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Honey-based synthesis of ZnO nanopowders and their cytotoxicity effects
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Original Research Paper
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Honey-based synthesis of ZnO nanopowders and their cytotoxicity effects
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Seyed Javad Hoseini a, Majid Darroudi b,c,⇑, Reza Kazemi Oskuee a,d, Leila Gholami e, Ali Khorsand Zak f
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a
Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Nuclear Medicine Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran c Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran d Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran e Neurogenic Inflammation Research Centre, School of Medicine, Mashhad University of Medical Sciences, Mashhad 9177948564, Iran f Nanotechnology Laboratory, Esfarayen University of Technology, Esfarayen 96619-98195, North Khorasan, Iran b
a r t i c l e
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Article history: Received 3 December 2013 Received in revised form 21 February 2015 Accepted 9 April 2015 Available online xxxx Keywords: ZnO Honey Greener Cytotoxicity Electron microscopy
a b s t r a c t The use of food and bio-derived products for the synthesis of different nanomaterials is of enormous interest to modern nanobiotechnology. We have developed a simple, novel, ‘‘greener’’, bio-directed, and low cost method for the synthesis of zinc oxide nanopowders (ZnO–NPs) by using honey as a food and bio-derived product. The prepared ZnO–NPs were characterized by UV–visible spectroscopy (UV– vis), field emission scanning electron microscopy (FESEM), thermogravimetric analysis and differential thermal analysis (TGA/DTA), and powder X-ray diffraction (PXRD). Spherical ZnO–NPs were synthesized at different calcination temperatures and FESEM images and its corresponding particle size distributions showed the formation of nanopowders in size of about 30 nm. The PXRD analysis revealed wurtzite hexagonal ZnO with preferential orientation at (1 0 1) reflection plane. In vitro cytotoxicity studies on neuro2A cells showed a dose dependent toxicity with non-toxic effect of a concentration up to 7.8 lg/mL. Ó 2015 Published by Elsevier B.V. on behalf of The Society of Powder Technology Japan. All rights reserved.
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1. Introduction ZnO nanopowders (ZnO–NPs) are an important inorganic semiconductor material with a hexagonal wurtzite crystal structure which has a wide and direct band gap (nearly 3.37 eV) and a large excitonic binding energy (60 meV) at ambient temperature [1,2]. The ZnO–NPs has numerous applications such as catalysis [3], piezoelectric devices [4], pigment [5], chemical sensors [6], and cosmetic materials especially for transparent UV protection [7]. There are wide reports available on the preparation of ZnO–NPs using different methodological approaches like solvothermal and hydrothermal synthesis [8–10], precipitation [11,12], polymerization method [13], laser ablation [14], sonochemical [15,16], flame spray synthesis [17], and sol–gel [18,19] methods. However, the utility of such methods suffers several drawbacks like the use of high temperature and pressure, toxic reagents, long reaction time, requirement of external additives as a specific stabilizer, base and ⇑ Corresponding author at: Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Tel.: +98 513 8002286; fax: +98 513 8002287. E-mail addresses: [email protected], [email protected] (M. Darroudi).
promoter during the reaction which limits the purity of the final product. It is important to prepare ZnO–NPs by a simple, low cost, and eco-friendly process that has potential to yield nanoparticles of uniform particles in size. The sol–gel method has gained lots of interest among researchers since it offers controlled consolidation, shape modulation, patterning of the nanostructures and a low processing temperature [20,21]. Recently, biomaterials have been used in the synthesis of ZnO–NPs such as plant extracts and derivatives [22–26]. Natural polymers such as gelatin, starch, chitosan, and different proteins are all interesting materials in synthesis of nanomaterials because they are biodegradable and bioabsorbable with degradation products that are non-toxic [27–29]. Newly, honey has been used in the field of nanotechnology to apply green chemistry rules and environmentally benign synthesis of nanopowders [30–32]. Honey is a sweet viscous which is fluid produced from honeybees, and is mainly composed of carbohydrates, enzymes, vitamins, minerals and antioxidants [33]. Honey mediated biological synthesis has lots of advantages over other types of biological methods, including avoidance of elaborate processes such as drying plant materials and the maintenance of cell cultures [32]. Many cytotoxicological assays give some insights about cytotoxicity induced by several nanomaterials [34]. However it is
http://dx.doi.org/10.1016/j.apt.2015.04.003 0921-8831/Ó 2015 Published by Elsevier B.V. on behalf of The Society of Powder Technology Japan. All rights reserved.
Please cite this article in press as: S.J. Hoseini et al., Honey-based synthesis of ZnO nanopowders and their cytotoxicity effects, Advanced Powder Technology (2015), http://dx.doi.org/10.1016/j.apt.2015.04.003
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necessary to determine the cytotoxicity of each nanomaterial individually because many parameters have been identified as being responsible for nanomaterials toxicity [35]. Moreover, the obtained data are not results of the same experimental, so it is difficult to compare with other cytotoxicity results [36]. Because of some medicinal applications of CeO2–NPs [37], in the present study we decided to evaluate the cytotoxicity of the prepared NPs to have better insight into the biological effects of them. ZnO–NPs are present in different products such as sunscreens. For this reason, in vitro studies have investigated exposure effects of on various cell lines (e.g., skin cells). Cytotoxicity studies of different ZnO–NPs in sizes and structures with respect to a variety of cells have been performed [38], but the safety of this material for humans and environment is still unclear. In many studies, in vitro studies using ZnO–NPs often show a small concentration range in which cell viability is reduced from almost 100% to almost zero. It is assumed that this is caused by free intracellular Zn2+ from dissolution of ZnO–NPs [39]. At acidic environment such as in lung lining fluid (surfactant) with increase of ZnO dissolution, leading to momentary increases in the concentration of Zn2+ and local toxicity [40]. Released Zn2+ due to the solubility of ZnO–NPs could be responsible for inducing inflammatory responses and necrosis [41]. For example, inhalation of 20 nm ZnO–NPs (2.5 mg/kg bw [body weight]) by rats twice daily (for 3 days) resulted in an increased Zn content in the liver and kidneys and histopathology results revealed damage in liver and lung tissues [42]. In this study, we attempted the fabrication of ZnO–NPs by using the sol–gel method and honey as a greener capping and/or stabilizing agent. The extensive number of carbohydrates, enzymes, and vitamins containing hydroxyl and amine groups in the honey matrix structure can facilitate the complexation of zinc cations (Zn2+) to an initial molecular matrix. This structure enables honey to coat and stabilize zinc species and finally ZnO–NPs while inhibiting their excessive aggregation or crystal growth. This method can be used as a bio-directed, facile, greener and economically feasible route to fabricate different nanopowders. In addition, we examined the cytotoxic effect of ZnO–NPs on neuro2A cells. In vitro cytotoxicity studies on neuro2A cells displayed a non-toxic effect of a concentration up to 7.8 lg/mL. Furthermore, it was found that the ZnO–NPs exerted a cytotoxic effect on the neuro2A cell line in upper concentrations.
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2. Materials and methods
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2.1. Materials and reagents
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All the materials used were of analytical grade and were used without any purification. Zinc nitrate was purchased from Merck (Germany) and honey was purchased from the local market (Ghayour–Mobarhan Honey Co., Mashhad – Iran). All glassware used in the laboratory experiments were cleaned with a fresh solution of HNO3/HCl (3:1, v/v), washed thoroughly with doubly distilled water, and dried before use. Double distilled water was used in all experiments. For the evaluation of metabolic activity, Neuro2A murine neuroblastoma cells (ATCC CCL-131, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (1 g/L glucose, 2 mM glutamine), supplemented with 10% FBS, streptomycin at 100 m g/mL, and penicillin at 100 U/mL. All cells were incubated at 37 °C in a humidified 5% CO2 atmosphere.
