Effects of the Absorption Behaviour of ZnO Nanoparticles on Cytotoxicity Measurements

1 Tissue Culture Research Laboratory, Centre of Synthesis and Chemical Biology, Institute of Science, Universiti Teknologi MARA, 40450 Shah Alam, Malaysia 2 Department of Pharmacognosy and Pharmaceutical Chemistry, School of Pharmacy, Faculty of Medicine, University of Sulaimani, Iraq 3 Nanomaterials Research Laboratory, Centre for Nanomaterials Research, Institute of Science, Universiti Teknologi MARA, 40450 Shah Alam, Malaysia 4Centre of Synthesis and Chemical Biology, Institute of Science, Universiti Teknologi MARA, 40450 Shah Alam, Malaysia


Introduction
The industrial use of nanoparticles in a wide variety of applications has been rapidly expanding in the last decade [1].Such applications include the use of zinc, titanium, magnesium, and other metallic oxide nanoparticles, thereby increasing the occupational and other environmental exposure of these nanoparticles to humans and other species [2].
Zinc oxide (ZnO) has properties such as wide band gap (3.37 eV), high exciton binding energy (60 meV), and a variety of morphologies [3].ZnO nanoparticles have unique properties including small size and correspondingly large-specific surface area to volume ratio.ZnO has been increasingly employed in a variety of industrial applications including production of wave filters, UV detectors, catalysts, paint, transparent conductive film, cosmetics, gas sensor and catalytic processes, solar cells and microelectronics [4][5][6][7][8][9][10][11][12], personal care products (toothpaste, beauty products, and sunscreens [13]), and textiles [14].Increased applications in industry will increase chances of exposure of the nanoparticles to humans.Thus, zinc oxide nanoparticles are the subject of much research because of their high probability exposure to human and environment [1].
In this study, we evaluated the possible contribution of ZnO nanoparticles absorption in the readings of cytotoxicity measurements.We also demonstrated cytotoxicity of two different sizes of spherically shaped ZnO nanoparticles in normal and cancer cell lines derived from different histological origin.Finally, we evaluated that the cytotoxicity of ZnO was particle size, concentration, and time dependent.

ZnO Nanoparticles.
The ZnO nanoparticles were synthesized using a method detailed elsewhere [30].Two groups of ZnO nanoparticles with the average particle size of around 85.7 nm and 190 nm named Z1 and Z2 were used, respectively.The ZnO nanoparticle size of one sample is twice that of the other sample.The absorption of the ZnO nanoparticles was measured using Perkin Elmer Lambda 750 UV-Vis-NIR spectrophotometer with a range of about 330 to 700 nm.The reflectance mode was used for the measurements.
The micrographs from the field emission transmission electron microscope (FETEM) are obtained from the JEOL JEM2100F.The microstructures can clearly be seen from the phase contrast image.
The stock solutions of ZnO nanoparticles (Z1 and Z2) were prepared in phosphate buffer saline (PBS, 0.01 M, Sigma, USA), with 100 mM stock concentration, sonicated for 30 minutes, and stored at 4 ∘ C. The nanoparticles stock concentrations were vigorously vortexed and then diluted with complete medium prior each experiment, resulting in a series of final concentrations ranging from 50 M to 10 mM.Dose response effects of ZnO nanoparticles to cancer and normal cell lines were evaluated at two different exposure times, 24 and 96 h.

Cell Lines and Culture Conditions.
Human medulloblastoma SH-SY5Y cell line was a gift from Dr. Carol Sanfeliu (Department of Pharmacology and Toxicology, Institute of Biological Research, Barcelona, Spain).Lymphoma U937 and lung cancer A549 cell lines were a gift from Dr. Mohamed Saifulaman (Faculty of Applied Sciences, UiTM, Malaysia).Normal lung Hs888Lu cell lines were purchased from American Type Culture Collection (ATCC, The Global Bioresource Centre, Manassas, USA).

