxicity of AgNPs treated with A549 cells Determination of cell viability using MTT dye is very common and efficient protocol for evaluating the cytotoxic potential of various nanoparticles [29]. MTT is a tetrazolium dye that undergoes reduction by the
mitochondrial enzymes to form a blue colored formazan and the intensity of this color is directly proportional to cell viability [30]. Cancer cells are highly metabolic and porous in nature and are known to internalize solutes rapidly compared to normal cells. Therefore, we hypothesized that sunflower oil coated AgNPs will also show internalization within
Fig. 4. FT-IR spectra of sunflower oil and sunflower oil capped AgNPs.
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Fig. 5. Phase contrast photomicrographs of A549 cells exposed to untreated (sunflower oil extract) and 10 μg/mL, 20 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL of sunflower oil capped AgNPs.
cancer cells. Interaction of cationic particles with negatively charged cancer cells has been well reported in the literature [22]. However, Dasgupta et al. have observed that neither size nor zeta potential alone determines the optimal cell response induced by NPs [23]. The components of cellular growth media used in vitro studies could interact with NPs and change their physiochemical properties. Thus size, aggregation state, surface charge and surface chemistry would be significantly modified via electrostatic screening which in turn could influence their ability to interact with or enter cells [24]. In the present study, exposure of AgNPs to A549 cells resulted in a dose dependent (10–100 μg/mL) cytotoxicity with the highest dose (100 μg/mL) recording N70% cell death (Fig. 6A). Leakage of an intracellular enzyme LDH following toxic insult is an indicative of loss of cellular integrity. Induction of cell death in A549 cells exposed to sunflower oil coated AgNPs was in compliment with a degree of LDH release from cells (Fig. 6B). For comparison, the cytotoxicity was also evaluated in 3T3L1 fibroblast cells. It was found that the extent of toxicity was less compared to that recorded in A549 cells (Supplementary data).
Nanoparticle mediated oxidative stress in cultured cells has been demonstrated by various research groups and the same has been attributed to the generation of reactive oxygen species [30,31]. In the present study, status of cellular oxidative stress was evaluated by using DCF-DA (2′, 7′-dichlorodihydrofluorescein diacetate) fluorescence dye. The results clearly indicated dose dependent heightened ROS generation in AgNPs exposed A549 cells (Fig. 7). Further, these ROS are known to attack polyunsaturated fatty acids (PUFAs) of plasma membrane and form peroxyl radicals, that eventually results in a chain reaction called as lipid peroxidation [32]. Eukaryotic cells antioxidant machinery to combat various oxidative stress conditions viz. enzymatic and non-enzymatic antioxidants. Often there exists an inverse relation between oxidative stress and content of cellular antioxidants. We recorded elevated levels of lipid peroxidation coupled with the lowered content of nonenzymatic antioxidant, reduced glutathione (Fig. 8A&B) and the same corroborates with the result of DCF-DA staining. Recently, different research groups have reported that nanoparticles of various dimensions and chemical compositions get localized in
Fig. 6. Effect of sunflower oil capped AgNP exposure to A549 cells on (A) cell viability and (B) LDH release. Results are expressed as mean ± S.E.M. for n = 3 (replicates).
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Fig. 7. Florescence photomicrographs of DCF-DA stained A549 cells exposed to vehicle (sunflower oil extract), and 10 μg/mL, 20 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL of sunflower oil capped AgNPs.
mitochondria and induce cellular apoptosis through disruption of the electron transport chain and ATP production [33]. We evaluated mitochondrial membrane potential using rhodamine 123 dye in order to
decipher mode of cell death following exposure to sunflower oil capped AgNPs. Surprisingly, there were non-significant alterations in the level of mitochondrial membrane potential (Fig. 8C). Thus, it can be
Fig. 8. Effect of sunflower oil capped AgNP exposure to A549 cells on (A) lipid peroxidation (LPO) and (B) reduced glutathione (GSH). Results are expressed as mean ± S.E.M. for n = 3 (replicates) and (C) effect of sunflower oil capped AgNP exposure to A549 cells on mitochondrial membrane potential and ROS generation. Results are expressed as mean ± S.E.M. for n = 3 (replicates).
