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Structural morphology and in vitro toxicity studies of nano- and micro-sized zinc oxide structures
Journal of Environmental Chemical Engineering 3 (2015) 436–444
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Journal of Environmental Chemical Engineering journal homepage: www.elsevier.com/locate/jece
Structural morphology and in vitro toxicity studies of nano- and micro-sized zinc oxide structures Ling Chuo Ann a, * , Shahrom Mahmud a , Azman Seeni b , Siti Khadijah Mohd Bakhori a , Amna Sirelkhatim a , Dasmawati Mohamad c , Habsah Hasan d a
Nano-optoelectronic Research (NOR) Laboratory, School of Physics, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Pulau Pinang, Malaysia School of Dental Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150 Kelantan, Malaysia d School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150 Kelantan, Malaysia b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 July 2014 Accepted 16 December 2014
The structural morphology and in vitro toxicity of nano- and micro-sized ZnO structures were investigated. All the ZnO samples were characterized to determine their morphologies, particle sizes, structures and optical bandgaps. Transmission electron microscopy and field-emission scanning electron microscopy results revealed that the morphologies of nano-sized ZnO-N1 and ZnO-N2 samples consisted of spherical and irregularly-shaped particles, respectively. The corresponding particle sizes were 20–40 nm and 50–80 nm, respectively. The morphologies of micro-sized ZnO-M1 and ZnO-M2 samples were found to be mostly rod-structures and plate-structures, respectively. Rod diameters were 40–100 nm whereas plate widths were 50–150 nm. ZnO-N1 had the highest toxicity towards the cells, causing the cell viability to be less than 70% for all concentration ranges. This phenomenon was due to the small particle size and release of zinc ions. Micro-sized ZnO-M1 and ZnO-M2 samples had toxicity limits of 0.3 mM at most. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Toxicity Zinc oxide Morphology Structural
Introduction Nanomaterials have specific physicochemical properties that are not evident in bulk samples. Compared to micro-sized materials, most of the unique properties of nanoparticles have been attributed to their high surface-to-volume ratio. Nanoparticles offer a large surface for adsorption, and sometimes a high reactivity in many processes [1,2]. Toxicology has become an important area of research in dealing with the interactions of nanomaterials, nanostructures and nanodevices with biological molecules and organisms [3]. The evaluation of the safety of nanoparticles provides useful information about their undesirable effects, and even contributes to the development of tools to prevent such effects. ZnO micro/nano-structures have attracted a great deal of attention due to their useful optoelectronic properties and novel applications in catalysis, paints, UV detectors, transparent conductive films, varistors, gas sensors, solar cells and cosmetic products [4–9]. Furthermore, ZnO nanoparticles are frequent
* Corresponding author. Tel.: +60 164708973. E-mail address: [email protected] (L.C. Ann). http://dx.doi.org/10.1016/j.jece.2014.12.015 2213-3437/ ã 2014 Elsevier Ltd. All rights reserved.
constituents or ingredients in many personal healthcare products such as cosmetics and sunscreens, as a result of their superior UV absorption and reflectance properties [10]. The expanding production and use of ZnO has led to the potential for its release into the environment. For instance, Gottschalk et al. [11] reported that ZnO nanoparticles were found with concentration of 10 ng/l in natural surface water and 430 ng/l in treated wastewater in Europe. Moreover, the concentration of ZnO nanoparticles was estimated to be 100 mg/l (water) and few mg/kg (soil) in UK environment [12]. The review by Daughton and Ternes revealed that the level of ZnO particles would increase continually due to the extensive application of these materials [13]. Recently, there were some research works that had been conducted to investigate the toxicity impact of ZnO nanoparticles towards variety of organisms, such as human cell lines [14,15], bacteria [16,17], algae [18,19], nematodes [20], and plants [21]. Nonetheless, data and information about the toxicological effects and mechanisms of ZnO nanoparticles are still limited. The results of many researchers showed different views on the toxicity mechanisms. One or more mechanisms possibly responsible for the toxicity effects in which the major mechanism may depend on the test organism species and the test media. More extensive research is required to reveal a deeper insight into the toxic action
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of this widespread material in order to justify the potential adverse effects on human, animals, microorganisms and the environment. This study evaluates the in vitro biocompatibility of micro- and nano-sized ZnO particles using L929 mouse fibroblast as cell model. Brayner et al. [22] reported that ZnO nanoparticles at a concentration of between 3 and 10 mM could lead to 100% inhibition and damage to bacterial cells. Therefore, the concentration of ZnO samples used in this study was manipulated from 0.1 to 0.5 mM, in order to justify the toxicity limit of the ZnO samples. In this study, four types of ZnO particles (two micro-sized and two nano-sized) with different morphologies were used in toxicology experiments to determine their effects on L929 mouse fibroblast cell lines. A comparison of their toxicity levels was performed and their potential mechanisms of toxicity were elucidated. Experimental details Two micro-sized ZnO samples and two nano-sized ZnO samples were used as the starting materials in this study. The micro-sized ZnO particles were synthesized through French process according to the oxidation of zinc metal in a factory, which was done in our previous work [23]. The nano-sized ZnO samples were commercial ZnO nanoparticles, purchased from Canada. Micro-sized ZnO particles were named ZnO-M1 and ZnO-M2 while nano-sized ZnO particles were named ZnO-N1 and ZnO-N2. All of the samples were of very high purity (>99%). The structural morphologies of the ZnO samples were investigated using a Phillips CM12 transmission electron microscope (TEM) and a FEI NovaNanoSEM 450 field-emission scanning electron microscope (FESEM). The percentage composition (atomic percentages) of the ZnO samples was examined through energydispersive X-ray spectroscopy (EDS) analysis. The crystalline
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structures of the samples were characterized using a PANalytical X’Pert PRO MED PW3040 high resolution X-ray diffractometer with Cu Ka radiation (l = 1.5406 Å). Optical absorption properties of ZnO samples were examined using a Shidmazu UV–vis 1800UV spectrophotometer. Optical bandgaps were obtained from the absorption spectra based on the UV-visible spectrum measurements. Besides, zeta potentials of the ZnO samples were studied using Zetasizer Nano-zs at room temperature. In the sample preparation, 0.01 g ZnO powder was suspended in 100 ml distilled water, which was then sonicated for 15 min to fully disperse ZnO agglomeration. Cytotoxicity tests were conducted to study the toxicity effects of ZnO samples towards L929 fibroblasts mouse cell lines. Initially, the cell lines were sub-cultured in Dulbecco’s modified Eagle medium supplemented with 10% foetal bovine serum. The cells were grown at 37 C in a 5% CO2 incubator and the sub-culture process was repeated 3–4 times until the cell lines were confluent in the T-25 cell culture flask. Subsequently, L929 cells were seeded into 6-wellplates and allowed to attach to the plates for 24 h. Twenty thousand cells were manipulated for seeding into each well. Dispersed ZnO particles with a concentration of 1 mM were used as a stock suspension and were sterilized using an autoclave. Then, the ZnO particles were diluted to 0.1, 0.2, 0.3, 0.4 and 0.5 mM by addition of culture media. The ZnO-media mixture was left in the incubator for 24 h so that the ZnO particles could react with and treat the media. After 24 h incubation, media in the 6-wellplates was discarded. The respective ZnO-media mixture was syringefiltered to remove the insoluble ZnO particles. Then, the treated media was added to the cells in the 6-well plates. Cells without ZnO particles were also prepared as negative controls in a respective experiment. Besides, positive control was prepared through the treatment of cells with calamine lotion. A calamine lotion contained high amount of ZnO powder, which dominated
Fig. 1. TEM images of (a) ZnO-N1; (b) ZnO-N2; (c) ZnO-M1 and (d) ZnO-M2.