Assay for Cytotoxic Activity.
Cells (1 × 10 5 cells/mL) were seeded in 96-well plates and left to grow overnight in humidified atmosphere containing 5% CO 2 at 37 ∘ C. On the following day, cells were treated with serial dilution of ZnO concentrations ranging from 50 M to 10 mM.After 24 h and 96 h cell viability was measured by CellTiter 96 AQ ueous Assay which uses the novel tetrazolium compound (3-(4,5-dimethyl-thiazol2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS, Promega, USA) using Glomax multidetection system (Promaga, USA) and read at 490 nm [31].Results were representative of at least three independent experiments and were expressed as percentage of the value observed with a control which contained no ZnO.

Assay for Neurotoxicity.
Retinoic acid (RA, Sigma, USA) will induce the differentiation of the neuroblastoma cells (SH-SY5Y) to behave like neuron-phenotypic cells [32,33].Approximately, 1 × 10 4 cells/mL were seeded in 96-well plates.After 24 h, RA was added at a final concentration of 10 M in complete EMEM-F12 media.The medium in the plate was changed at day 3 with fresh RA and cultures were ready to be tested on day 6.ZnO nanoparticles (both groups) were tested for their neurotoxicity effect.The nanoparticles serial dilutions in EMEM-F12 were made fresh prior to each test.For neurotoxicity, the differentiated SH-SY5Y cells in each well were tested with final concentrations of ZnO nanoparticles ranging from 50 M to 10 mM.The wells were agitated lightly and incubated for 24 h or 96 h.Following treatments, cell viability was assessed with Cell Titer 96 * Aqueous Non-Radioactive Cell Proliferation Assay (MTS, Promega, USA).The MTS assay is a colorimetric assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)2-(4 sulfophenyl)-2H-tetrazolium (MTS) by dehydrogenase enzymes found in metabolically active cells.The amount of colour formazan product is proportional to the number of viable cells.Briefly, 20 L MTS solution was added to each well and incubated in humidified incubator at 37 ∘ C in 5% CO 2 for 2-4 h in the dark.The quantity of formazan product present was determined by measuring the absorbance at 490 nm with a microtiter plate reader (GloMax Integrated Systems by Promega, USA).Values were expressed as the percentage of optical density of control cells (nontreated).

Interference of ZnO Nanoparticles with the Cell Viability
Measurements.ZnO absorption and its influence on the reading of the cytotoxicity measurements were studied.A nanoparticles-free assay was developed to investigate the direct interference of ZnO nanoparticles (Z1 or Z2) with cell viability assay.Cells (1 × 10 5 cells/mL) were seeded in 96-well plates and left to grow overnight.The following day, cells were treated with serial dilution of ZnO concentrations ranging from 50 M to 10 mM.After 24 or 96 h the supernatant containing excess of ZnO nanoparticles that did not attach to the cells or culture plates was disposed and gently the cells were washed with PBS twice.100 L of fresh media was then added to each well followed by 20 L MTS reagent.Thereafter, the absorbance for cell with ZnO and cell-free ZnO system was determined by reading at 490 nm wavelength of the MTS assay.Nontreated cultures were used as controls.

Interference of MTS Reagents with the Cell Viability
Measurements.Cells (1 × 10 5 cells/mL) were seeded in 96well plates and left to grow overnight.On the following day, cells were treated with serial dilution of ZnO concentrations ranging from 50 M to 10 mM.After 24 or 96 h the interference of MTS reagent with the cell viability assay was assessed by measuring the absorbance of treated culture in the absence of MTS reagent.The absorbance with and without MTS was measured at 490 nm.

Cell Morphology.
Interference of ZnO nanoparticles was also evident by morphological changes that appeared in cell lines.Prior to strong changes in metabolism or proliferation, cells often change their shape in response to toxic reagents.The changes in the morphology of cell lines treated with various ZnO nanoparticles concentrations for 24 and 96 hours were compared to that of corresponding cell-free ZnO and untreated cells, by confocal microscopy.Bright field images of cells were acquired with 40× objective using a Leica confocal microscope (Leica DMI 4000B, Wetzlar, Germany) equipped with a digital camera.

Statistical Analysis.
GraphPad PRISM version 5.0 program was used to create the graphs.All determinations were performed at least in triplicate.Means and standard deviations were determined.All comparisons were made using two-tailed Student's t-test, ( *  < 0.05; * *  < 0.01; * * *  < 0.001) using GraphPad PRISM version 5.0. of the ZnO samples is spherical in shape as can be seen in the scanning electron microscopy (SEM) results in Figure 2. From the micrographs, it is obvious that the particle size of the ZnO has an average size of 85.7 nm (sample Z1) and 190 nm (sample Z2).