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hypothesized that sunflower oil capped AgNPs cause cellular death of A549 cells without altering mitochondrial integrity and probably via necrotic pathway. 4. Conclusions This study is the first report on the synthesis of sunflower oil capped silver nanoparticles with hexagonal shape. Presently recorded necrotic cell death of A549 cells exposed to AgNPs could be attributed to its novel shape and further studies are required to interpret exact mode of action therein. In conclusion, our study displays a time- and dosedependent impairment of cell viability after incubation with AgNP. The extent of cytotoxicity was found to be greater in A549 cells compared to 3T3L1 cells. This might offer the possibility of future sunflower oil capped AgNP applications in clinical practice. However, the mechanism of cell stress induction and cytotoxicity needs to be further analyzed in order to be able to predict possible health risks. Acknowledgement The authors are grateful to Dr. P.S. Nagar of Department of Botany for the useful suggestions and SAIF Shillong for the transmission electron microscopy studies. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.08.019. References [1] K.B. Narayanan, N. Sakthivel, Adv. Colloid Interface Sci. 156 (2010) 1. [2] P. PeiGan, S. HanNg, Y. Huang, S.F. YauLi, Bioresour. Technol. 113 (2012) 132. [3] S. Iravani, Green Chem. 13 (2011) 2638.
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[4] H.T. Song, J.S. Choi, Y.M. Huh, S. Kim, Y.W. Jun, J.S. Suh, J. Cheon, J. Am. Chem. Soc. 127 (2005) 9992. [5] E.C. Da Silva, M.G.A. Da Silva, S.M.P. Meneghetti, G. Machado, A.R.C. Alencar, J.M. Hickmann, M.R. Menegetti, J. Nanoparticle Res. 1 (2008) 201. [6] R. Zamiri, A. Zakari, H. Abbastabar, M. Darroudi, M.S. Husin, M. Mahdi, Int. J. Nanomedicine 6 (2011) 565. [7] G.H. Crapiste, M.I.V. Brevedan, A.A. Carelli, J. Am. Oil Chem. Soc. 76 (1999) 1437. [8] A. Kamal-Eldin, L. Appelqvist, Lipids 31 (1996) 671. [9] N. Barnaby, S. Yu, K. Fath, A. Tsiola, O. Khalpari, I.A. Banerjee, Nanotechnology 22 (2011) 225605 (10 pp.). [10] V.F. Puntes, K.M. Krishnan, A.P. Alivisatos, Science 291 (2001) 2115. [11] C.J. Carter, C.J. Ackerson, D.L. Feldheim, ACS Nano 4 (2010) 3883. [12] C.J. Murphy, Science 298 (2003) 2139. [13] M. Valodkar, A. Pal, S. Thakore, J. Alloys Compd. 509 (2011) 523. [14] C. Tang, W. Sun, J. Lu, W. Yan, J. Colloid Interface Sci. 416 (2014) 86. [15] M.C. Thounaojam, R.N. Jadeja, M. Valodkar, P.S. Nagar, R.V. Devkar, S. Thakore, Food Chem. Toxicol. 49 (2011) 2990. [16] J.A. Buege, S.D. Aust, Methods Enzymol. 52 (1978) 302. [17] M.S. Moron, J.W. Kepierre, B. Mannervick, Biochim. Biophys. Acta 582 (1979) 67. [18] R.N. Jadeja, M.C. Thounaojam, R.V. Devkar, A.V. Ramachandran, Food Chem. Toxicol. 49 (2011) 1195. [19] M. Valodkar, R.N. Jadeja, M.C. Thounaojam, R.V. Devkar, S. Thakore, Mater. Chem. Phys. 128 (2011) 83. [20] A. Thomas, Weinheim: Wiley-VCH (2002) 198. [21] F. Aladedunye, R. Przybylski, Food Chem. 141 (2013) 2373. [22] A.M. Alkilany, C.J. Murphy, J. Nanoparticle Res. 12 (2010) 2313. [23] H.K. Patra, A. Dasgupta, Nanomedicine Nanotechnol. 8 (2011) 115. [24] V.S. Nikolov, A. Wasan, Adv. Colloid Interface Sci. 134–135 (2007) 268. [25] E. Koren, R. Kohen, I.A. Ginsburg, J. Agric. Food Chem. 57 (2009) 7644. [26] J.A. Jacob, H.S. Mahal, N. Biswas, T. Mukherjee, S. Kapoor, Langmuir 24 (2008) 528. [27] S. Casal, R. Malheiro, A. Sendas, B. Oliveira, J.A. Pereira, Food Chem. Toxicol. 48 (2010) 2972. [28] D. Firestone, Phys. Chem. Characteristics of Oils, Fats, and Waxes, 1999. 