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approximately 20% of the mixture (100 ml). All of the cell preparation and toxicity tests were performed in duplicate. The mixture of ZnO and cells was incubated in a CO2 incubator for 24 h. To look into the effect of ZnO particles on the L929 cell lines, the morphologies of the cells were observed after 24, 48 and 72 h of incubation using an Olympus CKX41 optical light microscope. In order to calculate the percentage of viable cells, cell counting was performed after 72 h incubation. The ZnO-cell mixture was transferred into a conical tube and centrifuged at 1500 rpm. The supernatant was discarded and 1 ml of media was added into
the tube. Then, the mixture was mixed with trypan blue at a 1:1 ratio. Some of the stained cells were placed onto a Neubauer chamber and the number of viable cells was counted. Results and discussion Morphology and crystalline properties Fig. 1 shows some TEM images of the ZnO samples. Of the four ZnO samples examined, all of them exhibited different
Fig. 2. FESEM images and EDS spectrum of: (a and b) ZnO-N1; (c and d) ZnO-N2; (e and f) ZnO-M1 and (g and h) ZnO-M2.
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Fig. 3. (a) XRD patterns of ZnO samples; (b) optical absorption of ZnO samples.
morphologies and particle sizes. Nano-sized ZnO-N1 and ZnON2 samples possessed spherical and irregular-shaped particles, respectively. The corresponding particle sizes were 20–40 nm (ZnO-N1) and 50–80 nm (ZnO-N2). For the micro-sized ZnOM1 and ZnO-M2 samples, the major morphologies consisted of rod-structures and plate/slab-structures, respectively. The rodstructures had a diameter size of 40–100 nm while the platestructures were 50–150 nm wide. Other morphologies also existed in both ZnO-M1 and ZnO-M2 samples, including hexagonal drumshaped particles and irregularly-shaped particles. The particles’ sizes and shapes were further examined using FESEM imaging. Fig. 2 shows images obtained after FESEM and EDS analysis of the ZnO samples. The FESEM images clearly showed three-dimensional ZnO structures, which were consistent with the TEM images. The major difference among the four samples was determined to be in their sizes and structures. Furthermore, EDS results revealed that the ZnO powder samples were of high purity, with different atomic ratios of oxygen and zinc atoms. Nano-sized ZnO-N1 and ZnO-N2 samples possessed lower O:Zn ratios compared to those of micro-sized ZnO-M1 and ZnOM2 samples. These results were most likely due to the different sizes of the particles, where nano-sized particles tended to have lower O:Zn ratios while micro-sized particles exhibited higher O: Zn ratios. The rod structures also had a relatively greater oxygen content compared to zinc atoms, as reported in our previous work [24,25]. Therefore, many multi-sized rod structures in ZnOM1 samples contributed to the higher O:Zn ratio in the respective samples. Structural, optical and surface properties Fig. 3a shows the X-ray diffraction (XRD) patterns of the ZnO samples. The XRD parameters are listed in Table 1. All of the ZnO samples exhibited well-defined diffraction peaks that correlated with a ZnO wurtzite structure, with the absence of any impurities. The XRD data were indexed using the international crystallographic data table JCPDS 800074. The intense peaks that had high reflective intensities were composed of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (11 0), and (1 0 3) planes. Using the highest intensity (1 0 1)
plane, the peak positions of all of the ZnO samples were compared to the reference peak: 36.207 (JCPDS 800074). The reflective intensities of ZnO-M1 (1682 a.u.) and ZnO-M2 (1728 a.u.) were higher than those of ZnO-N1 (451 a.u.) and ZnO-N2 (604 a.u.). This was probably due to the larger particles of ZnO having preferable crystalline atomic orientations compared to smaller-sized particles. An increased number of defects and increased strain in nano-sized particles could also have been factors that suppressed the intensities of the spectra [26]. In order to investigate the average crystallite sizes of ZnO particles, full-wave half maximum (FWHM) data were obtained from the XRD results. ZnO-N1 possessed the highest value of FWHM (0.3936 ), indicating the smallest particle sizes. ZnON2 showed a slightly smaller magnitude of FWHM (0.2952 ), which indicated a larger particle size compared to ZnO-N1. ZnOM1 and ZnO-M2 exhibited similar FWHM values (0.1968 ), because of the multiple particle sizes in both of the samples, ranging from nano- to micro-sized. Therefore, the XRD data merely showed the average crystallite sizes of ZnO-M1 and ZnO-M2. Based on the FWHM values obtained, the average crystallite sizes were calculated using Scherer’s equation: t¼
0:9l FWHMcosu
(1)
where t is crystallite size, l is wavelength of X-ray radiation (1.5406 Å), FWHM is peak width at half-maximum intensity, and u is peak position. Notably, ZnO-N1 possessed the smallest crystallite size (t = 24.3 nm), followed by ZnO-N2 (t = 34.1 nm). The uniform sizes of ZnO-N1 and ZnO-N2 gave rise to repeatable crystallite sizes with respect to XRD values. Nonetheless, the calculated crystallite size of ZnO-M1 (t = 56.2 nm) was very similar to that of ZnO-M2 (t = 56.9 nm). This outcome was due to the wide range of particle sizes in ZnO-M1 and ZnO-M2. Therefore, the results from TEM imaging were more reliable for predicting the particle sizes of ZnO-M1 and ZnO-M2, which ranged from 40–100 nm for ZnO-M1 to 50–150 nm for ZnO-M2. The existence of nano-sized and micro-sized ZnOM1 and ZnO-M2 particles should be taken into account when studying the properties of these two samples.
Table 1 Structural parameters, optical bandgaps and zeta potentials of ZnO samples. Samples
Peak position (1 0 1) ( )
Intensity (1 0 1) (a.u.)
FWHM ( )
Crystallite sizes (nm)
d-spacings (Å)
Optical bandgap (eV)
Zeta potential (mV)
ZnO-N1 ZnO-N2 ZnO-M1 ZnO-M2
36.218 36.206 36.221 36.194
451 604 1682 1728
0.3936 0.2952 0.1968 0.1968
24.3 34.1 56.2 56.9
2.4803 2.4811 2.4816 2.4818
3.074 3.109 3.237 3.223
19.2 28.1 30.3 31.5
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both the micro-sized ZnO samples exhibited low zinc ion dissolution.