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2.2. Synthesis of ZnO–NPs
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To prepare 5.0 g of ZnO–NPs, 18.25 g of Zn(NO3)2.6H2O was dissolved in 30 mL of distilled water and then stirred for 30 min.
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Meanwhile, 25 g of honey was dissolved in 50 mL of distilled water and stirred for 15 min at room temperature to achieve a clear honey solution. Afterward, the zinc nitrate solution was added to the honey solution, and had the container was place in an oil bath with a temperature at 60 °C. At beginning, we have a yellow color solution which is turn to light brown gel after the heating process at 60 °C for 6 h. The light brown color obtained gel was divided into 4 parts to be individually heated at a rate of 4 °C/min up to the respective temperatures of 200 (Z1), 400 (Z2), 600 (Z3), and 800 °C (Z4), then the product was maintained for 2 h at the specified temperature in air to obtain ZnO–NPs.
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2.3. Characterization of ZnO–NPs
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The prepared ZnO–NPs were characterized by powder X-ray diffraction (PXRD), thermogravimetric analysis and differential thermal analysis (TGA/DTA, TA Instruments, USA), UV–visible spectroscopy (UV–vis), and field emission scanning electron microscopy (FESEM). The phase evaluation and crystalline structure of the ZnO–NPs were investigated by PXRD (Philips, X’pert, Cu Ka). The FESEM observations and UV–vis studies were carried out on a Carl Zeiss Supra 55VP electron microscope and Thermo Fisher Scientific (Evolution 300Ò) spectrophotometer, respectively. The particle size of nanopowders was determined using the UTHSCSA Image Tool Version 3.00 program and their particle size distribution was calculated by SPSS software Version 18.
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2.4. Evaluation of cytotoxicity effect
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The cytotoxicity of ZnO–NPs was evaluated by the method using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [43]. Briefly, neuro2A cells were seeded at a density of 1 104 cells per well in 96-well plates and incubated for 24 h. Thereafter, the cells were treated with various concentrations of nanopowders in the presence of 10% FBS. The calcined ZnO–NPs (Z3) was suspended in a stock solution at 5 lg/mL in a solution of dimethyl sulfoxide (DMSO)/double distilled water. After 24 h of incubation, 20 ll of 5 mg/mL MTT in the PBS buffer was added to each well, and the cells were further incubated for 4 h at 37 °C. The medium containing unreacted dye was discarded, and 100 ll of DMSO was added to dissolve the formazan crystal formed by live cells. Optical absorbance was measured at 590 nm (reference wavelength 630 nm) using a microplate reader (Statfax-2100, Awareness Technology, USA), and cell viability was expressed as a percent relative to untreated control cells. Values of metabolic activity are presented as mean ± SD of triplicate.
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Fig. 1. Lab photo of honey-based synthesized ZnO–NPs at different calcination temperatures.
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Fig. 2. The TGA/DTA curves of initial gel (Zinc nitrate + Honey) from 20 to 1000 °C.
Fig. 3. UV–vis spectrum and band gap estimation (inset) of ZnO–NPs (Z3). (A: Absorbance and E: Photon energy).
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3. Results and discussion
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This section reports the results of the synthesized ZnO–NPs in aqueous the honey solutions. As shown in Fig. 1, the color of sol– gel derived ZnO–NPs due to the increased calcination temperature changed from black to white. The thermogravimetric analysis and differential thermal analysis (TGA/DTA) curves of the as-prepared gel by the sol–gel method in a honey environment are presented in Fig. 2. The heating process was started at about 20 °C, and then increased up to 1000 °C along with a temperature rate change of 10 °C/min. The TGA curve descends until it becomes horizontal around 470 °C, and about 88.5% weight loss was observed during the heating process. The TGA/ DTA traces show three main regions having an initial loss of water in the first weight loss between 20 and 160 °C (39.1%), bends of Ed1, Ed2, and Ed3. Ed1 is related to the evaporation of the initial water. Ed2 and Ed3 can be related to the decomposition of the aromatic bonds of honey. The second weight loss from 160 °C to 385 °C (35.4%) is attributed to the decomposition of chemically
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Fig. 4. The PXRD patterns of honey-based synthesized ZnO–NPs in air at different temperatures.
bound groups, which corresponds to bend Ed4. The third step from 385 to 460 °C (14%) is related to the formation and decomposition of the pyrochlore phases along with the formation of ZnO pure phases indicated by bend Ed5. No weight loss between 460 and 1000 °C was detected on the TGA curve, which indicates the formation of nanocrystalline ZnO as the decomposition product [19,44–46].
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Typical room temperature UV–vis absorption spectrum of the ZnO–NPs is shown in Fig. 3. The ZnO–NPs (Z3) was dispersed in water with concentration of 0.1 wt.% and then the solution was used to perform the UV–vis measurement. The spectrum reveals a characteristic absorption peak of ZnO at a wavelength of 372 nm which can be assigned to the intrinsic band-gap absorption of ZnO due to the electron transitions from the valence band to the conduction band (O2p ? Zn3d) [47]. In addition, this sharp peak shows that the particles are in nano-size and the particle size distribution is narrow. It is clearly shown that the maximum peak in the absorbance spectrum does not correspond to the true optical band gap of ZnO–NPs (Z3). A common way to obtain the band gap from absorbance spectra is to find the first derivative of the absorbance with respect to the photon energy and find the maximum in the derivative spectrum at the lower energy sides [48,49]. The derivative of the absorbance of ZnO–NPs (Z3) is shown in the inset of Fig. 3, which indicates a band gap of 3.2 eV for the ZnO–NPs. The good absorption of the ZnO–NPs in the UV region proves the applicability of this product in medical applications such as sun-screen protectors or antiseptic in ointments [50]. The PXRD patterns of Z1 to Z4 which heated at different temperatures are shown in Fig. 4, respectively. All of the detectable peaks with Miller indices (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4), (2 0 2), (1 0 4), (2 0 3), and (2 1 0) can be indexed to the ZnO wurtzite hexagonal structure (JCPDS # 36-1451) with preferential orientation at (1 0 1) reflection plane [51]. As it is confirmed by the FESEM images at different magnification of ZnO–NPs (Z3) (Fig. 5), the crystalline size of the obtained samples is in nanoscale which was implied by the
broadening of the peaks, and through achieving such fine and small sizes, a satisfying result was gained. The morphology of prepared ZnO–NPs by FESEM analyze is shown in Fig. 5. The nanopowders possess a spherical and uniform shape in size about 30 nm. The as-prepared ZnO–NPs were calcined at different temperatures for 2 h, PXRD peaks became sharper with increasing calcination temperatures and full width at half maximum (FWHM) decreased, indicating that the crystallinity of ZnO–NPs is accelerated by the calcination process. Moreover, no other peaks related to an impurity regarding the prepared ZnO–NPs at different calcination temperatures, indicating that the final nano powders were relatively pure. The results of in vitro cytotoxicity studies after 24 h of incubation with different concentrations of nanopowders, ranging from 0 to 500 lg/mL, are shown in Fig. 6. As the results showed, for concentration above 7.8 lg/mL the metabolic activity was decreased in a concentration dependent manner meaning that metabolic activity started to decrease from 7.8 lg/mL and reached its maximal decreasing in 500 lg/mL. It has been reported that there is a relationships between cytotoxicity and physicochemical properties of ZnO–NPs such as particle size and surface charge [52]. Baek et al. in their study on comparison of two different sizes of ZnO–NPs (20 and 70 nm), had reported higher cytotoxic effects of smaller particles compared to larger particles. Similarly, Hanley et al. observed that smaller ZnO–NPs are more toxic to mammalian cells than larger ZnO–NPs [53] and also the shape of the nanostructures has strong influence on cellular toxicity [54]. Heng et al. have shown that the particle to cell ratio plays a crucial role for particle’s toxicity [55]. In addition, it has been described that the cytotoxic effect
Fig. 5. FESEM images of ZnO–NPs (Z3) (with mean diameter equal to 29.1 ± 9.11 nm) at different magnification (5000 (a), 50,000 (b), and 130,000 (c)) and the particle size distribution of the ZnO–NPs (d).