100. [29] M. Valodkar, R.N. Jadeja, M.C. Thounaojam, R.V. Devkar, S. Thakore, Mater. Sci. Eng. C 31 (2011) 1723. [30] V. Sharma, D. Anderson, A. Dhawan, J. Biomed. Nanotechnol. 7 (2011) 98. [31] Q. Saquib, A.A. Al-Khedhairy, M.A. Siddiqui, F.M. Abou-Tarboush, A. Azam, J. Musarrat, Toxicol. in Vitro 26 (2012) 351. [32] R.N. Jadeja, M.C. Thounaojam, D.K. Patel, R.V. Devkar, A.V. Ramachandran, Cardiovasc. Toxicol. 10 (2010) 174. [33] C.S. Costa, J.V.V. Ronconi, J.F. Daufenbach, C.L. Gonc, G.T. Rezin, E.L. Streck, M.M.S. Paula, Mol. Cell. Biochem. 342 (2010) 51.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Sunflower oil mediated biomimetic synthesis and cytotoxicity of monodisperse hexagonal silver nanoparticles Sonal Thakore a,⁎, Puran Singh Rathore a, Ravirajsinh N. Jadeja b, Menaka Thounaojam b, Ranjitsinh V. Devkar b a b
Department of Chemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 14 July 2014 Accepted 2 August 2014 Available online 11 August 2014 Keywords: Hexagonal nanoparticles Cytotoxicity Sunflower oil Biosynthesis Silver nanoparticles
a b s t r a c t In this work, sunflower oil was utilized for the biomimetic synthesis of silver (Ag) nanoparticles (NPs), leading to highly mono-dispersed hexagonal-shaped silver nanoparticles (NPs) at various concentrations. It was found that the biomolecules of the oil not only have the capability to reduce silver ions, due to its extended phenolic system, but also appear to recognize and affect the Ag nanocrystal growth on the (110) face, leading to hexagonal growth of the NPs of 50 nm size. Initially, some spherical AgNPs of less than 10 nm diameter were observed; however, over a longer period of time, a majority of hexagonal-shaped nanocrystals were formed. The one step synthesis can be extended for other metals. The as prepared sunflower oil capped AgNPs being completely free of toxic chemicals can be directly utilized for in vitro studies and offer a more rational approach for cellular applications. The NP solution exhibited dose-dependent cytotoxicity in human lung carcinoma cells and physiologically relevant cell model (3T3L1cells). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Over the past several years, the biological synthesis of nanoparticles (NPs) has been reported by several research groups. Compared to microorganisms [1], a plant-mediated synthesis of NPs is simple and cost-effective [2]. Nanoparticles produced by plants are more stable and the rate of synthesis is faster than that of microorganisms. There are many reviews that discussed plant components used in synthesis of metal nanoparticles [3]. The advantages of plant-derived materials based biosynthesis have created interest in researchers to investigate the possible mechanism of metal nanoparticle formation. Among various plant materials, the studies reported with oils as stabilizing or reducing agent are very few and the process is tedious too. NPs using fatty acids (oleic and lauric acid) and vegetable oils as stabilizers have been synthesized in the past [4–6]. Unlike aqueous plant extracts fatty acids of oils consist of amphiphilic molecules with polar carboxylic groups which are able to adsorb NPs and their non-polar long carbon chain prevents NP agglomeration through steric repulsion [6]. The other advantage of using edible oils is that the as synthesized nanoparticle suspension can be utilized for nanomedicinal tests as well as application. For instance the sunflower oil contains tocopherols that are related to the oxidative stability of the oil [7]. Tocopherols are also an important lipid oxidation inhibitors in food and biological
⁎ Corresponding author. Tel.: +91 265 2795552. E-mail address: [email protected] (S. Thakore).