The lattice spacing was obtained from Bragg’s equation: d¼
nl 2sinu
(2)
where l (1.5406 Å) is X-ray wavelength, and u is diffraction angle. For nano-sized ZnO samples, the lattice spacing was 2.4803 Å (ZnO-N1) and 2.4811 Å (ZnO-N2). ZnO-M1 and ZnO-M2 samples had larger d-spacing compared to ZnO-N1 and ZnO-N2, which were 2.4816 and 2.4818 Å, respectively. The lowest d-spacing value seen in the ZnO-N1 sample revealed that there was likely tensile stress in the samples, due to the lower O:Zn ratio and higher oxygen vacancies [27]. Conversely, ZnO-M1 and ZnO-M2 might have experienced lower tensile stress due to the higher O:Zn ratio, leading to higher d-spacing values. This result was consistent with the results from EDS analysis which revealed a lower O:Zn ratio for ZnO-N1 but higher O:Zn ratio for both ZnO-M1 and ZnOM2 samples. Fig. 3b shows the optical absorption of ZnO samples. The optical bandgaps were calculated from the equation: ðahvÞ2 ¼ Aðhv Eg Þ
(3)
where a is the absorption coefficient, h is the Planck’s constant, v is the frequency and Eg is the bandgap. All of the ZnO samples exhibited strong absorption in the ultraviolet region. The sharp edges of UV absorption spectra revealed good crystallinity of ZnO powder samples. The optical bandgaps of nano-sized ZnO-N1 (Eg = 3.074 eV) and ZnO-N2 (Eg = 3.109 eV) were lower than that of the ZnO bulk (Eg = 3.37 eV). The observed difference in optical bandgaps may originate from the crystals’ defects, especially oxygen vacancies and zinc interstitials [28]. This phenomenon agreed well with the EDS results shown in Fig. 2 wherein ZnON1 and ZnO-N2 exhibited a lower content of oxygen atoms, possibly correlating to the oxygen vacancies or zinc interstitial formation. On the other hand, the optical bandgaps for the microsized ZnO-M1 (Eg = 3.237 eV) and ZnO-M2 (Eg = 3.223 eV) were larger than those of ZnO-N1 and ZnO-N2. There are some rodstructures in both ZnO-M1 and ZnO-M2 samples, giving rise to a possible formation of oxygen interstitial and zinc vacancies [25]. The EDS analyses (Fig. 2) also revealed consistent results where both samples contained higher atomic percentages of oxygen atoms compared to zinc atoms. However, the slightly smaller magnitude of Eg of ZnO-M1 and ZnO-M2 samples compared to ZnO bulk (Eg = 3.37 eV) revealed the decreasing optical bandgap, owing to the oxygen interstitial and zinc vacancies suppressing the interband excitonic recombination [29]. Remarkably, the particle size and morphology are two key factors in altering the elemental contents in the crystalline structure as well as in affecting the optical property of the ZnO samples. On the other hand, the surface charges of ZnO particles in the suspension were examined through zeta potential measurement, as listed in Table 1. The positive signs of the potentials indicated a tendency of obtaining positively charged on the ZnO particle surfaces. Among the ZnO samples, ZnO-N1 exhibited the lowest zeta potential (19.2 mV), revealing the least amount of positively charges on the particles. This phenomenon was explained by the high level of zinc ion (Zn2+) release in the suspension, causing less zinc on ZnO particles. ZnO-N1 was regarded as less stable particles in the suspension form owing to highly dissolution of zinc ion and low magnitude of zeta potential. Meanwhile, ZnON2 possessed zeta potential of 28.1 mV, indicating higher surface positively charges and lower zinc ion dissolution. Micro-sized ZnO-M1 and ZnO-M2 showed high zeta potentials, which are 30.3 and 31.5 mV, respectively. The particles of ZnO-M1 and ZnOM2 had high stability in the suspension, where many positively charged zinc ions localized on the ZnO particles. Consequently,
Cytotoxicity tests The implementation of in vitro assays to measure cellular viability is very important for gaining insight into the potential toxicity of nanomaterials. We evaluated the toxicity of ZnO samples through microscopy imaging and Neubauer viable cells counting. Fig. 4 shows the optical microscopy of L929 cell lines being treated with 0.1 and 0.5 mM ZnO-N1 and ZnO-N2. The images revealed the different toxicity responses of the ZnO samples on the cell lines after 24 h, 48 h and 72 h of treatment. Fig. 4a shows the negative control, used to compare the cell growth of treated and untreated cell lines. Besides, positive control was shown in Fig. 5a through the calamine lotion treatment with the cell lines. The high concentration of ZnO in the calamine lotion (about 20%) could induce 100% fatality towards the L929 cell lines. The toxicity impacts of ZnO samples are concentration dependent. Use of 0.5 mM ZnO-N1 and ZnO-N2 gave rise to increased cell death compared with 0.1 mM samples. Fig. 4b and d shows that the majority of the cell lines remained healthy and proliferated (about 70%) after being treated with 0.1 mM ZnO-N1 and ZnO-N2 samples. However, 0.5 mM ZnO-N1 and ZnO-N2 samples induced an acute lethal effect on the cells whereby many cells died after treatment, as indicated by the red arrows in Fig. 4. From the microscopic observation, the dead cells appeared to be “dull” and had a round shape. Only about 5% of cells were observed to proliferate in ZnON1 treatment after 24 h, 48 h and 72 h incubations (Fig. 4c). Conversely, only a small number of cells (about 20%) could proliferate in the 0.5 mM ZnO-N2 treatment after 24 h incubation (Fig. 4e). The cell proliferation in 0.5 mM ZnO-N2 treatment increased to about 40% and 60% after 48 and 72 h incubations, respectively. The amount of dead cells seen after treatment with ZnO-N1 was found to be greater than for the ZnO-N2 treatment, showing that ZnO-N1 had a greater toxicity. Fig. 5b–e shows the optical microscope images of L929 cell lines after treatment with ZnO-M1 and ZnO-M2 samples. Both of the ZnO samples exhibited similar effects on the cells, where 0.1 mM ZnO-M1 and ZnO-M2 had little toxicity towards the cells while 0.5 mM ZnO caused some damage to the cells. Low ZnO concentrations led to increased proliferation of the cell lines after 48 and 72 h incubation, as can be seen in Fig. 5b and d. Meanwhile, some of the cells’ proliferative activities deteriorated when treated with 0.5 mM ZnO, as indicated by the red arrows in Fig. 5c and e. This observation suggested that some cell lines (about 30%) could have succumbed to the toxicity of 0.5 mM ZnO-M1 and ZnO-M2, giving rise to the fatality of the cell lines. Assessing the toxicity of ZnO samples is of utmost important. The use of in vitro assays to test cellular viability was performed to evaluate the potential toxicity of the ZnO samples. Cell counting was conducted using a Neubauer chamber and the percentage of viable cells was calculated based on a negative control, using the equation: viable cells=control Þ 100% Percentage viable cells ¼ ð control
(4)
According to the criteria of the Nanotechnology Characterisation Laboratory in Frederick, USA [30], a nanoparticle is considered to be non-toxic if the cell viability at 48 h is higher than 75%. In this study, we determined the non-toxic level to be above 70% after 72 h, considering the longer contact time of the cells during ZnO treatment. Fig. 6 shows the percentage of viable cells after being treated with different concentrations (0.1–0.5 mM) of ZnO samples. Different toxicity levels were noted from the cell viability results,
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Fig. 4. Light microscopic images of L929 cell lines under treatment of: (a) negative control; (b) 0.1 mM ZnO-N1; (c) 0.5 mM ZnO-N1; (d) 0.1 mM ZnO-N2 and (e) 0.5 mM ZnON2.
for cell lines treated with the ZnO samples. The toxicities of all of the ZnO samples were concentration dependent. The percentage of viable cells decreased when the concentration of ZnO samples increased. Among the ZnO samples, ZnO-N1 exhibited the highest toxicity towards the L929 mouse cell lines. Concentrations of 0.1 and 0.2 mM ZnO-N1 led to percentage cell viabilities of 52% and 23%, which are lower than the non-toxic level of 70%. Moreover,
concentrations of 0.4 and 0.5 mM caused the fatality of all of the cells. This result was consistent with the images obtained from light microscopy (Fig. 4c) where most of the cell lines were destroyed when treated with 0.5 mM ZnO-N1. Therefore, ZnON1 had induced an acute lethal effect on the cells at concentrations above 0.1 mM. In contrast, ZnO-N2 exerted a lower toxicity on the cells and gave rise to greater amounts of viable cells compared to
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Fig. 5. Light microscopic images of L929 cell lines under treatment of: (a) positive control (b) 0.1 mM ZnO-M1; (c) 0.5 mM ZnO-M1; (d) 0.1 mM ZnO-M2 and (e) 0.5 mM ZnOM2.