Results and Discussion
The microstructure of the nano-ZnO can be clearly seen from the TEM images showing spherical geometries in Figure 3.The size is as estimated earlier using SEM.This is because SEM can give a wider field of view enabling measurements of size on more number of particles giving a statistically better accuracy for average size.

UV-Vis of ZnO.
The reflectance measurement of ZnO is shown in Figure 4.It can be seen that the absorption edge of the materials is about 390 nm.The measurement for the toxicity studies is at 490 nm.Even though the absorption edge is at 390 nm, there is still about 1.3% of absorption of light at 490 nm.This absorption at 490 nm will affect the toxicity measurements made in the experiments, especially when the density of ZnO in the samples is high.

Studies of the Effects and Interference of Nanoparticles
with Cytotoxicity Data.Absorbance reading at 490 nm shows an increase in cell viability compared to the control (nontreated) after treatment with ZnO nanoparticles above 1 mM concentration (Figures 8-10), which is not logical.Thus, questions arise as to the effect of the ZnO nanoparticles themselves and/or MTS reagent on the measurements for the absorbance at 490 nm.Therefore, a nanoparticles-free assay was developed to investigate the direct interference of ZnO nanoparticles with absorbance measurements and then with cell viability assay.The results show a significant decrease (P's < 0.01) in the relative percentage absorbance after removing ZnO nanoparticles from the culture system compared with the system in the presence of ZnO nanoparticles at the concentrations of more than 1 mM (Figure 5).The cells treated with ZnO nanoparticles concentrations equal or less than 1 mM do not show significant change under the ZnO  2009) addressed the cytotoxicity effects of high concentration ranges of ZnO nanoparticles between 1.5-10 mM in glioma cell lines and normal human astrocytes [41], but the interference of ZnO nanoparticles with the absorbance measurement at 490 nm was not investigated or reported; it should be noted that they examined cytotoxicity of ZnO nanoparticles using lactate dehydrogenase (LDH) release.Our findings showed an interference of ZnO nanoparticles with absorbance readings at 490 nm at concentrations above 1 mM (Figure 5) and these results are novel.

Studies of the Effects and Interference of MTS Reagent
with Cytotoxicity Data.The interference of MTS reagent with the cell viability assay was also assessed by measuring the absorbance of treated cell lines in the absence of MTS reagent.Our novel findings with regard to MTS interference demonstrated the dramatic increase (P's < 0.01) in absorbance reading at 490 nm in the absence of MTS reagents at concentrations above 1 mM compared to the control cells (no ZnO).No increase in absorbance measurements was observed below 1 mM ZnO nanoparticles (Figure 6).
This observation is suggesting that MTS reagent is not involved and the nanoparticles themselves may be interfering in the increase in absorbance reading.The results demonstrated that after the elimination of ZnO nanoparticles from the culture condition, the percentages of relative absorbance significantly decrease at the concentrations above 1 mM.This has not been reported previously.