http://dx.doi.org/10.1016/j.msec.2014.08.019 0928-4931/© 2014 Elsevier B.V. All rights reserved.
systems [8]. The medicinal value of sunflower oil can be enhanced by conjugation with nanoparticles. In spite of all this it is very necessary to develop a method that can afford control over size distribution, shape and crystallinity of nanoparticles, as it allows for tunability of the properties of the nanomaterials for various device fabrications [9]. Shape control of nanomaterials can be attained by controlling the growth of specific faces of the nanocrystals during the nucleation stage, which in turn allows the growth along a specific crystal axis over a period of time [10]. In most of the cases, the growth of nanocrystals towards a specific face is achieved by capping in the presence of organic surfactants or specific peptide sequences [11,12] or by hydrothermal and photochemical methods [13,14]. Herein, we report for the first time a simple synthesis of highly monodisperse hexagonal AgNPs in sunflower oil medium and their application as anticancer agents.
2. Materials and methods 2.1. Chemicals Silver nitrate (AgNO3), ethanol and petroleum ether were purchased from Merck Mumbai, India. All the solutions were prepared using double-distilled deionized water. The solution of AgNO3 was prepared using ethanol. Sunflower oil (Helianthus annuus L.) was obtained from a local store.
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2.2. Synthesis of nanoparticles
2.7. Measurement of intracellular reduced glutathione
In a typical process, 10 mL of sunflower oil and petroleum ether (1:1) was mixed with 0.6 mL of 0.01 M alcoholic AgNO3 solution with stirring in a 25 mL cap Erlenmeyer flask. It was heated in a domestic microwave (MW) oven operated at 100% power of 1350 W and frequency of 2450 MHz for about 30 s. The formation of AgNPs was marked by the characteristic pale yellow coloration of the solution. After microwave heating traces of petroleum ether and alcohol were evaporated under vacuum and the solution was kept in a refrigerator thermo-stated at 4 °C for ~48 h. The addition of petroleum ether was essential for homogeneous mixing of sunflower oil with an alcoholic AgNO3 solution.
Reduced glutathione estimation in the cell lysate was performed by the method of Moron et al. [17]. A549 cells (1 × 105 cells/well) were maintained in 6 well plates as described earlier for 24 h. At the end of the experimental period, Cell lysate was mixed with 25% of trichloroacetic acid, and centrifuged at 3000 rpm for 15 min. Supernatants were collected in microcentrifuge tubes and diluted up to 1 mL with 0.2 M sodium phosphate buffer (pH 8.0) and 2.0 mL of 0.6 mM dithionitrobenzoic acid was added and incubated at room temperature for 10 min and absorbance was read at 405 nm. 2.8. Measurement of intracellular ROS generation
2.3. Characterization of nanoparticles Characteristic optical properties of the NP solutions were recorded using a PerkinElmer Lambda 35 UV–vis spectrophotometer. FT-IR spectra of sunflower oil and vacuum dried sunflower oil capped AgNPs were recorded as KBr pellet on the Perkin Elmer RX1 model in the range of 4000–400 cm−1. Size and shape of the NPs were determined by using TEM on a Philips, Holland Technai 20 model operating at 200 kV. The sample for TEM was prepared by putting one drop of the suspension onto standard carbon-coated copper grids and then drying under an IR lamp for 30 min. The particle size distribution and zeta potential were measured using a 90 Plus DLS unit from Brookhaven (Holtsville, USA).