ZnO-N1. The percentage of viable cells was above the non-toxic level (70%) at ZnO-N2 concentrations of 0.1 mM. The concentration of ZnO-N2 that initiated the killing effect on the cells was 0.2 mM, and the cell viability was obviously greater than that of ZnO-N1. The percentage of viable cells reduced to 40 % after treatment with 0.5 mM ZnO-N2. Furthermore, light microscope imaging (Fig. 4d and e) revealed that the majority of cell lines could grow robustly under 0.1 mM ZnO-N2 treatment whereas some cell lines were killed when treated with 0.5 mM ZnO-N2.
On the other hand, ZnO-M1 and ZnO-M2 samples were less toxic and the resultant cell viabilities were higher than those of the ZnO-N1 and ZnO-N2 samples. Both ZnO-M1 and ZnO-M2 samples only exhibited lethal effects at concentrations of 0.4 mM, which was lower than non-toxic level (70%). After 0.5 mM ZnO treatment, cell viabilities reduced to 54% (ZnO-M1) and 53% (ZnO-M2). This result suggested that the threshold concentration of ZnO-M1 and ZnO-M2 samples to be used for non-toxic application was 0.3 mM at most. This quantitative result was supported by the previous
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Fig. 6. Percentage of viable cells after 72 h treatment with different concentration of ZnO samples.
microscope images (Fig. 5) wherein no dead cells were identified in 0.1 mM ZnO samples but some unhealthy/dead cells were found in the 0.5 mM ZnO samples. Toxicity mechanisms of ZnO To date, the health risks and potential hazards of both micro- and nano-sized ZnO particles have not been assessed and are somewhat concerning; they have become a subject of discussion [31–33]. However, there is no consistent conclusion about the toxicity of ZnO and the impact of particle size and morphology on ZnO toxicity. Several studies have been performed to assess the toxicities of the nanoparticles using different cellular systems and test methods [34– 36]. Some researchers found that different sizes of ZnO nanoparticles exhibited comparable cytotoxicity on different cells, while the IC50 after 24 h was about 10–20 mg/ml [37,38]. Conversely, Lee et al. reported that rod-shaped ZnO particles could trigger greater toxicity than spherical ZnO particles [39]. In this study, the morphology and size of ZnO particles were discovered to be the key factors causing different toxicity effects. Because the ZnO in the media culture was filtered before being mixed with cell lines, direct contact between the ZnO particles and the cells was avoided. Therefore, two chemical mechanisms involving free radicals generated by ZnO particles were proposed. Firstly, release of Zn2+ ion from ZnO could be an important contributor to toxicity. Typically, the amount of zinc ions in the suspension is affected by the solubility of ZnO. The dissolution of Zn ions depends on the dosage of ZnO used in the distilled water, whereby the use of 0.5 mM ZnO samples would induce the highest toxicity by releasing increased levels of Zn ions compared with lower concentrations of ZnO. Deng et al. [40] observed that ZnO nanoparticles and ZnCl2 had a comparable toxic effect on mouse NSCs, indicating that a similar quantity of Zn2+ was released from both solutions. Further, Fukui et al. [41] suggested that an increase in intracellular Zn2+ induced higher intracellular ROS levels and oxidative stress in rat lung cells. The cell viability and lactate dehydrogenase (LDH) level depended on the level of zinc ions in the suspension. Furthermore, smaller ZnO particle sizes would result in greater toxicity compared to larger particles sizes. Nanosized ZnO-N1 and ZnO-N2 samples possessed surface-to-volume ratios and so could potentially release more Zn2+ into the suspensions, triggering greater levels of toxicity towards cells. Zeta potential (Table 1) showed the consistent result wherein ZnON1 probably exhibited the highest dissolution of zinc ions due to the smallest positive value of zeta potential. Secondly, based on the potential redox properties present in biological systems, as described by Ai et al. [42], it was also possible
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that chronological oxidation–reduction processes may have occurred at ZnO particle surfaces to generate reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH). Even when small amounts of ZnO particles are involved in the interactions, large amounts of ROS would be released. A concentration of ZnO particles greater than 5 mg/ml could increase the intracellular ROS levels [38]. The production of ROS from ZnO samples likely caused disruption of cellular function or disorganization of membranes. Furthermore, different morphologies of ZnO particles were believed to release different amounts of ROS, as shown in our previous works [25,43], which suggested that rod-structures possess greater amounts of oxygen atoms on the particles’ surfaces and generated relatively high quantities of ROS. The toxicity effect of ZnO-M1 was possibly due to the presence of rod-structures, which could produce more ROS in the suspension. However, the effect of particle size seemed to be more dominant than the particle morphology, as can be seen in the cell viability analysis, shown in Fig. 6. Due to larger rod-shaped particles (40–100 nm) in ZnO-M1 compared to the sphericalshaped particles (20 nm) present in ZnO-N1, nano-sized ZnON1 resulted in greater toxicity compared to micro-sized ZnO-M1. On the other hand, rod-shaped structures in ZnO-M1 and plateshaped structures in ZnO-M2 were comparable in terms of toxicity effects, probably due to the presence of multiple particle sizes. Conclusion ZnO particles with different particle sizes and morphological features were used to investigate the in vitro cytotoxicity towards L929 mouse fibroblast cell lines. Micro-sized ZnO-M1 and ZnOM2 samples exhibited lower toxicity compared to the nano-sized ZnO-N1 and ZnO-N2 samples. The limit of toxicity for ZnO-M1 and ZnO-M2 samples was at most 0.3 mM, while for ZnO-N2 was 0.1 mM at most. The release of zinc ions and the generation of ROS from the particles were suggested to be potential mechanisms of toxicity towards the cell lines. Acknowledgements The authors thank the financial support from Universiti Sains Malaysia through Research University (Individual) grant (1001/ PFIZIK/814174) and supporting grant RU-PRGS (1001/PFIZIK/ 846077). Assistance from NOR laboratory and AMDI laboratory is also acknowledged. The authors declare no conflict of interest related to this study. References [1] P.J. Borm, W. Kreyling, Toxicological hazards of inhaled nanoparticles – potential implications for drug delivery, J. Nanosci. Nanotechnol. 4 (5) (2004) 521–531, doi:http://dx.doi.org/10.1166/jnn.2004.081. 15503438. [2] N. Iqbal, M.R.A. Kadir, N.H. Mahmood, N. Salim, G.R.A. Froemming, H.R. Balaji, T. Kamarul, Characterization, antibacterial and in vitro compatibility of zinc– silver doped hydroxyapatite nanoparticles prepared through microwave synthesis, Ceram. Int. 40 (3) (2014) 4507–4513, doi:http://dx.doi.org/10.1016/j. ceramint.2013.08.125. [3] S.M. Hussain, L.K. Braydich-Stolle, A.M. Schrand, R.C. Murdock, K.O. Yu, D.M. Mattie, J.J. Schlager, M. Terrones, Toxicity evaluation for safe use of nanomaterials: recent achievements and technical challenges, Adv. Mater. 21 (16) (2009) 1549–1559, doi:http://dx.doi.org/10.1002/adma.200801395. [4] G. Ramakrishna, H.N. Ghosh, Effect of particle size on the reactivity of quantum size ZnO nanoparticles and charge-transfer dynamics with adsorbed catechols, Langmuir 19 (7) (2003) 3006–3012, doi:http://dx.doi.org/10.1021/ la020828u. [5] S.Y. Bae, H.W. Seo, J. Park, Vertically aligned sulfur-doped ZnO nanowires synthesized via chemical vapor deposition, J. Phys. Chem. B 108 (17) (2004) 5206–5210, doi:http://dx.doi.org/10.1021/jp036720k. [6] E. Comini, G. Faglia, G. Sberveglieri, Z. Pan, Z.L. Wang, Stable and highly sensitive gas sensors based on semiconducting oxide nanobelts, Appl. Phys. Lett. 81 (10) (2002) 1869–1871, doi:http://dx.doi.org/10.1063/1.1504867.