Cell Morphology.
Interference of ZnO nanoparticles was also evident by morphological changes that appeared in cell lines.The current study therefore routinely included bright field microscopic analysis of cell cultures.After 96 h incubation with Z1 nanoparticles at 1 mM concentration, SH-SY5Y cells have undergone morphological changes into spherical shape, gained in volume, and formed clusters in media after detachment from cell culture plate (Figure 7(c)).Cellular shrinkage and detachment from the surface of the plate as well as increase in cell death at doses 1 mM and 5 mM were also observed in normal lung cells and neuron-like cell (not shown).In contrast, lung cancer (A549) cell lines treated with 1 mM of ZnO nanoparticles (Z1) and incubated for 96 h exhibited no visible morphological changes (Figure 7(d)).
The numbers of dead cells increase with increasing nanoparticles concentration to 5 mM in both SH-SY5Y and A549 cells (Figures 7(e) and 7(f)).While a significant increase in absorbance reading at wavelength of 490 nm was observed at selected concentration (Figures 8(a) and 9(a)).
Figures 7(g) and 7(h) show the dramatic decrease in the cell number after removing ZnO nanoparticles and dead cells from the culture system compared with the system in the presence of ZnO particles at the concentration of 1 mM (Figures 7(c) and 7(d)).The absorbance readings dropped dramatically in the sample where ZnO nanoparticles have been removed (Figure 5).
In this study we found out that no effects are observed for lung cancer (A549) cell lines exposed to 1 mM of ZnO nanoparticles, Z1 and Z2 (85.7 nm group and 190 nm group, resp.), and incubated for 24 or 96 h.In contrast, other studies have described that the cell viability in A549 cell lines was reduced by 75-80% between 18 and 25 g/mL (0.2 and 0.3 mM) with ZnO particles (70 and 420 nm) between 6 and 12 h [23].The more pronounced toxicity was observed at 24 h [23].Recently, Kim and coworkers (2010) reported that ZnO nanoparticles exhibited the cytotoxicity in terms of cell proliferation, cell viability, and membrane integrity in A549 cells [22].
We have shown that Z1 nanoparticles did induce approximately 50-70% decrease in cell survival in normal lung cells Hs888Lu at 1 mM after 24 and 96 h, respectively.ZnO nanoparticles activity has not been reported previously in such cells by any research group.While, Kim and coworkers (2010) demonstrated the cytotoxic effect of ZnO nanoparticles in another normal lung cell types (L-132).
Cell lines are treated with ZnO nanoparticles concentrations ranging from 50 M to 10 mM of average particle sizes 85.7 nm (Z1) and 190 nm (Z2) and incubated for 24 or 96 hours.Our data indicated that exposure to either size of ZnO nanoparticles samples Z1 and Z2 induced different toxic effects in the human cell lines tested in this study (Figures 8-10).ZnO nanoparticles do not show toxicity in lung cancer cells A549, and they almost maintain 90-100% cell viability up to 1 mM concentration after exposure to either size of ZnO nanoparticles for 24 or 96 h (Figure 8(a)).
We further examined the cytotoxic effect of the ZnO nanoparticles in normal lung cells Hs888Lu.Cells were exposed to both particle sizes Z1 and Z2 for 24 and 96 h (Figure 8(b)).It seems that the difference in particle sizes behaves differently in normal lung tissue.Z1 particles induced almost 50% decrease in cell viability after 24 h incubation at 0.8 mM and 1 mM concentrations (P's < 0.01).Some cytotoxic effect was already observed after 96 h treatment with the same particles at 0.2 mM (data not shown), but the most pronounced effect was observed between 0.8 mM to 1 mM concentrations with 70% (P's < 0.01) viability loss (Figure 8(b)).Z2 particles were less potent in Hs888Lu cell lines, reducing viability by 20% after 96 h treatment at 0.8 mM and 1 mM concentrations (P's < 0.05), while no effects were observed after 24 h incubation.
The cytotoxic responses of different cell lines to ZnO nanoparticles were found to be different.To assess if ZnO nanoparticles may exert cytotoxicity on human neuron cells similar to those of cancer and normal lung cells, we also studied the cytotoxicity and neurotoxicity effects of ZnO nanoparticles on neuroblastoma (SH-SY5Y) and neuron-like cells (differentiated SH-SY5Y).Exposure of SH-SY5Y and differentiated SH-SY5Y cells to ZnO nanoparticles Z2 for 24 h at 1 mM concentration induces a decrease in cell survival to 60% and 80% (P's < 0.01), respectively (Figure 9).The longer incubation for 96 h significantly decreases the cell survival to less than 50% in both cell lines at 0.8−1 mM concentrations (P's < 0.01).
It seems that smaller particles average size of 85.7 nm (Z1) has exhibited more cytotoxic activity in both cell lines, where potent cytotoxic effect had been observed at a low concentration of 0.4 mM with Z1 nanoparticles.Some effects of Z2 were observed at 0.8 mM in both SH-SY5Y (P's < 0.01) and differentiated SH-SY5Y (P's < 0.05) cell lines after 96 h.Exposure of these cells to Z1 particles for 96 h at concentrations ranging between 0.4 mM and 1 mM induced a decrease (P's < 0.01) in cell survival to almost 30-40% (Figure 9).Chen and colleagues reported that ZnO and iron oxide (Fe 2 O 3 ) nanoparticles do not induce significant decrease of cell viability in SH-SY5Y cell lines at a concentration range of 0.01-100 M for 48 h [20].This is in agreement with our finding for SH-SY5Y and differentiated SH-SY5Y, where no effects were observed after exposure to ZnO nanoparticles at concentration 100 M for 24 and 96 h (data not shown).From our literature review no studies were reported on nanoparticles cytotoxicity in neuron-like (differentiated SH-SY5Y) cell lines.
Another human cell line from different histological origin, lymphoma (U937), was tested for its sensitivity to ZnO nanoparticles.The lymphoma cell lines treated with ZnO nanoparticles were under the same conditions as described above for the other cell lines.U937 cell lines almost maintain 95-100% cell viability up to 1 mM concentration after exposure to ZnO nanoparticles Z1 and Z2 for 24 h (Figure 10).While the longer incubation for 96 h significantly decreases the cell survival to 40-50% with either size of ZnO nanoparticles at concentrations ranging between 0.8 and 1 mM (P's < 0.01).Cytotoxicity of ZnO nanoparticles of either size did not show significant differences from each other in the U937 cell lines at concentrations ranging between 0.8 and 1 mM.From our literature review no studies were reported on nanoparticles cytotoxicity in lymphoma (U937) cell lines.into the cells will be facilitated and thus the detrimental effect on them more pronounced.The duration of exposure is one of the important parameters in any in vitro cytotoxicity assay.Thus, longer exposure periods may be necessary for screening the effects of metal oxide nanoparticles.The results in Figures 8-10 prove that the longer the incubation time with ZnO nanoparticles is (either group) the more harmful the effect to the cell lines is.This is in agreement with previous studies that revealed a time dependent cytotoxicity of A549 [23], human epidermal (A431) [21], human mesothelioma MSTO-211H, and rodent 3T3 fibroblast cells [42] on exposure to ZnO nanoparticles.Similar cytotoxic effects were obtained in the glioma cell lines (LN18, LN229) and normal human astrocytes, treated with 10 mM ZnO nanoparticles for 24 and 72 h [41].