A549 cells (0.5 × 105 cells/well) were grown on cover slips using 12 well cell culture plates for 16 h. At the end of the incubation period, cells in cover slips were incubated with 7.5 μM CM-H2DCFDA at 37 °C for 30 min in the dark [18]. Cells were observed with a Leica DMRB fluorescence microscope. 2.9. Morphological analysis of A549 cells treated with AgNPs A549 cells (1.0 × 105 cells/well) were maintained in 6 well plates (Tarson India Pvt Ltd) for a period of 24 h in the presence of AgNPs (10–100 μg/mL) or control. At the end of experimental period, cells were fixed in 4% p-formaldehyde for 10 min, mounted in glycerin and examined under a Leica DMIL inverted microscope (40 ×) and photographed [19].
2.4. Maintenance of A549 cells 2.10. Measurement of mitochondrial membrane potential Human lung carcinoma cells (A549) obtained from National Centre for Cell Sciences, Pune, India were seeded (1 × 105 cells/25 mm T Flask) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic/anti-mycotic solution at 37 °C with 5% CO2 (Thermo scientific, forma II water jacketed CO2 incubator). Cells were subsequently sub-cultured every third day by trypsinization with trypsin phosphate versus glucose solution (TPVG). All the reagents were sterile filtered through 0.22 μ filter (Laxbro Bio-Medical Aids Pvt. Ltd) prior to use for the experiment.
The changes in mitochondrial membrane potential were measured using the fluorescent cationic dye Rhodamine 123 (rho123) as per method described in previous reports [18]. A549 cells (1.0 × 105 cells/ well) were maintained in 6 well plates (Tarson, India Pvt Ltd) for a period of 24 h as described earlier. The cells were then incubated with 1 μM rho123 for 10 min at 37 °C. The fluorescence was determined at excitation and emission wavelengths of 485 and 530 nm, respectively, using a spectrofluorometer (Jasco FP-6350) and expressed as fluorescence intensity units (FIU).
2.5. Cytotoxicity assay
2.11. Statistical analysis
Mitochondrial MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay, as an index of cytotoxicity and antiproliferative, was performed as mentioned earlier [15]. For the cell viability assay, A549 cells (5.0 × 103 cells/well) were maintained in 96 well plates (Tarson India Pvt Ltd) for a period of 24 h. In the presence of sunflower oil as well as oil containing AgNPs (10–100 μg/mL). At the end of the incubation period, 10 μL of MTT (5 mg/mL) was added to the wells and the plates were incubated for 4 h at 37 °C. At the end of 4 h, culture media were discarded and all the wells were washed with phosphate buffer saline (HiMedia Pvt Ltd, Mumbai, India). This was followed by the addition of 150 μL of dimethyl sulphoxide (DMSO) and incubated for 30 min with constant shaking. Absorbance was read at 540 nm in ELX800 Universal Microplate Reader (Bio-Tek instruments, Inc., Winooski, VT).
Data was analyzed for statistical significance using one way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test and the results were expressed as mean ± S.E.M. using GraphPad Prism version 3.0 for Windows, Graph Pad Software, San Diego California USA. 3. Result and discussion 3.1. Composition of sunflower oil Sunflower oil has a perfect chemical composition for reduction as well as stabilization of AgNPs. Typical constituents of sunflower oil are mainly triglycerides of palmitic acid (4–9%), stearic acid (1–7%), oleic acid (14–40%) and linoleic acid (48–74%). The oil also contains lecithin, tocopherols, carotenoids, wax derivatives and other phenolics like 4hydroxybenzaldehyde and oryzanol [20,21].