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Contents lists available at ScienceDirect
Journal of Environmental Chemical Engineering journal homepage: www.elsevier.com/locate/jece
Structural morphology and in vitro toxicity studies of nano- and micro-sized zinc oxide structures Ling Chuo Ann a, * , Shahrom Mahmud a , Azman Seeni b , Siti Khadijah Mohd Bakhori a , Amna Sirelkhatim a , Dasmawati Mohamad c , Habsah Hasan d a
Nano-optoelectronic Research (NOR) Laboratory, School of Physics, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Pulau Pinang, Malaysia School of Dental Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150 Kelantan, Malaysia d School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150 Kelantan, Malaysia b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 July 2014 Accepted 16 December 2014
The structural morphology and in vitro toxicity of nano- and micro-sized ZnO structures were investigated. All the ZnO samples were characterized to determine their morphologies, particle sizes, structures and optical bandgaps. Transmission electron microscopy and field-emission scanning electron microscopy results revealed that the morphologies of nano-sized ZnO-N1 and ZnO-N2 samples consisted of spherical and irregularly-shaped particles, respectively. The corresponding particle sizes were 20–40 nm and 50–80 nm, respectively. The morphologies of micro-sized ZnO-M1 and ZnO-M2 samples were found to be mostly rod-structures and plate-structures, respectively. Rod diameters were 40–100 nm whereas plate widths were 50–150 nm. ZnO-N1 had the highest toxicity towards the cells, causing the cell viability to be less than 70% for all concentration ranges. This phenomenon was due to the small particle size and release of zinc ions. Micro-sized ZnO-M1 and ZnO-M2 samples had toxicity limits of 0.3 mM at most. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Toxicity Zinc oxide Morphology Structural
Introduction Nanomaterials have specific physicochemical properties that are not evident in bulk samples. Compared to micro-sized materials, most of the unique properties of nanoparticles have been attributed to their high surface-to-volume ratio. Nanoparticles offer a large surface for adsorption, and sometimes a high reactivity in many processes [1,2]. Toxicology has become an important area of research in dealing with the interactions of nanomaterials, nanostructures and nanodevices with biological molecules and organisms [3]. The evaluation of the safety of nanoparticles provides useful information about their undesirable effects, and even contributes to the development of tools to prevent such effects. ZnO micro/nano-structures have attracted a great deal of attention due to their useful optoelectronic properties and novel applications in catalysis, paints, UV detectors, transparent conductive films, varistors, gas sensors, solar cells and cosmetic products [4–9]. Furthermore, ZnO nanoparticles are frequent
* Corresponding author. Tel.: +60 164708973. E-mail address: [email protected] (L.C. Ann). http://dx.doi.org/10.1016/j.jece.2014.12.015 2213-3437/ ã 2014 Elsevier Ltd. All rights reserved.
constituents or ingredients in many personal healthcare products such as cosmetics and sunscreens, as a result of their superior UV absorption and reflectance properties [10]. The expanding production and use of ZnO has led to the potential for its release into the environment. For instance, Gottschalk et al. [11] reported that ZnO nanoparticles were found with concentration of 10 ng/l in natural surface water and 430 ng/l in treated wastewater in Europe. Moreover, the concentration of ZnO nanoparticles was estimated to be 100 mg/l (water) and few mg/kg (soil) in UK environment [12]. The review by Daughton and Ternes revealed that the level of ZnO particles would increase continually due to the extensive application of these materials [13]. Recently, there were some research works that had been conducted to investigate the toxicity impact of ZnO nanoparticles towards variety of organisms, such as human cell lines [14,15], bacteria [16,17], algae [18,19], nematodes [20], and plants [21]. Nonetheless, data and information about the toxicological effects and mechanisms of ZnO nanoparticles are still limited. The results of many researchers showed different views on the toxicity mechanisms. One or more mechanisms possibly responsible for the toxicity effects in which the major mechanism may depend on the test organism species and the test media. More extensive research is required to reveal a deeper insight into the toxic action
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of this widespread material in order to justify the potential adverse effects on human, animals, microorganisms and the environment. This study evaluates the in vitro biocompatibility of micro- and nano-sized ZnO particles using L929 mouse fibroblast as cell model. Brayner et al. [22] reported that ZnO nanoparticles at a concentration of between 3 and 10 mM could lead to 100% inhibition and damage to bacterial cells. Therefore, the concentration of ZnO samples used in this study was manipulated from 0.1 to 0.5 mM, in order to justify the toxicity limit of the ZnO samples. In this study, four types of ZnO particles (two micro-sized and two nano-sized) with different morphologies were used in toxicology experiments to determine their effects on L929 mouse fibroblast cell lines. A comparison of their toxicity levels was performed and their potential mechanisms of toxicity were elucidated. Experimental details Two micro-sized ZnO samples and two nano-sized ZnO samples were used as the starting materials in this study. The micro-sized ZnO particles were synthesized through French process according to the oxidation of zinc metal in a factory, which was done in our previous work [23]. The nano-sized ZnO samples were commercial ZnO nanoparticles, purchased from Canada. Micro-sized ZnO particles were named ZnO-M1 and ZnO-M2 while nano-sized ZnO particles were named ZnO-N1 and ZnO-N2. All of the samples were of very high purity (>99%). The structural morphologies of the ZnO samples were investigated using a Phillips CM12 transmission electron microscope (TEM) and a FEI NovaNanoSEM 450 field-emission scanning electron microscope (FESEM). The percentage composition (atomic percentages) of the ZnO samples was examined through energydispersive X-ray spectroscopy (EDS) analysis. The crystalline
437
structures of the samples were characterized using a PANalytical X’Pert PRO MED PW3040 high resolution X-ray diffractometer with Cu Ka radiation (l = 1.5406 Å). Optical absorption properties of ZnO samples were examined using a Shidmazu UV–vis 1800UV spectrophotometer. Optical bandgaps were obtained from the absorption spectra based on the UV-visible spectrum measurements. Besides, zeta potentials of the ZnO samples were studied using Zetasizer Nano-zs at room temperature. In the sample preparation, 0.01 g ZnO powder was suspended in 100 ml distilled water, which was then sonicated for 15 min to fully disperse ZnO agglomeration. Cytotoxicity tests were conducted to study the toxicity effects of ZnO samples towards L929 fibroblasts mouse cell lines. Initially, the cell lines were sub-cultured in Dulbecco’s modified Eagle medium supplemented with 10% foetal bovine serum. The cells were grown at 37 C in a 5% CO2 incubator and the sub-culture process was repeated 3–4 times until the cell lines were confluent in the T-25 cell culture flask. Subsequently, L929 cells were seeded into 6-wellplates and allowed to attach to the plates for 24 h. Twenty thousand cells were manipulated for seeding into each well. Dispersed ZnO particles with a concentration of 1 mM were used as a stock suspension and were sterilized using an autoclave. Then, the ZnO particles were diluted to 0.1, 0.2, 0.3, 0.4 and 0.5 mM by addition of culture media. The ZnO-media mixture was left in the incubator for 24 h so that the ZnO particles could react with and treat the media. After 24 h incubation, media in the 6-wellplates was discarded. The respective ZnO-media mixture was syringefiltered to remove the insoluble ZnO particles. Then, the treated media was added to the cells in the 6-well plates. Cells without ZnO particles were also prepared as negative controls in a respective experiment. Besides, positive control was prepared through the treatment of cells with calamine lotion. A calamine lotion contained high amount of ZnO powder, which dominated
Fig. 1. TEM images of (a) ZnO-N1; (b) ZnO-N2; (c) ZnO-M1 and (d) ZnO-M2.