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Fig. 6. Cell viability of neuro2A cells measured by the MTT assay for ZnO–NPs (Z3).
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of ZnO–NPs also depends upon the proliferation rate of mammalian cells [56,57]. Furthermore, Müller et al. showed that pHtriggered intracellular release of zinc ions (Zn2+) is responsible for the toxicity of ZnO nanowires [58]. In the mentioned studies also found significant reduction in viability detected by the MTT assay as the study presented here at approximately the same concentrations as tested here. The same report [59] showed that the decrease in viability as measured by the MTT assay reaches its maximum at around 45–55% when compared to the control treatment at a range of concentration about 10–20 lg/mL and does not decrease further if the exposure concentration is raised to about 40–60 lg/mL. In the study presented here, a higher response could be reached with higher concentrations. Due to these effects, ZnO– NPs for their implications can be used in cancer therapy [60].
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4. Conclusion
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A food based, bio-directed, facile, eco-friendly, and economically feasible synthetic method has been applied for the synthesis of ZnO–NPs via sol–gel route by using aqueous honey solutions. From FESEM and PXRD results, it was observed that the calcined ZnO–NPs at above 200 °C exhibited the high homogeneity with the wurtzite structure. The typical band gap was estimated from UV–vis spectrum and was obtained to be about 3.2 eV. In vitro cytotoxicity studies on neuro2A cells, a dose dependent toxicity with non-toxic effect of concentration below 7.8 lg/mL was shown.
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References
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[1] Z. Deng, M. Chen, G. Gu, L. Wu, A Facile method to fabricate ZnO hollow spheres and their photocatalytic property, J. Phys. Chem. B 112 (2008) 16–22. [2] Z.L. Wang, ZnO nanowire and nanobelt platform for nanotechnology, Mater. Sci. Eng. R-Rep. 64 (2009) 33–71. [3] W.F. Elseviers, H. Verelst, Transition metal oxides for hot gas desulphurization, Fuel 78 (1999) 601–612. [4] M.-P. Lu, J. Song, M.-Y. Lu, M.-T. Chen, Y. Gao, L.-J. Chen, Z.L. Wang, Piezoelectric nanogenerator using p-type ZnO nanowire arrays, Nano Lett. 9 (2009) 1223–1227. [5] M. Rostami, S. Rasouli, B. Ramezanzadeh, A. Askari, Electrochemical investigation of the properties of Co doped ZnO nanoparticle as a corrosion inhibitive pigment for modifying corrosion resistance of the epoxy coating, Corros. Sci. 88 (2014) 387–399. [6] T. Yumak, F. Kuralay, M. Muti, A. Sinag, A. Erdem, S. Abaci, Preparation and characterization of zinc oxide nanoparticles and their sensor applications for electrochemical monitoring of nucleic acid hybridization, Colloids Surf., B 86 (2011) 397–403. [7] M.J. Osmond, M.J. Mccall, Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard, Nanotoxicology 4 (2010) 15–41. [8] R. Razali, A.K. Zak, W.H.A. Majid, M. Darroudi, Solvothermal synthesis of microsphere ZnO nanostructures in DEA media, Ceram. Int. 37 (2011) 3657– 3663.
5
[9] Q. Li, Z. Kang, B. Mao, E. Wang, C. Wang, C. Tian, S. Li, One-step polyoxometalate-assisted solvothermal synthesis of ZnO microspheres and their photoluminescence properties, Mater. Lett. 62 (2008) 2531–2534. [10] H.Y. Xu, H. Wang, Y.C. Zhang, W.L. He, M.K. Zhu, B. Wang, H. Yan, Hydrothermal synthesis of zinc oxide powders with controllable morphology, Ceram. Int. 30 (2004) 93–97. [11] C.L. Kuo, C.L. Wang, H.H. Ko, W.S. Hwang, K.M. Chang, W.L. Li, H.H. Huang, Y.H. Chang, M.C. Wang, Synthesis of zinc oxide nanocrystalline powders for cosmetic applications, Ceram. Int. 36 (2010) 693–698. [12] A.S. Lanje, S.J. Sharma, R.S. Ningthoujam, J.-S. Ahn, R.B. Pode, Low temperature dielectric studies of zinc oxide (ZnO) nanoparticles prepared by precipitation method, Adv. Powder Technol. 24 (2013) 331–335. [13] P. Jajarmi, Fabrication of pure ZnO nanoparticles by polymerization method, Mater. Lett. 63 (2009) 2646–2648. [14] R. Zamiri, A. Zakaria, H.A. Ahangar, M. Darroudi, A.K. Zak, G.P.C. Drummen, Aqueous starch as a stabilizer in zinc oxide nanoparticle synthesis via laser ablation, J. Alloys Compd. 516 (2012) 41–48. [15] C. Deng, H. Hu, G. Shao, C. Han, Facile template-free sonochemical fabrication of hollow ZnO spherical structures, Mater. Lett. 64 (2010) 852–855. [16] A. Khorsand Zak, W.H.A. Majid, H.Z. Wang, Ramin Yousefi, A. Moradi Golsheikh, Z.F. Ren, Sonochemical synthesis of hierarchical ZnO nanostructures, Ultrason. Sonochem. 20 (2013) 395–400. [17] T. Rudin, K. Wegner, S.E. Pratsinis, Towards carbon-free flame spray synthesis of homogeneous oxide nanoparticles from aqueous solutions, Adv. Powder Technol. 24 (2013) 632–642. [18] A.K. Zak, W.H.A. Majid, M. Darroudi, R. Yousefi, Synthesis and characterization of ZnO nanoparticles prepared in gelatin media, Mater. Lett. 65 (2011) 70–73. [19] A. Khorsand Zak, W.H.Abd. Majid, M.R. Mahmoudian, Majid Darroudi, Ramin Yousefi, Starch-stabilized synthesis of ZnO nanopowders at low temperature and optical properties study, Adv. Powder Technol. 24 (2013) 618–624. [20] B. Sunandan, D. Joydeep, Hydrothermal growth of ZnO nanostructures, Sci. Technol. Adv. Mater. 10 (2009) 013001. [21] E.G. Lori, D.Y. Benjamin, L. Matt, Z. David, Y. Peidong, Solution grown zinc oxide nanowires, Inorg. Chem. 45 (2006) 7535–7543. [22] N.A. Samat, R.M. Nor, Sol–gel synthesis of zinc oxide nanoparticles using Citrus aurantifolia extracts, Ceram. Int. 39 (2013) S545–S548. [23] G. Sangeetha, S. Rajeshwari, R. Venckatesh, Green synthesis of zinc oxide nanoparticles by Aloe barbadensis miller leaf extract: structure and optical properties, Mater. Res. Bull. 46 (2011) 2560–2566. [24] R.P. Singh, V.K. Shukla, R.S. Yadav, P.K. Sharma, P.K. Singh, A.C. Pandey, Biological approach of zinc oxide nanoparticles formation and its characterization, Adv. Mater. Lett. 2 (2011) 313–317. [25] V. Rana, P. Rai, A.K. Tiwary, R.S. Singh, J.F. Kennedy, C.J. Knill, Modified gums: approaches and applications in drug delivery, Carbohydr. Polym. 