Conclusion
The data from the present study demonstrate that the presence of ZnO at high concentrations affected the cytotoxicity measurements due to the absorption characteristic of ZnO nanoparticles.The data revealed that the ZnO nanoparticles with an average particle size of around 85.7 nm and 190 nm induced cytotoxicity towards U937, SH-SY5Y, differentiated SH-SY5Y, and Hs888Lu cell lines.The ZnO nanoparticles were found inactive in lung cancer cell line (A549).Moreover, our data have also indicated that the cytotoxicity of ZnO was particle size, concentrations, and time dependent.Hence, the results of this study demonstrated that a ZnO nanoparticle at concentrations above 1 mM has a profound influence on the cytotoxic effects of nanoparticles in different cell lines.Further studies will attempt to investigate the underlying mechanisms of this phenomenon.

Figure 7 :
Figure 7: Morphological changes of A549 and SH-SY5Y cell lines after 96 h incubation in Z1 (85.7 nm) nanoparticles: (a) and (b), control cells in regular cell culture (no nanoparticles); (c) and (d), cell lines treated with 1 mM of Z1 nanoparticles; (e) and (f), cell lines treated with 5 mM of Z1; (g) and (h), cell lines treated with 5 mM of Z1 followed by disposing ZnO.
ZnO nanoparticles.This is in agreement with the study demonstrated byLin and coworkers (2009)stating that cell viability was not particle size dependent at all particle diameters (70 and 420 nm) in A549 cell lines at 24 h incubation.The size dependent results of our work can be explained by the fact that for small particle size ZnO, diffusion Figure 10: The response of lymphoma cells (U937) exposed to ZnO nanoparticles Z1 (85.7 nm) and Z2 (190 nm) for 24 or 96 h.Control represents untreated cell lines.Values are means ± SD ( = 3) and all comparisons were made using two-tailed Student's t-test ( *  < 0.05; * *  < 0.01; * * *  < 0.001).