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approximately 20% of the mixture (100 ml). All of the cell preparation and toxicity tests were performed in duplicate. The mixture of ZnO and cells was incubated in a CO2 incubator for 24 h. To look into the effect of ZnO particles on the L929 cell lines, the morphologies of the cells were observed after 24, 48 and 72 h of incubation using an Olympus CKX41 optical light microscope. In order to calculate the percentage of viable cells, cell counting was performed after 72 h incubation. The ZnO-cell mixture was transferred into a conical tube and centrifuged at 1500 rpm. The supernatant was discarded and 1 ml of media was added into
the tube. Then, the mixture was mixed with trypan blue at a 1:1 ratio. Some of the stained cells were placed onto a Neubauer chamber and the number of viable cells was counted. Results and discussion Morphology and crystalline properties Fig. 1 shows some TEM images of the ZnO samples. Of the four ZnO samples examined, all of them exhibited different
Fig. 2. FESEM images and EDS spectrum of: (a and b) ZnO-N1; (c and d) ZnO-N2; (e and f) ZnO-M1 and (g and h) ZnO-M2.
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Fig. 3. (a) XRD patterns of ZnO samples; (b) optical absorption of ZnO samples.
morphologies and particle sizes. Nano-sized ZnO-N1 and ZnON2 samples possessed spherical and irregular-shaped particles, respectively. The corresponding particle sizes were 20–40 nm (ZnO-N1) and 50–80 nm (ZnO-N2). For the micro-sized ZnOM1 and ZnO-M2 samples, the major morphologies consisted of rod-structures and plate/slab-structures, respectively. The rodstructures had a diameter size of 40–100 nm while the platestructures were 50–150 nm wide. Other morphologies also existed in both ZnO-M1 and ZnO-M2 samples, including hexagonal drumshaped particles and irregularly-shaped particles. The particles’ sizes and shapes were further examined using FESEM imaging. Fig. 2 shows images obtained after FESEM and EDS analysis of the ZnO samples. The FESEM images clearly showed three-dimensional ZnO structures, which were consistent with the TEM images. The major difference among the four samples was determined to be in their sizes and structures. Furthermore, EDS results revealed that the ZnO powder samples were of high purity, with different atomic ratios of oxygen and zinc atoms. Nano-sized ZnO-N1 and ZnO-N2 samples possessed lower O:Zn ratios compared to those of micro-sized ZnO-M1 and ZnOM2 samples. These results were most likely due to the different sizes of the particles, where nano-sized particles tended to have lower O:Zn ratios while micro-sized particles exhibited higher O: Zn ratios. The rod structures also had a relatively greater oxygen content compared to zinc atoms, as reported in our previous work [24,25]. Therefore, many multi-sized rod structures in ZnOM1 samples contributed to the higher O:Zn ratio in the respective samples. Structural, optical and surface properties Fig. 3a shows the X-ray diffraction (XRD) patterns of the ZnO samples. The XRD parameters are listed in Table 1. All of the ZnO samples exhibited well-defined diffraction peaks that correlated with a ZnO wurtzite structure, with the absence of any impurities. The XRD data were indexed using the international crystallographic data table JCPDS 800074. The intense peaks that had high reflective intensities were composed of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (11 0), and (1 0 3) planes. Using the highest intensity (1 0 1)
plane, the peak positions of all of the ZnO samples were compared to the reference peak: 36.207 (JCPDS 800074). The reflective intensities of ZnO-M1 (1682 a.u.) and ZnO-M2 (1728 a.u.) were higher than those of ZnO-N1 (451 a.u.) and ZnO-N2 (604 a.u.). This was probably due to the larger particles of ZnO having preferable crystalline atomic orientations compared to smaller-sized particles. An increased number of defects and increased strain in nano-sized particles could also have been factors that suppressed the intensities of the spectra [26]. In order to investigate the average crystallite sizes of ZnO particles, full-wave half maximum (FWHM) data were obtained from the XRD results. ZnO-N1 possessed the highest value of FWHM (0.3936 ), indicating the smallest particle sizes. ZnON2 showed a slightly smaller magnitude of FWHM (0.2952 ), which indicated a larger particle size compared to ZnO-N1. ZnOM1 and ZnO-M2 exhibited similar FWHM values (0.1968 ), because of the multiple particle sizes in both of the samples, ranging from nano- to micro-sized. Therefore, the XRD data merely showed the average crystallite sizes of ZnO-M1 and ZnO-M2. Based on the FWHM values obtained, the average crystallite sizes were calculated using Scherer’s equation: t¼
0:9l FWHMcosu
(1)
where t is crystallite size, l is wavelength of X-ray radiation (1.5406 Å), FWHM is peak width at half-maximum intensity, and u is peak position. Notably, ZnO-N1 possessed the smallest crystallite size (t = 24.3 nm), followed by ZnO-N2 (t = 34.1 nm). The uniform sizes of ZnO-N1 and ZnO-N2 gave rise to repeatable crystallite sizes with respect to XRD values. Nonetheless, the calculated crystallite size of ZnO-M1 (t = 56.2 nm) was very similar to that of ZnO-M2 (t = 56.9 nm). This outcome was due to the wide range of particle sizes in ZnO-M1 and ZnO-M2. Therefore, the results from TEM imaging were more reliable for predicting the particle sizes of ZnO-M1 and ZnO-M2, which ranged from 40–100 nm for ZnO-M1 to 50–150 nm for ZnO-M2. The existence of nano-sized and micro-sized ZnOM1 and ZnO-M2 particles should be taken into account when studying the properties of these two samples.
Table 1 Structural parameters, optical bandgaps and zeta potentials of ZnO samples. Samples
Peak position (1 0 1) ( )
Intensity (1 0 1) (a.u.)
FWHM ( )
Crystallite sizes (nm)
d-spacings (Å)
Optical bandgap (eV)
Zeta potential (mV)
ZnO-N1 ZnO-N2 ZnO-M1 ZnO-M2
36.218 36.206 36.221 36.194
451 604 1682 1728
0.3936 0.2952 0.1968 0.1968
24.3 34.1 56.2 56.9
2.4803 2.4811 2.4816 2.4818
3.074 3.109 3.237 3.223
19.2 28.1 30.3 31.5
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both the micro-sized ZnO samples exhibited low zinc ion dissolution.