83 (2011) 1031–1047. [26] M. Darroudi, Z. Sabouri, R. Kazemi Oskuee, A. Khorsand Zak, H. Kargar, M.H.N. Abd Hamid, Sol–gel synthesis, characterization, and neurotoxicity effect of zinc oxide nanoparticles using gum tragacanth, Ceram. Int. 39 (2013) 9195– 9199. [27] M. Darroudi, M.B. Ahmad, R. Zamiri, A.K. Zak, A.H. Abdullah, N.A. Ibrahim, Time-dependent effect in green synthesis of silver nanoparticles, Int. J. Nanomed. 6 (2011) 677–681. [28] M. Darroudi, M. Sarani, R. Kazemi Oskuee, A. Khorsand Zak, H.A. Hosseini, L. Gholami, Green synthesis and evaluation of metabolic activity of starch mediated nanoceria, Ceram. Int. 40 (2014) 2041–2045. [29] R. Salehi, M. Arami, N.M. Mahmoodi, H. Bahrami, S. Khorramfar, Novel biocompatible composite (Chitosan–zinc oxide nanoparticle): preparation, characterization and dye adsorption properties, Colloids Surf., B 80 (2010) 86– 93. [30] D. Philip, Honey mediated green synthesis of gold nanoparticles, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 73 (2009) 650–653. [31] D. Philip, Honey mediated green synthesis of silver nanoparticles, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 75 (2010) 1078–1081. [32] R. Venu, T.S. Ramulu, S. Anandakumar, V.S. Rani, C.G. Kim, Bio-directed synthesis of platinum nanoparticles using aqueous honey solutions and their catalytic applications, Colloids Surf. A: Physicochem. Eng. Asp. 384 (2011) 733–738. [33] D.W. Ball, The chemical composition of honey, J. Chem. Educ. 84 (2007) 1643– 1646. [34] K. Soto, K.M. Garza, L.E. Murr, Cytotoxic effects of aggregated nanomaterials, Acta Biomater. 3 (2007) 351–358. [35] S. Park, Y.K. Lee, M. Jung, K.H. Kim, N. Chung, E.K. Ahn, Y. Lim, K.H. Lee, Cellular toxicity of various inhalable metal nanoparticles on human alveolar epithelial cells, Inhalation Toxicol. 19 (2007) 59–65. [36] K. Soto, A. Carrasco, T.G. Powell, L.E. Murr, K.M. Garza, Biological effects of nanoparticulate materials, Mater. Sci. Eng. C 26 (2006) 1421–1427. [37] G. Renu, V.V. Divya Rani, S.V. Nair, K.R.V. Subramanian, V. Lakshmanan, Development of cerium oxide nanoparticles and its cytotoxicity in prostate cancer cells, Adv. Sci. Lett. 6 (2012) 17–25. [38] L. Nie, L. Gao, P. Feng, J. Zhang, X. Fu, Y. Liu, X.Y. Prof, T.W. Prof, Threedimensional functionalized tetrapod-like ZnO nanostructures for Plasmid DNA delivery, Small 2 (2006) 621–625. [39] R.J. Vandebriel, W.H.D. Jong, A review of mammalian toxicity of ZnO nanoparticles, Nanotechnol., Sci. Appl. 5 (2012) 61–71. [40] M.J. Osmond, M.J. McCall, Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard, Nanotoxicology 4 (2010) 15–41.
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[41] R. Landsiedel, L. Ma-Hock, A. Kroll, D. Hahn, J. Schnekenburger, K. Wiench, W. Wohlleben, Testing metal-oxide nanomaterials for human safety, Adv. Mater. 22 (2010) 2601–2627. [42] L. Wang, W. Ding, F. Zhang, Acute toxicity of ferric oxide and zinc oxide nanoparticles in rats, J. Nanosci. Nanotechnol. 10 (2010) 8617–8624. [43] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [44] C.P. Rezende, J.B. Da Silva, N.D.S. Mohallem, Influence of drying on the characteristics of zinc oxide nanoparticles, Braz. J. Phys. 39 (2009) 248–251. [45] M. Darroudi, Z. Sabouri, R. Kazemi Oskuee, H. Kargar, H.A. Hosseini, Neuronal toxicity of biopolymer-template synthesized ZnO nanoparticles, Nanomed. J. 1 (2014) 88–93. [46] M. Darroudi, Z. Sabouri, R. Kazemi Oskuee, A. Khorsand Zak, H. Kargar, M.H.N.A. Hamid, Green chemistry approach for the synthesis of ZnO nanopowders and their cytotoxic effects, Ceram. Int. 40 (2014) 4827–4831. [47] A. Khorsand Zak, R. Yousefi, W.H.A. Majid, M.R. Muhamad, Facile synthesis and X-ray peak broadening studies of Zn1 xMgxO nanoparticles, Ceram. Int. 38 (2012) 2059–2064. [48] A. Khorsand Zak, R. Razali, W.H.A. Majid, M. Darroudi, Synthesis and characterization of a narrow size distribution of zinc oxide nanoparticles, Int. J. Nanomed. 6 (2011) 1399–1403. [49] M. Darroudi, M. Hakimi, M. Sarani, R. Kazemi Oskuee, A. Khorsand Zak, L. Gholami, Facile synthesis, characterization, and evaluation of neurotoxicity effect of cerium oxide nanoparticles, Ceram. Int. 39 (2013) 6917–6921. [50] F. Harding, Breast Cancer: Cause–Prevention–Cure, Tekline Publishing, Aylesbury, 2006. [51] Joint Committee on Powder Diffraction Standards Diffraction Data File, JCPDS International Center for Diffraction Data No. 36-1451, 1991.
[52] M. Baek, M.K. Kim, H.J. Cho, J.A. Lee, J. Yu, H.E. Chung, S.J. Choi, Factors influencing the cytotoxicity of zinc oxide nanoparticles: particle size and surface charge, J. Phys.: Conf. Ser. 304 (2011) 012044. [53] C. Hanley, A. Thurber, C. Hanna, A. Punnoose, J. Zhang, D.G. Wingett, The influences of cell type and ZnO nanoparticle size on immune cell cytotoxicity and cytokine induction, Nanoscale Res. Lett. 4 (2009) 1409–1420. [54] I.L. Hsiao, Y.J. Huang, Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells, Sci. Total Environ. 409 (2011) 1219–1228. [55] B.C. Heng, X. Zhao, S. Xiong, K.W. Ng, F.Y.C. Boey, J.S.-C. Loo, Cytotoxicity of zinc oxide (ZnO) nanoparticles is influenced by cell density and culture format, Arch. Toxicol. 85 (2011) 695–704. [56] L. Taccola, V. Raffa, C. Riggio, O. Vittorio, M.C. Iorio, R. Pietrabissa, A. Cuschieri, Zinc oxide nanoparticles as selective killers of proliferating cells, Int. J. Nanomed. 6 (2011) 1129–1140. [57] M. Premanathan, K. Karthikeyan, K. Jeyasubramanian, G. Manivannan, Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation, Nanomed.: Nanotechnol. Biol. Med. 7 (2011) 184–192. [58] K.H. Müller, J. Kulkarni, M. Motskin, A. Goode, P. Winship, J.N. Skepper, M.P. Ryan, A.E. Poeter, PH-dependent toxicity of high aspect ratio ZnO nanowires in macrophages due to intracellular dissolution, ACS Nano 4 (2010) 6767–6779. [59] W. Lin, Y.-W. Huang, X.-D. Zhou, Toxicity of cerium oxide nanoparticles in human lung cancer cells, Int. J. Toxicol. 25 (2006) 451–457. [60] M.J. Akhtar, M. Ahamed, S. Kumar, M.M. Khan, J. Ahmad, et al., Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species, Int. J. Nanomed. 7 (2012) (2012) 845–857.