2.6. Lipid peroxidation assay
3.2. Optical properties of sunflower oil capped AgNPs
A549 cells (1 × 105 cells/well) were maintained in 6 well plates as described earlier for 24 h. Subsequently, cells were collected from the plate with a cell scraper (Tarson India Pvt Ltd) into a 2 mL centrifuge tube. Lipid peroxidation was assayed in the cell suspension using TBA–TCA–HCL reagent [16].
Digital photograph shows change in oil color due to formation of AgNPs (Fig. 1). The characteristic absorption peak in UV–vis absorption spectra (Fig. 2) due to the surface-plasmon resonance (SPR) of Ag colloids was observed at 428 nm. This further supports the formation of the AgNPs.
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An intense band is observed at 3420 cm−1, due to the associated \OH (stretching) group present in phenolic compounds as well as fatty acids. A significant band at 1748 cm−1 is due to carbonyl groups of triglyceride, tocopherols and other phenolics. The band at 2929 cm−1 is due to asymmetric and symmetric stretch of C\H groups of alkanes, which are abundantly found in the sunflower oil and 3008 cm− 1 for C_C (stretching) alkene. The IR spectra of the oil obtained after formation of AgNPs show the presence of peak at 3420 cm−1 shifted to 3473 cm−1 accompanied by a decrease in intensity of peak (Fig. 4B). Also, a shift in the peaks at 1020 and 1180 cm−1 to 1091 and 1111 cm−1 respectively indicates that the phenolic \OH groups participated in the bio-reduction of AgNPs. Other bands which shifted during reduction were 1748 cm−1 to 1719 cm−1 due to changes in C_O band and 1460 cm−1 to 1417 cm− 1 due to changes in C_C band. 3.5. DLS and zeta potential of sunflower oil capped AgNPs Fig. 1. Digital photograph showing formation of AgNPs.
3.3. Transmission electron microscopy study of sunflower oil capped AgNPs The TEM image in Fig. 3A of sunflower oil capped AgNPs before cold storage at 4 °C reveals the presence of spherical or semi-hexagonal particles of size less than or around 10 nm, whereas, Fig. 3B shows the TEM image of sunflower oil capped AgNPs after cold storage wherein hexagonal nanoparticles along with some spherical particles of smaller diameter are clearly visible. A high magnification TEM image in Fig. 3C suggests that the size of the hexagonal AgNPs is about 40–50 nm, while the spherical particles have a diameter of around 10 nm. It is possible that during cold storage the spherical particles gradually grow into larger hexagonal particles. Inset of Fig. 3B gives an electron diffraction (ED) pattern of the single hexagonal silver nanoparticle. Analyzing these diffraction patterns suggested that AgNPs have an fcc crystal structure. This indicates that the hexagonal silver NP is a single crystal grown along the [110] direction and enclosed by the [200] and [220] planes at the top and side surfaces, respectively.
3.4. FT-IR spectroscopy study of sunflower oil capped AgNPs The presence of triglyceride and various phenolic compounds in the oil is verified by using FT-IR spectroscopy. As clearly visible in the IR spectra of sunflower oil (Fig. 4A), the spectra exhibited a band at 1020 and 1180 cm−1 which is a depiction of 1° and 2° alcoholic groups and phenolic derivatives. The bands visible at 622 to 789 cm−1 signify the presence of R\CH group and aromatic C\H bending. The bands at 1381 cm−1 due to ether linkage and 1460 cm− 1 are due to aromatic C_C stretching. The band at 1630 cm−1 is for C_C bending of alkene.