The lattice spacing was obtained from Bragg’s equation: d¼
nl 2sinu
(2)
where l (1.5406 Å) is X-ray wavelength, and u is diffraction angle. For nano-sized ZnO samples, the lattice spacing was 2.4803 Å (ZnO-N1) and 2.4811 Å (ZnO-N2). ZnO-M1 and ZnO-M2 samples had larger d-spacing compared to ZnO-N1 and ZnO-N2, which were 2.4816 and 2.4818 Å, respectively. The lowest d-spacing value seen in the ZnO-N1 sample revealed that there was likely tensile stress in the samples, due to the lower O:Zn ratio and higher oxygen vacancies [27]. Conversely, ZnO-M1 and ZnO-M2 might have experienced lower tensile stress due to the higher O:Zn ratio, leading to higher d-spacing values. This result was consistent with the results from EDS analysis which revealed a lower O:Zn ratio for ZnO-N1 but higher O:Zn ratio for both ZnO-M1 and ZnOM2 samples. Fig. 3b shows the optical absorption of ZnO samples. The optical bandgaps were calculated from the equation: ðahvÞ2 ¼ Aðhv Eg Þ
(3)
where a is the absorption coefficient, h is the Planck’s constant, v is the frequency and Eg is the bandgap. All of the ZnO samples exhibited strong absorption in the ultraviolet region. The sharp edges of UV absorption spectra revealed good crystallinity of ZnO powder samples. The optical bandgaps of nano-sized ZnO-N1 (Eg = 3.074 eV) and ZnO-N2 (Eg = 3.109 eV) were lower than that of the ZnO bulk (Eg = 3.37 eV). The observed difference in optical bandgaps may originate from the crystals’ defects, especially oxygen vacancies and zinc interstitials [28]. This phenomenon agreed well with the EDS results shown in Fig. 2 wherein ZnON1 and ZnO-N2 exhibited a lower content of oxygen atoms, possibly correlating to the oxygen vacancies or zinc interstitial formation. On the other hand, the optical bandgaps for the microsized ZnO-M1 (Eg = 3.237 eV) and ZnO-M2 (Eg = 3.223 eV) were larger than those of ZnO-N1 and ZnO-N2. There are some rodstructures in both ZnO-M1 and ZnO-M2 samples, giving rise to a possible formation of oxygen interstitial and zinc vacancies [25]. The EDS analyses (Fig. 2) also revealed consistent results where both samples contained higher atomic percentages of oxygen atoms compared to zinc atoms. However, the slightly smaller magnitude of Eg of ZnO-M1 and ZnO-M2 samples compared to ZnO bulk (Eg = 3.37 eV) revealed the decreasing optical bandgap, owing to the oxygen interstitial and zinc vacancies suppressing the interband excitonic recombination [29]. Remarkably, the particle size and morphology are two key factors in altering the elemental contents in the crystalline structure as well as in affecting the optical property of the ZnO samples. On the other hand, the surface charges of ZnO particles in the suspension were examined through zeta potential measurement, as listed in Table 1. The positive signs of the potentials indicated a tendency of obtaining positively charged on the ZnO particle surfaces. Among the ZnO samples, ZnO-N1 exhibited the lowest zeta potential (19.2 mV), revealing the least amount of positively charges on the particles. This phenomenon was explained by the high level of zinc ion (Zn2+) release in the suspension, causing less zinc on ZnO particles. ZnO-N1 was regarded as less stable particles in the suspension form owing to highly dissolution of zinc ion and low magnitude of zeta potential. Meanwhile, ZnON2 possessed zeta potential of 28.1 mV, indicating higher surface positively charges and lower zinc ion dissolution. Micro-sized ZnO-M1 and ZnO-M2 showed high zeta potentials, which are 30.3 and 31.5 mV, respectively. The particles of ZnO-M1 and ZnOM2 had high stability in the suspension, where many positively charged zinc ions localized on the ZnO particles. Consequently,
Cytotoxicity tests The implementation of in vitro assays to measure cellular viability is very important for gaining insight into the potential toxicity of nanomaterials. We evaluated the toxicity of ZnO samples through microscopy imaging and Neubauer viable cells counting. Fig. 4 shows the optical microscopy of L929 cell lines being treated with 0.1 and 0.5 mM ZnO-N1 and ZnO-N2. The images revealed the different toxicity responses of the ZnO samples on the cell lines after 24 h, 48 h and 72 h of treatment. Fig. 4a shows the negative control, used to compare the cell growth of treated and untreated cell lines. Besides, positive control was shown in Fig. 5a through the calamine lotion treatment with the cell lines. The high concentration of ZnO in the calamine lotion (about 20%) could induce 100% fatality towards the L929 cell lines. The toxicity impacts of ZnO samples are concentration dependent. Use of 0.5 mM ZnO-N1 and ZnO-N2 gave rise to increased cell death compared with 0.1 mM samples. Fig. 4b and d shows that the majority of the cell lines remained healthy and proliferated (about 70%) after being treated with 0.1 mM ZnO-N1 and ZnO-N2 samples. However, 0.5 mM ZnO-N1 and ZnO-N2 samples induced an acute lethal effect on the cells whereby many cells died after treatment, as indicated by the red arrows in Fig. 4. From the microscopic observation, the dead cells appeared to be “dull” and had a round shape. Only about 5% of cells were observed to proliferate in ZnON1 treatment after 24 h, 48 h and 72 h incubations (Fig. 4c). Conversely, only a small number of cells (about 20%) could proliferate in the 0.5 mM ZnO-N2 treatment after 24 h incubation (Fig. 4e). The cell proliferation in 0.5 mM ZnO-N2 treatment increased to about 40% and 60% after 48 and 72 h incubations, respectively. The amount of dead cells seen after treatment with ZnO-N1 was found to be greater than for the ZnO-N2 treatment, showing that ZnO-N1 had a greater toxicity. Fig. 5b–e shows the optical microscope images of L929 cell lines after treatment with ZnO-M1 and ZnO-M2 samples. Both of the ZnO samples exhibited similar effects on the cells, where 0.1 mM ZnO-M1 and ZnO-M2 had little toxicity towards the cells while 0.5 mM ZnO caused some damage to the cells. Low ZnO concentrations led to increased proliferation of the cell lines after 48 and 72 h incubation, as can be seen in Fig. 5b and d. Meanwhile, some of the cells’ proliferative activities deteriorated when treated with 0.5 mM ZnO, as indicated by the red arrows in Fig. 5c and e. This observation suggested that some cell lines (about 30%) could have succumbed to the toxicity of 0.5 mM ZnO-M1 and ZnO-M2, giving rise to the fatality of the cell lines. Assessing the toxicity of ZnO samples is of utmost important. The use of in vitro assays to test cellular viability was performed to evaluate the potential toxicity of the ZnO samples. Cell counting was conducted using a Neubauer chamber and the percentage of viable cells was calculated based on a negative control, using the equation: viable cells=control Þ 100% Percentage viable cells ¼ ð control
(4)
According to the criteria of the Nanotechnology Characterisation Laboratory in Frederick, USA [30], a nanoparticle is considered to be non-toxic if the cell viability at 48 h is higher than 75%. In this study, we determined the non-toxic level to be above 70% after 72 h, considering the longer contact time of the cells during ZnO treatment. Fig. 6 shows the percentage of viable cells after being treated with different concentrations (0.1–0.5 mM) of ZnO samples. Different toxicity levels were noted from the cell viability results,
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Fig. 4. Light microscopic images of L929 cell lines under treatment of: (a) negative control; (b) 0.1 mM ZnO-N1; (c) 0.5 mM ZnO-N1; (d) 0.1 mM ZnO-N2 and (e) 0.5 mM ZnON2.
for cell lines treated with the ZnO samples. The toxicities of all of the ZnO samples were concentration dependent. The percentage of viable cells decreased when the concentration of ZnO samples increased. Among the ZnO samples, ZnO-N1 exhibited the highest toxicity towards the L929 mouse cell lines. Concentrations of 0.1 and 0.2 mM ZnO-N1 led to percentage cell viabilities of 52% and 23%, which are lower than the non-toxic level of 70%. Moreover,
concentrations of 0.4 and 0.5 mM caused the fatality of all of the cells. This result was consistent with the images obtained from light microscopy (Fig. 4c) where most of the cell lines were destroyed when treated with 0.5 mM ZnO-N1. Therefore, ZnON1 had induced an acute lethal effect on the cells at concentrations above 0.1 mM. In contrast, ZnO-N2 exerted a lower toxicity on the cells and gave rise to greater amounts of viable cells compared to
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Fig. 5. Light microscopic images of L929 cell lines under treatment of: (a) positive control (b) 0.1 mM ZnO-M1; (c) 0.5 mM ZnO-M1; (d) 0.1 mM ZnO-M2 and (e) 0.5 mM ZnOM2.