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Contents lists available at ScienceDirect
Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt
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Original Research Paper
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Honey-based synthesis of ZnO nanopowders and their cytotoxicity effects
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Seyed Javad Hoseini a, Majid Darroudi b,c,⇑, Reza Kazemi Oskuee a,d, Leila Gholami e, Ali Khorsand Zak f
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a
Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Nuclear Medicine Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran c Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran d Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran e Neurogenic Inflammation Research Centre, School of Medicine, Mashhad University of Medical Sciences, Mashhad 9177948564, Iran f Nanotechnology Laboratory, Esfarayen University of Technology, Esfarayen 96619-98195, North Khorasan, Iran b
a r t i c l e
i n f o
Article history: Received 3 December 2013 Received in revised form 21 February 2015 Accepted 9 April 2015 Available online xxxx Keywords: ZnO Honey Greener Cytotoxicity Electron microscopy
a b s t r a c t The use of food and bio-derived products for the synthesis of different nanomaterials is of enormous interest to modern nanobiotechnology. We have developed a simple, novel, ‘‘greener’’, bio-directed, and low cost method for the synthesis of zinc oxide nanopowders (ZnO–NPs) by using honey as a food and bio-derived product. The prepared ZnO–NPs were characterized by UV–visible spectroscopy (UV– vis), field emission scanning electron microscopy (FESEM), thermogravimetric analysis and differential thermal analysis (TGA/DTA), and powder X-ray diffraction (PXRD). Spherical ZnO–NPs were synthesized at different calcination temperatures and FESEM images and its corresponding particle size distributions showed the formation of nanopowders in size of about 30 nm. The PXRD analysis revealed wurtzite hexagonal ZnO with preferential orientation at (1 0 1) reflection plane. In vitro cytotoxicity studies on neuro2A cells showed a dose dependent toxicity with non-toxic effect of a concentration up to 7.8 lg/mL. Ó 2015 Published by Elsevier B.V. on behalf of The Society of Powder Technology Japan. All rights reserved.
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1. Introduction ZnO nanopowders (ZnO–NPs) are an important inorganic semiconductor material with a hexagonal wurtzite crystal structure which has a wide and direct band gap (nearly 3.37 eV) and a large excitonic binding energy (60 meV) at ambient temperature [1,2]. The ZnO–NPs has numerous applications such as catalysis [3], piezoelectric devices [4], pigment [5], chemical sensors [6], and cosmetic materials especially for transparent UV protection [7]. There are wide reports available on the preparation of ZnO–NPs using different methodological approaches like solvothermal and hydrothermal synthesis [8–10], precipitation [11,12], polymerization method [13], laser ablation [14], sonochemical [15,16], flame spray synthesis [17], and sol–gel [18,19] methods. However, the utility of such methods suffers several drawbacks like the use of high temperature and pressure, toxic reagents, long reaction time, requirement of external additives as a specific stabilizer, base and ⇑ Corresponding author at: Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Tel.: +98 513 8002286; fax: +98 513 8002287. E-mail addresses: [email protected], [email protected] (M. Darroudi).
promoter during the reaction which limits the purity of the final product. It is important to prepare ZnO–NPs by a simple, low cost, and eco-friendly process that has potential to yield nanoparticles of uniform particles in size. The sol–gel method has gained lots of interest among researchers since it offers controlled consolidation, shape modulation, patterning of the nanostructures and a low processing temperature [20,21]. Recently, biomaterials have been used in the synthesis of ZnO–NPs such as plant extracts and derivatives [22–26]. Natural polymers such as gelatin, starch, chitosan, and different proteins are all interesting materials in synthesis of nanomaterials because they are biodegradable and bioabsorbable with degradation products that are non-toxic [27–29]. Newly, honey has been used in the field of nanotechnology to apply green chemistry rules and environmentally benign synthesis of nanopowders [30–32]. Honey is a sweet viscous which is fluid produced from honeybees, and is mainly composed of carbohydrates, enzymes, vitamins, minerals and antioxidants [33]. Honey mediated biological synthesis has lots of advantages over other types of biological methods, including avoidance of elaborate processes such as drying plant materials and the maintenance of cell cultures [32]. Many cytotoxicological assays give some insights about cytotoxicity induced by several nanomaterials [34]. However it is
http://dx.doi.org/10.1016/j.apt.2015.04.003 0921-8831/Ó 2015 Published by Elsevier B.V. on behalf of The Society of Powder Technology Japan. All rights reserved.
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necessary to determine the cytotoxicity of each nanomaterial individually because many parameters have been identified as being responsible for nanomaterials toxicity [35]. Moreover, the obtained data are not results of the same experimental, so it is difficult to compare with other cytotoxicity results [36]. Because of some medicinal applications of CeO2–NPs [37], in the present study we decided to evaluate the cytotoxicity of the prepared NPs to have better insight into the biological effects of them. ZnO–NPs are present in different products such as sunscreens. For this reason, in vitro studies have investigated exposure effects of on various cell lines (e.g., skin cells). Cytotoxicity studies of different ZnO–NPs in sizes and structures with respect to a variety of cells have been performed [38], but the safety of this material for humans and environment is still unclear. In many studies, in vitro studies using ZnO–NPs often show a small concentration range in which cell viability is reduced from almost 100% to almost zero. It is assumed that this is caused by free intracellular Zn2+ from dissolution of ZnO–NPs [39]. At acidic environment such as in lung lining fluid (surfactant) with increase of ZnO dissolution, leading to momentary increases in the concentration of Zn2+ and local toxicity [40]. Released Zn2+ due to the solubility of ZnO–NPs could be responsible for inducing inflammatory responses and necrosis [41]. For example, inhalation of 20 nm ZnO–NPs (2.5 mg/kg bw [body weight]) by rats twice daily (for 3 days) resulted in an increased Zn content in the liver and kidneys and histopathology results revealed damage in liver and lung tissues [42]. In this study, we attempted the fabrication of ZnO–NPs by using the sol–gel method and honey as a greener capping and/or stabilizing agent. The extensive number of carbohydrates, enzymes, and vitamins containing hydroxyl and amine groups in the honey matrix structure can facilitate the complexation of zinc cations (Zn2+) to an initial molecular matrix. This structure enables honey to coat and stabilize zinc species and finally ZnO–NPs while inhibiting their excessive aggregation or crystal growth. This method can be used as a bio-directed, facile, greener and economically feasible route to fabricate different nanopowders. In addition, we examined the cytotoxic effect of ZnO–NPs on neuro2A cells. In vitro cytotoxicity studies on neuro2A cells displayed a non-toxic effect of a concentration up to 7.8 lg/mL. Furthermore, it was found that the ZnO–NPs exerted a cytotoxic effect on the neuro2A cell line in upper concentrations.
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2. Materials and methods
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2.1. Materials and reagents
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All the materials used were of analytical grade and were used without any purification. Zinc nitrate was purchased from Merck (Germany) and honey was purchased from the local market (Ghayour–Mobarhan Honey Co., Mashhad – Iran). All glassware used in the laboratory experiments were cleaned with a fresh solution of HNO3/HCl (3:1, v/v), washed thoroughly with doubly distilled water, and dried before use. Double distilled water was used in all experiments. For the evaluation of metabolic activity, Neuro2A murine neuroblastoma cells (ATCC CCL-131, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (1 g/L glucose, 2 mM glutamine), supplemented with 10% FBS, streptomycin at 100 m g/mL, and penicillin at 100 U/mL. All cells were incubated at 37 °C in a humidified 5% CO2 atmosphere.
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2.2. Synthesis of ZnO–NPs
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To prepare 5.0 g of ZnO–NPs, 18.25 g of Zn(NO3)2.6H2O was dissolved in 30 mL of distilled water and then stirred for 30 min.