The DLS (supporting information) of sunflower oil capped AgNPs before cold storage at 4 °C showed a single narrow distribution with average particle size in the range of 1–21 nm, whereas, after cold storage, the average particle size of NPs was 22–72 nm (supporting information). The measurement of zeta potential reveals that the oil as well as oil capped NPs showed negative zeta potential values (supporting information). 3.6. Probable mechanism of formation of sunflower oil capped AgNPs It has been reported that polyphenols and phenolic derivatives exhibit chelating properties towards transition and redox metals [25] and in some cases this aspect has been shown to play an important role in the formation of metal nanoparticles [26]. Sunflower seed oil is a good source of phenolics mainly α-tocopherol (Vitamin-E). It appears that during MW heating the tocopherols may develop an affinity towards silver ions and consequently may form silver nanoparticles. αTocopherol being an important constituent having reducing properties may be responsible for synthesis of AgNPs. It is reported in literature that during heating of oil α-tocopherol is degraded sharply to αtocopherylquinone [27]. It is possible that during microwave heating the conversion from α-tocopherol to α-tocopherylquinone might have occurred. This generally happens in two successive univalent steps; the initial step is reversible and consists of the production of a phenoxyl radical which forms an unstable oxidation–reduction system with the phenol. In a second step the phenoxyl radical gives αtocopherylquinone irreversibly and an electron which facilitate the reduction of the Ag ion as shown in scheme-S1 (Supplementary data) [28]. Initially NPs having a spherical shape or semi-hexagonal shape were formed, but over a longer period of time (After 48 h.), most of the NPs adapted hexagonal shape. Although the exact mechanism is not known we hypothesize that free fatty acids and phenolic compounds attach to the silver nuclei, leading to lower surface energy of the (110) face during cold storage. Thus the components of sunflower oil don't only have the capability to reduce silver ions, but also appear to recognize and affect the Ag nanocrystal growth on the (110) face, leading to the formation of hexagon-shaped Ag nanocrystals. 3.7. Morphological evaluation of AgNPs treated with A549 cells
Fig. 2. UV–vis absorption spectra of sunflower oil capped AgNPs.
Morphological evaluation of cultured cells following toxic insult is a very useful parameter to study cell death [29]. As shown in Fig. 5, there was a dose dependent alteration in cell shape and morphology of A549 cells treated with AgNPs. Majority of the cells appeared to be rounded and shrunken with higher doses of AgNPs compared to the control cells which depicted a characteristic spindle to oblong cellular morphology (Fig. 5).
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Fig. 3. TEM images of sunflower oil capped AgNPs (A) before cold storage, (B) after cold storage (inset showing the electron diffraction pattern) and (C) high resolution image after cold storage.
3.8. Cytotoxicity of AgNPs treated with A549 cells Determination of cell viability using MTT dye is very common and efficient protocol for evaluating the cytotoxic potential of various nanoparticles [29]. MTT is a tetrazolium dye that undergoes reduction by the
mitochondrial enzymes to form a blue colored formazan and the intensity of this color is directly proportional to cell viability [30]. Cancer cells are highly metabolic and porous in nature and are known to internalize solutes rapidly compared to normal cells. Therefore, we hypothesized that sunflower oil coated AgNPs will also show internalization within
Fig. 4. FT-IR spectra of sunflower oil and sunflower oil capped AgNPs.
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Fig. 5. Phase contrast photomicrographs of A549 cells exposed to untreated (sunflower oil extract) and 10 μg/mL, 20 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL of sunflower oil capped AgNPs.
cancer cells. Interaction of cationic particles with negatively charged cancer cells has been well reported in the literature [22]. However, Dasgupta et al. have observed that neither size nor zeta potential alone determines the optimal cell response induced by NPs [23]. The components of cellular growth media used in vitro studies could interact with NPs and change their physiochemical properties. Thus size, aggregation state, surface charge and surface chemistry would be significantly modified via electrostatic screening which in turn could influence their ability to interact with or enter cells [24]. In the present study, exposure of AgNPs to A549 cells resulted in a dose dependent (10–100 μg/mL) cytotoxicity with the highest dose (100 μg/mL) recording N70% cell death (Fig. 6A). Leakage of an intracellular enzyme LDH following toxic insult is an indicative of loss of cellular integrity. Induction of cell death in A549 cells exposed to sunflower oil coated AgNPs was in compliment with a degree of LDH release from cells (Fig. 6B). For comparison, the cytotoxicity was also evaluated in 3T3L1 fibroblast cells. It was found that the extent of toxicity was less compared to that recorded in A549 cells (Supplementary data).