ZnO-N1. The percentage of viable cells was above the non-toxic level (70%) at ZnO-N2 concentrations of 0.1 mM. The concentration of ZnO-N2 that initiated the killing effect on the cells was 0.2 mM, and the cell viability was obviously greater than that of ZnO-N1. The percentage of viable cells reduced to 40 % after treatment with 0.5 mM ZnO-N2. Furthermore, light microscope imaging (Fig. 4d and e) revealed that the majority of cell lines could grow robustly under 0.1 mM ZnO-N2 treatment whereas some cell lines were killed when treated with 0.5 mM ZnO-N2.
On the other hand, ZnO-M1 and ZnO-M2 samples were less toxic and the resultant cell viabilities were higher than those of the ZnO-N1 and ZnO-N2 samples. Both ZnO-M1 and ZnO-M2 samples only exhibited lethal effects at concentrations of 0.4 mM, which was lower than non-toxic level (70%). After 0.5 mM ZnO treatment, cell viabilities reduced to 54% (ZnO-M1) and 53% (ZnO-M2). This result suggested that the threshold concentration of ZnO-M1 and ZnO-M2 samples to be used for non-toxic application was 0.3 mM at most. This quantitative result was supported by the previous
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Fig. 6. Percentage of viable cells after 72 h treatment with different concentration of ZnO samples.
microscope images (Fig. 5) wherein no dead cells were identified in 0.1 mM ZnO samples but some unhealthy/dead cells were found in the 0.5 mM ZnO samples. Toxicity mechanisms of ZnO To date, the health risks and potential hazards of both micro- and nano-sized ZnO particles have not been assessed and are somewhat concerning; they have become a subject of discussion [31–33]. However, there is no consistent conclusion about the toxicity of ZnO and the impact of particle size and morphology on ZnO toxicity. Several studies have been performed to assess the toxicities of the nanoparticles using different cellular systems and test methods [34– 36]. Some researchers found that different sizes of ZnO nanoparticles exhibited comparable cytotoxicity on different cells, while the IC50 after 24 h was about 10–20 mg/ml [37,38]. Conversely, Lee et al. reported that rod-shaped ZnO particles could trigger greater toxicity than spherical ZnO particles [39]. In this study, the morphology and size of ZnO particles were discovered to be the key factors causing different toxicity effects. Because the ZnO in the media culture was filtered before being mixed with cell lines, direct contact between the ZnO particles and the cells was avoided. Therefore, two chemical mechanisms involving free radicals generated by ZnO particles were proposed. Firstly, release of Zn2+ ion from ZnO could be an important contributor to toxicity. Typically, the amount of zinc ions in the suspension is affected by the solubility of ZnO. The dissolution of Zn ions depends on the dosage of ZnO used in the distilled water, whereby the use of 0.5 mM ZnO samples would induce the highest toxicity by releasing increased levels of Zn ions compared with lower concentrations of ZnO. Deng et al. [40] observed that ZnO nanoparticles and ZnCl2 had a comparable toxic effect on mouse NSCs, indicating that a similar quantity of Zn2+ was released from both solutions. Further, Fukui et al. [41] suggested that an increase in intracellular Zn2+ induced higher intracellular ROS levels and oxidative stress in rat lung cells. The cell viability and lactate dehydrogenase (LDH) level depended on the level of zinc ions in the suspension. Furthermore, smaller ZnO particle sizes would result in greater toxicity compared to larger particles sizes. Nanosized ZnO-N1 and ZnO-N2 samples possessed surface-to-volume ratios and so could potentially release more Zn2+ into the suspensions, triggering greater levels of toxicity towards cells. Zeta potential (Table 1) showed the consistent result wherein ZnON1 probably exhibited the highest dissolution of zinc ions due to the smallest positive value of zeta potential. Secondly, based on the potential redox properties present in biological systems, as described by Ai et al. [42], it was also possible
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that chronological oxidation–reduction processes may have occurred at ZnO particle surfaces to generate reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH). Even when small amounts of ZnO particles are involved in the interactions, large amounts of ROS would be released. A concentration of ZnO particles greater than 5 mg/ml could increase the intracellular ROS levels [38]. The production of ROS from ZnO samples likely caused disruption of cellular function or disorganization of membranes. Furthermore, different morphologies of ZnO particles were believed to release different amounts of ROS, as shown in our previous works [25,43], which suggested that rod-structures possess greater amounts of oxygen atoms on the particles’ surfaces and generated relatively high quantities of ROS. The toxicity effect of ZnO-M1 was possibly due to the presence of rod-structures, which could produce more ROS in the suspension. However, the effect of particle size seemed to be more dominant than the particle morphology, as can be seen in the cell viability analysis, shown in Fig. 6. Due to larger rod-shaped particles (40–100 nm) in ZnO-M1 compared to the sphericalshaped particles (20 nm) present in ZnO-N1, nano-sized ZnON1 resulted in greater toxicity compared to micro-sized ZnO-M1. On the other hand, rod-shaped structures in ZnO-M1 and plateshaped structures in ZnO-M2 were comparable in terms of toxicity effects, probably due to the presence of multiple particle sizes. Conclusion ZnO particles with different particle sizes and morphological features were used to investigate the in vitro cytotoxicity towards L929 mouse fibroblast cell lines. Micro-sized ZnO-M1 and ZnOM2 samples exhibited lower toxicity compared to the nano-sized ZnO-N1 and ZnO-N2 samples. The limit of toxicity for ZnO-M1 and ZnO-M2 samples was at most 0.3 mM, while for ZnO-N2 was 0.1 mM at most. The release of zinc ions and the generation of ROS from the particles were suggested to be potential mechanisms of toxicity towards the cell lines. Acknowledgements The authors thank the financial support from Universiti Sains Malaysia through Research University (Individual) grant (1001/ PFIZIK/814174) and supporting grant RU-PRGS (1001/PFIZIK/ 846077). Assistance from NOR laboratory and AMDI laboratory is also acknowledged. The authors declare no conflict of interest related to this study. References [1] P.J. Borm, W. Kreyling, Toxicological hazards of inhaled nanoparticles – potential implications for drug delivery, J. Nanosci. Nanotechnol. 4 (5) (2004) 521–531, doi:http://dx.doi.org/10.1166/jnn.2004.081. 15503438. [2] N. Iqbal, M.R.A. Kadir, N.H. Mahmood, N. Salim, G.R.A. Froemming, H.R. Balaji, T. Kamarul, Characterization, antibacterial and in vitro compatibility of zinc– silver doped hydroxyapatite nanoparticles prepared through microwave synthesis, Ceram. Int. 40 (3) (2014) 4507–4513, doi:http://dx.doi.org/10.1016/j. ceramint.2013.08.125. [3] S.M. Hussain, L.K. Braydich-Stolle, A.M. Schrand, R.C. Murdock, K.O. Yu, D.M. Mattie, J.J. Schlager, M. Terrones, Toxicity evaluation for safe use of nanomaterials: recent achievements and technical challenges, Adv. Mater. 21 (16) (2009) 1549–1559, doi:http://dx.doi.org/10.1002/adma.200801395. [4] G. Ramakrishna, H.N. Ghosh, Effect of particle size on the reactivity of quantum size ZnO nanoparticles and charge-transfer dynamics with adsorbed catechols, Langmuir 19 (7) (2003) 3006–3012, doi:http://dx.doi.org/10.1021/ la020828u. [5] S.Y. Bae, H.W. Seo, J. Park, Vertically aligned sulfur-doped ZnO nanowires synthesized via chemical vapor deposition, J. Phys. Chem. B 108 (17) (2004) 5206–5210, doi:http://dx.doi.org/10.1021/jp036720k. [6] E. Comini, G. Faglia, G. Sberveglieri, Z. Pan, Z.L. Wang, Stable and highly sensitive gas sensors based on semiconducting oxide nanobelts, Appl. Phys. Lett. 81 (10) (2002) 1869–1871, doi:http://dx.doi.org/10.1063/1.1504867.
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