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Meanwhile, 25 g of honey was dissolved in 50 mL of distilled water and stirred for 15 min at room temperature to achieve a clear honey solution. Afterward, the zinc nitrate solution was added to the honey solution, and had the container was place in an oil bath with a temperature at 60 °C. At beginning, we have a yellow color solution which is turn to light brown gel after the heating process at 60 °C for 6 h. The light brown color obtained gel was divided into 4 parts to be individually heated at a rate of 4 °C/min up to the respective temperatures of 200 (Z1), 400 (Z2), 600 (Z3), and 800 °C (Z4), then the product was maintained for 2 h at the specified temperature in air to obtain ZnO–NPs.
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2.3. Characterization of ZnO–NPs
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The prepared ZnO–NPs were characterized by powder X-ray diffraction (PXRD), thermogravimetric analysis and differential thermal analysis (TGA/DTA, TA Instruments, USA), UV–visible spectroscopy (UV–vis), and field emission scanning electron microscopy (FESEM). The phase evaluation and crystalline structure of the ZnO–NPs were investigated by PXRD (Philips, X’pert, Cu Ka). The FESEM observations and UV–vis studies were carried out on a Carl Zeiss Supra 55VP electron microscope and Thermo Fisher Scientific (Evolution 300Ò) spectrophotometer, respectively. The particle size of nanopowders was determined using the UTHSCSA Image Tool Version 3.00 program and their particle size distribution was calculated by SPSS software Version 18.
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2.4. Evaluation of cytotoxicity effect
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The cytotoxicity of ZnO–NPs was evaluated by the method using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [43]. Briefly, neuro2A cells were seeded at a density of 1 104 cells per well in 96-well plates and incubated for 24 h. Thereafter, the cells were treated with various concentrations of nanopowders in the presence of 10% FBS. The calcined ZnO–NPs (Z3) was suspended in a stock solution at 5 lg/mL in a solution of dimethyl sulfoxide (DMSO)/double distilled water. After 24 h of incubation, 20 ll of 5 mg/mL MTT in the PBS buffer was added to each well, and the cells were further incubated for 4 h at 37 °C. The medium containing unreacted dye was discarded, and 100 ll of DMSO was added to dissolve the formazan crystal formed by live cells. Optical absorbance was measured at 590 nm (reference wavelength 630 nm) using a microplate reader (Statfax-2100, Awareness Technology, USA), and cell viability was expressed as a percent relative to untreated control cells. Values of metabolic activity are presented as mean ± SD of triplicate.
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Fig. 1. Lab photo of honey-based synthesized ZnO–NPs at different calcination temperatures.
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Fig. 2. The TGA/DTA curves of initial gel (Zinc nitrate + Honey) from 20 to 1000 °C.
Fig. 3. UV–vis spectrum and band gap estimation (inset) of ZnO–NPs (Z3). (A: Absorbance and E: Photon energy).
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3. Results and discussion
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This section reports the results of the synthesized ZnO–NPs in aqueous the honey solutions. As shown in Fig. 1, the color of sol– gel derived ZnO–NPs due to the increased calcination temperature changed from black to white. The thermogravimetric analysis and differential thermal analysis (TGA/DTA) curves of the as-prepared gel by the sol–gel method in a honey environment are presented in Fig. 2. The heating process was started at about 20 °C, and then increased up to 1000 °C along with a temperature rate change of 10 °C/min. The TGA curve descends until it becomes horizontal around 470 °C, and about 88.5% weight loss was observed during the heating process. The TGA/ DTA traces show three main regions having an initial loss of water in the first weight loss between 20 and 160 °C (39.1%), bends of Ed1, Ed2, and Ed3. Ed1 is related to the evaporation of the initial water. Ed2 and Ed3 can be related to the decomposition of the aromatic bonds of honey. The second weight loss from 160 °C to 385 °C (35.4%) is attributed to the decomposition of chemically
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Fig. 4. The PXRD patterns of honey-based synthesized ZnO–NPs in air at different temperatures.
bound groups, which corresponds to bend Ed4. The third step from 385 to 460 °C (14%) is related to the formation and decomposition of the pyrochlore phases along with the formation of ZnO pure phases indicated by bend Ed5. No weight loss between 460 and 1000 °C was detected on the TGA curve, which indicates the formation of nanocrystalline ZnO as the decomposition product [19,44–46].
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Typical room temperature UV–vis absorption spectrum of the ZnO–NPs is shown in Fig. 3. The ZnO–NPs (Z3) was dispersed in water with concentration of 0.1 wt.% and then the solution was used to perform the UV–vis measurement. The spectrum reveals a characteristic absorption peak of ZnO at a wavelength of 372 nm which can be assigned to the intrinsic band-gap absorption of ZnO due to the electron transitions from the valence band to the conduction band (O2p ? Zn3d) [47]. In addition, this sharp peak shows that the particles are in nano-size and the particle size distribution is narrow. It is clearly shown that the maximum peak in the absorbance spectrum does not correspond to the true optical band gap of ZnO–NPs (Z3). A common way to obtain the band gap from absorbance spectra is to find the first derivative of the absorbance with respect to the photon energy and find the maximum in the derivative spectrum at the lower energy sides [48,49]. The derivative of the absorbance of ZnO–NPs (Z3) is shown in the inset of Fig. 3, which indicates a band gap of 3.2 eV for the ZnO–NPs. The good absorption of the ZnO–NPs in the UV region proves the applicability of this product in medical applications such as sun-screen protectors or antiseptic in ointments [50]. The PXRD patterns of Z1 to Z4 which heated at different temperatures are shown in Fig. 4, respectively. All of the detectable peaks with Miller indices (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4), (2 0 2), (1 0 4), (2 0 3), and (2 1 0) can be indexed to the ZnO wurtzite hexagonal structure (JCPDS # 36-1451) with preferential orientation at (1 0 1) reflection plane [51]. As it is confirmed by the FESEM images at different magnification of ZnO–NPs (Z3) (Fig. 5), the crystalline size of the obtained samples is in nanoscale which was implied by the
broadening of the peaks, and through achieving such fine and small sizes, a satisfying result was gained. The morphology of prepared ZnO–NPs by FESEM analyze is shown in Fig. 5. The nanopowders possess a spherical and uniform shape in size about 30 nm. The as-prepared ZnO–NPs were calcined at different temperatures for 2 h, PXRD peaks became sharper with increasing calcination temperatures and full width at half maximum (FWHM) decreased, indicating that the crystallinity of ZnO–NPs is accelerated by the calcination process. Moreover, no other peaks related to an impurity regarding the prepared ZnO–NPs at different calcination temperatures, indicating that the final nano powders were relatively pure. The results of in vitro cytotoxicity studies after 24 h of incubation with different concentrations of nanopowders, ranging from 0 to 500 lg/mL, are shown in Fig. 6. As the results showed, for concentration above 7.8 lg/mL the metabolic activity was decreased in a concentration dependent manner meaning that metabolic activity started to decrease from 7.8 lg/mL and reached its maximal decreasing in 500 lg/mL. It has been reported that there is a relationships between cytotoxicity and physicochemical properties of ZnO–NPs such as particle size and surface charge [52]. Baek et al. in their study on comparison of two different sizes of ZnO–NPs (20 and 70 nm), had reported higher cytotoxic effects of smaller particles compared to larger particles. Similarly, Hanley et al. observed that smaller ZnO–NPs are more toxic to mammalian cells than larger ZnO–NPs [53] and also the shape of the nanostructures has strong influence on cellular toxicity [54]. Heng et al. have shown that the particle to cell ratio plays a crucial role for particle’s toxicity [55]. In addition, it has been described that the cytotoxic effect
Fig. 5. FESEM images of ZnO–NPs (Z3) (with mean diameter equal to 29.1 ± 9.11 nm) at different magnification (5000 (a), 50,000 (b), and 130,000 (c)) and the particle size distribution of the ZnO–NPs (d).
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Fig. 6. Cell viability of neuro2A cells measured by the MTT assay for ZnO–NPs (Z3).