Nanoparticle mediated oxidative stress in cultured cells has been demonstrated by various research groups and the same has been attributed to the generation of reactive oxygen species [30,31]. In the present study, status of cellular oxidative stress was evaluated by using DCF-DA (2′, 7′-dichlorodihydrofluorescein diacetate) fluorescence dye. The results clearly indicated dose dependent heightened ROS generation in AgNPs exposed A549 cells (Fig. 7). Further, these ROS are known to attack polyunsaturated fatty acids (PUFAs) of plasma membrane and form peroxyl radicals, that eventually results in a chain reaction called as lipid peroxidation [32]. Eukaryotic cells antioxidant machinery to combat various oxidative stress conditions viz. enzymatic and non-enzymatic antioxidants. Often there exists an inverse relation between oxidative stress and content of cellular antioxidants. We recorded elevated levels of lipid peroxidation coupled with the lowered content of nonenzymatic antioxidant, reduced glutathione (Fig. 8A&B) and the same corroborates with the result of DCF-DA staining. Recently, different research groups have reported that nanoparticles of various dimensions and chemical compositions get localized in
Fig. 6. Effect of sunflower oil capped AgNP exposure to A549 cells on (A) cell viability and (B) LDH release. Results are expressed as mean ± S.E.M. for n = 3 (replicates).
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Fig. 7. Florescence photomicrographs of DCF-DA stained A549 cells exposed to vehicle (sunflower oil extract), and 10 μg/mL, 20 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL of sunflower oil capped AgNPs.
mitochondria and induce cellular apoptosis through disruption of the electron transport chain and ATP production [33]. We evaluated mitochondrial membrane potential using rhodamine 123 dye in order to
decipher mode of cell death following exposure to sunflower oil capped AgNPs. Surprisingly, there were non-significant alterations in the level of mitochondrial membrane potential (Fig. 8C). Thus, it can be
Fig. 8. Effect of sunflower oil capped AgNP exposure to A549 cells on (A) lipid peroxidation (LPO) and (B) reduced glutathione (GSH). Results are expressed as mean ± S.E.M. for n = 3 (replicates) and (C) effect of sunflower oil capped AgNP exposure to A549 cells on mitochondrial membrane potential and ROS generation. Results are expressed as mean ± S.E.M. for n = 3 (replicates).
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hypothesized that sunflower oil capped AgNPs cause cellular death of A549 cells without altering mitochondrial integrity and probably via necrotic pathway. 4. Conclusions This study is the first report on the synthesis of sunflower oil capped silver nanoparticles with hexagonal shape. Presently recorded necrotic cell death of A549 cells exposed to AgNPs could be attributed to its novel shape and further studies are required to interpret exact mode of action therein. In conclusion, our study displays a time- and dosedependent impairment of cell viability after incubation with AgNP. The extent of cytotoxicity was found to be greater in A549 cells compared to 3T3L1 cells. This might offer the possibility of future sunflower oil capped AgNP applications in clinical practice. However, the mechanism of cell stress induction and cytotoxicity needs to be further analyzed in order to be able to predict possible health risks. Acknowledgement The authors are grateful to Dr. P.S. Nagar of Department of Botany for the useful suggestions and SAIF Shillong for the transmission electron microscopy studies. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.08.019. References [1] K.B. Narayanan, N. Sakthivel, Adv. Colloid Interface Sci. 156 (2010) 1. [2] P. PeiGan, S. HanNg, Y. Huang, S.F. YauLi, Bioresour. Technol. 113 (2012) 132. [3] S. Iravani, Green Chem. 13 (2011) 2638.
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