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of ZnO–NPs also depends upon the proliferation rate of mammalian cells [56,57]. Furthermore, Müller et al. showed that pHtriggered intracellular release of zinc ions (Zn2+) is responsible for the toxicity of ZnO nanowires [58]. In the mentioned studies also found significant reduction in viability detected by the MTT assay as the study presented here at approximately the same concentrations as tested here. The same report [59] showed that the decrease in viability as measured by the MTT assay reaches its maximum at around 45–55% when compared to the control treatment at a range of concentration about 10–20 lg/mL and does not decrease further if the exposure concentration is raised to about 40–60 lg/mL. In the study presented here, a higher response could be reached with higher concentrations. Due to these effects, ZnO– NPs for their implications can be used in cancer therapy [60].
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A food based, bio-directed, facile, eco-friendly, and economically feasible synthetic method has been applied for the synthesis of ZnO–NPs via sol–gel route by using aqueous honey solutions. From FESEM and PXRD results, it was observed that the calcined ZnO–NPs at above 200 °C exhibited the high homogeneity with the wurtzite structure. The typical band gap was estimated from UV–vis spectrum and was obtained to be about 3.2 eV. In vitro cytotoxicity studies on neuro2A cells, a dose dependent toxicity with non-toxic effect of concentration below 7.8 lg/mL was shown.
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References
272 273 274 275 276 277 278 279 280 281 282 283 284
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[1] Z. Deng, M. Chen, G. Gu, L. Wu, A Facile method to fabricate ZnO hollow spheres and their photocatalytic property, J. Phys. Chem. B 112 (2008) 16–22. [2] Z.L. Wang, ZnO nanowire and nanobelt platform for nanotechnology, Mater. Sci. Eng. R-Rep. 64 (2009) 33–71. [3] W.F. Elseviers, H. Verelst, Transition metal oxides for hot gas desulphurization, Fuel 78 (1999) 601–612. [4] M.-P. Lu, J. Song, M.-Y. Lu, M.-T. Chen, Y. Gao, L.-J. Chen, Z.L. Wang, Piezoelectric nanogenerator using p-type ZnO nanowire arrays, Nano Lett. 9 (2009) 1223–1227. [5] M. Rostami, S. Rasouli, B. Ramezanzadeh, A. Askari, Electrochemical investigation of the properties of Co doped ZnO nanoparticle as a corrosion inhibitive pigment for modifying corrosion resistance of the epoxy coating, Corros. Sci. 88 (2014) 387–399. [6] T. Yumak, F. Kuralay, M. Muti, A. Sinag, A. Erdem, S. Abaci, Preparation and characterization of zinc oxide nanoparticles and their sensor applications for electrochemical monitoring of nucleic acid hybridization, Colloids Surf., B 86 (2011) 397–403. [7] M.J. Osmond, M.J. Mccall, Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard, Nanotoxicology 4 (2010) 15–41. [8] R. Razali, A.K. Zak, W.H.A. Majid, M. Darroudi, Solvothermal synthesis of microsphere ZnO nanostructures in DEA media, Ceram. Int. 37 (2011) 3657– 3663.
5
[9] Q. Li, Z. Kang, B. Mao, E. Wang, C. Wang, C. Tian, S. Li, One-step polyoxometalate-assisted solvothermal synthesis of ZnO microspheres and their photoluminescence properties, Mater. Lett. 62 (2008) 2531–2534. [10] H.Y. Xu, H. Wang, Y.C. Zhang, W.L. He, M.K. Zhu, B. Wang, H. Yan, Hydrothermal synthesis of zinc oxide powders with controllable morphology, Ceram. Int. 30 (2004) 93–97. [11] C.L. Kuo, C.L. Wang, H.H. Ko, W.S. Hwang, K.M. Chang, W.L. Li, H.H. Huang, Y.H. Chang, M.C. Wang, Synthesis of zinc oxide nanocrystalline powders for cosmetic applications, Ceram. Int. 36 (2010) 693–698. [12] A.S. Lanje, S.J. Sharma, R.S. Ningthoujam, J.-S. Ahn, R.B. Pode, Low temperature dielectric studies of zinc oxide (ZnO) nanoparticles prepared by precipitation method, Adv. Powder Technol. 24 (2013) 331–335. [13] P. Jajarmi, Fabrication of pure ZnO nanoparticles by polymerization method, Mater. Lett. 63 (2009) 2646–2648. [14] R. Zamiri, A. Zakaria, H.A. Ahangar, M. Darroudi, A.K. Zak, G.P.C. Drummen, Aqueous starch as a stabilizer in zinc oxide nanoparticle synthesis via laser ablation, J. Alloys Compd. 516 (2012) 41–48. [15] C. Deng, H. Hu, G. Shao, C. Han, Facile template-free sonochemical fabrication of hollow ZnO spherical structures, Mater. Lett. 64 (2010) 852–855. [16] A. Khorsand Zak, W.H.A. Majid, H.Z. Wang, Ramin Yousefi, A. Moradi Golsheikh, Z.F. Ren, Sonochemical synthesis of hierarchical ZnO nanostructures, Ultrason. Sonochem. 20 (2013) 395–400. [17] T. Rudin, K. Wegner, S.E. Pratsinis, Towards carbon-free flame spray synthesis of homogeneous oxide nanoparticles from aqueous solutions, Adv. Powder Technol. 24 (2013) 632–642. [18] A.K. Zak, W.H.A. Majid, M. Darroudi, R. Yousefi, Synthesis and characterization of ZnO nanoparticles prepared in gelatin media, Mater. Lett. 65 (2011) 70–73. [19] A. Khorsand Zak, W.H.Abd. Majid, M.R. Mahmoudian, Majid Darroudi, Ramin Yousefi, Starch-stabilized synthesis of ZnO nanopowders at low temperature and optical properties study, Adv. Powder Technol. 24 (2013) 618–624. [20] B. Sunandan, D. Joydeep, Hydrothermal growth of ZnO nanostructures, Sci. Technol. Adv. Mater. 10 (2009) 013001. [21] E.G. Lori, D.Y. Benjamin, L. Matt, Z. David, Y. Peidong, Solution grown zinc oxide nanowires, Inorg. Chem. 45 (2006) 7535–7543. [22] N.A. Samat, R.M. Nor, Sol–gel synthesis of zinc oxide nanoparticles using Citrus aurantifolia extracts, Ceram. Int. 39 (2013) S545–S548. [23] G. Sangeetha, S. Rajeshwari, R. Venckatesh, Green synthesis of zinc oxide nanoparticles by Aloe barbadensis miller leaf extract: structure and optical properties, Mater. Res. Bull. 46 (2011) 2560–2566. [24] R.P. Singh, V.K. Shukla, R.S. Yadav, P.K. Sharma, P.K. Singh, A.C. Pandey, Biological approach of zinc oxide nanoparticles formation and its characterization, Adv. Mater. Lett. 2 (2011) 313–317. [25] V. Rana, P. Rai, A.K. Tiwary, R.S. Singh, J.F. Kennedy, C.J. Knill, Modified gums: approaches and applications in drug delivery, Carbohydr. Polym. 83 (2011) 1031–1047. [26] M. Darroudi, Z. Sabouri, R. Kazemi Oskuee, A. Khorsand Zak, H. Kargar, M.H.N. Abd Hamid, Sol–gel synthesis, characterization, and neurotoxicity effect of zinc oxide nanoparticles using gum tragacanth, Ceram. Int. 39 (2013) 9195– 9199. [27] M. Darroudi, M.B. Ahmad, R. Zamiri, A.K. Zak, A.H. Abdullah, N.A. Ibrahim, Time-dependent effect in green synthesis of silver nanoparticles, Int. J. Nanomed. 6 (2011) 677–681. [28] M. Darroudi, M. Sarani, R. Kazemi Oskuee, A. Khorsand Zak, H.A. Hosseini, L. Gholami, Green synthesis and evaluation of metabolic activity of starch mediated nanoceria, Ceram. Int. 40 (2014) 2041–2045. [29] R. Salehi, M. Arami, N.M. Mahmoodi, H. Bahrami, S. Khorramfar, Novel biocompatible composite (Chitosan–zinc oxide nanoparticle): preparation, characterization and dye adsorption properties, Colloids Surf., B 80 (2010) 86– 93. [30] D. Philip, Honey mediated green synthesis of gold nanoparticles, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 73 (2009) 650–653. [31] D. Philip, Honey mediated green synthesis of silver nanoparticles, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 75 (2010) 1078–1081. [32] R. Venu, T.S. Ramulu, S. Anandakumar, V.S. Rani, C.G. Kim, Bio-directed synthesis of platinum nanoparticles using aqueous honey solutions and their catalytic applications, Colloids Surf. A: Physicochem. Eng. Asp. 384 (2011) 733–738. [33] D.W. Ball, The chemical composition of honey, J. Chem. Educ. 84 (2007) 1643– 1646. [34] K. Soto, K.M. Garza, L.E. Murr, Cytotoxic effects of aggregated nanomaterials, Acta Biomater. 3 (2007) 351–358. [35] S. Park, Y.K. Lee, M. Jung, K.H. Kim, N. Chung, E.K. Ahn, Y. Lim, K.H. Lee, Cellular toxicity of various inhalable metal nanoparticles on human alveolar epithelial cells, Inhalation Toxicol. 19 (2007) 59–65. [36] K. Soto, A. Carrasco, T.G. Powell, L.E. Murr, K.M. Garza, Biological effects of nanoparticulate materials, Mater. Sci. Eng. C 26 (2006) 1421–1427. [37] G. Renu, V.V. Divya Rani, S.V. Nair, K.R.V. Subramanian, V. Lakshmanan, Development of cerium oxide nanoparticles and its cytotoxicity in prostate cancer cells, Adv. Sci. Lett. 6 (2012) 17–25. [38] L. Nie, L. Gao, P. Feng, J. Zhang, X. Fu, Y. Liu, X.Y. Prof, T.W. Prof, Threedimensional functionalized tetrapod-like ZnO nanostructures for Plasmid DNA delivery, Small 2 (2006) 621–625. [39] R.J. Vandebriel, W.H.D. Jong, A review of mammalian toxicity of ZnO nanoparticles, Nanotechnol., Sci. Appl. 5 (2012) 61–71. [40] M.J. Osmond, M.J. McCall, Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard, Nanotoxicology 4 (2010) 15–41.
Please cite this article in press as: S.J. Hoseini et al., Honey-based synthesis of ZnO nanopowders and their cytotoxicity effects, Advanced Powder Technology (2015), http://dx.doi.org/10.1016/j.apt.2015.04.003
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[41] R. Landsiedel, L. Ma-Hock, A. Kroll, D. Hahn, J. Schnekenburger, K. Wiench, W. Wohlleben, Testing metal-oxide nanomaterials for human safety, Adv. Mater. 22 (2010) 2601–2627. [42] L. Wang, W. Ding, F. Zhang, Acute toxicity of ferric oxide and zinc oxide nanoparticles in rats, J. Nanosci. Nanotechnol. 10 (2010) 8617–8624. [43] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [44] C.P. Rezende, J.B. Da Silva, N.D.S. Mohallem, Influence of drying on the characteristics of zinc oxide nanoparticles, Braz. J. Phys. 39 (2009) 248–251. [45] M. Darroudi, Z. Sabouri, R. Kazemi Oskuee, H. Kargar, H.A. Hosseini, Neuronal toxicity of biopolymer-template synthesized ZnO nanoparticles, Nanomed. J. 1 (2014) 88–93. [46] M. Darroudi, Z. Sabouri, R. Kazemi Oskuee, A. Khorsand Zak, H. Kargar, M.H.N.A. Hamid, Green chemistry approach for the synthesis of ZnO nanopowders and their cytotoxic effects, Ceram. Int. 40 (2014) 4827–4831. [47] A. Khorsand Zak, R. Yousefi, W.H.A. Majid, M.R. Muhamad, Facile synthesis and X-ray peak broadening studies of Zn1 xMgxO nanoparticles, Ceram. Int. 38 (2012) 2059–2064. [48] A. Khorsand Zak, R. Razali, W.H.A. Majid, M. Darroudi, Synthesis and characterization of a narrow size distribution of zinc oxide nanoparticles, Int. J. Nanomed. 6 (2011) 1399–1403. [49] M. Darroudi, M. Hakimi, M. Sarani, R. Kazemi Oskuee, A. Khorsand Zak, L. Gholami, Facile synthesis, characterization, and evaluation of neurotoxicity effect of cerium oxide nanoparticles, Ceram. Int. 39 (2013) 6917–6921. [50] F. Harding, Breast Cancer: Cause–Prevention–Cure, Tekline Publishing, Aylesbury, 2006. [51] Joint Committee on Powder Diffraction Standards Diffraction Data File, JCPDS International Center for Diffraction Data No. 36-1451, 1991.
[52] M. Baek, M.K. Kim, H.J. Cho, J.A. Lee, J. Yu, H.E. Chung, S.J. Choi, Factors influencing the cytotoxicity of zinc oxide nanoparticles: particle size and surface charge, J. Phys.: Conf. Ser. 304 (2011) 012044. [53] C. Hanley, A. Thurber, C. Hanna, A. Punnoose, J. Zhang, D.G. Wingett, The influences of cell type and ZnO nanoparticle size on immune cell cytotoxicity and cytokine induction, Nanoscale Res. Lett. 4 (2009) 1409–1420. [54] I.L. Hsiao, Y.J. Huang, Effects of various physicochemical characteristics on the toxicities of ZnO and TiO2 nanoparticles toward human lung epithelial cells, Sci. Total Environ. 409 (2011) 1219–1228. [55] B.C. Heng, X. Zhao, S. Xiong, K.W. Ng, F.Y.C. Boey, J.S.-C. Loo, Cytotoxicity of zinc oxide (ZnO) nanoparticles is influenced by cell density and culture format, Arch. Toxicol. 85 (2011) 695–704. [56] L. Taccola, V. Raffa, C. Riggio, O. Vittorio, M.C. Iorio, R. Pietrabissa, A. Cuschieri, Zinc oxide nanoparticles as selective killers of proliferating cells, Int. J. Nanomed. 6 (2011) 1129–1140. [57] M. Premanathan, K. Karthikeyan, K. Jeyasubramanian, G. Manivannan, Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation, Nanomed.: Nanotechnol. Biol. Med. 7 (2011) 184–192. [58] K.H. Müller, J. Kulkarni, M. Motskin, A. Goode, P. Winship, J.N. Skepper, M.P. Ryan, A.E. Poeter, PH-dependent toxicity of high aspect ratio ZnO nanowires in macrophages due to intracellular dissolution, ACS Nano 4 (2010) 6767–6779. [59] W. Lin, Y.-W. Huang, X.-D. Zhou, Toxicity of cerium oxide nanoparticles in human lung cancer cells, Int. J. Toxicol. 25 (2006) 451–457. [60] M.J. Akhtar, M. Ahamed, S. Kumar, M.M. Khan, J. Ahmad, et al., Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species, Int. J. Nanomed. 7 (2012) (2012) 845–857.
Please cite this article in press as: S.J. Hoseini et al., Honey-based synthesis of ZnO nanopowders and their cytotoxicity effects, Advanced Powder Technology (2015), http://dx.doi.org/10.1016/j.apt.2015.04.003
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