Toxicology in Vitro 28 (2014) 1349–1358
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells Sß ükran Sß eker a,b, A. Eser Elçin a,b, Tug˘rul Yumak b, Ali Sınag˘ b, Y. Murat Elçin a,b,⇑ a b
Ankara University Stem Cell Institute, Ankara 06100, Turkey Ankara University Faculty of Science, Ankara 06100, Turkey
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i n f o
Article history: Received 31 January 2014 Accepted 25 June 2014 Available online 9 July 2014 Keywords: Nanoparticles Toxicity Zinc oxide Impedance measurement Periodontal ligament fibroblasts Dermal fibroblasts
a b s t r a c t The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7–8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun–Emmet–Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 lg/mL for 6, 24 and 48 h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of 50–100 lg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The unique size-dependent properties of nanomaterials make them very attractive for potential applications within the biomedical, commercial and environmental sectors (Aili and Stevens, 2010; Harishchandra et al., 2010). Due to their superior electrical, thermal, mechanical, and imaging properties, engineered nanomaterials have found applications such as in cell imaging (Zhong et al., 2012), drug delivery (Panyam and Labhasetwar, 2012), cancer therapy (Xiao et al., 2012) and piezoelectric (Seker et al., 2010), Abbreviations: NP, nanoparticle; ZnO, zinc oxide; hPDLF, human periodontal ligament fibroblast; mDF, mouse dermal fibroblast; AFM, atomic force microscopy; BET, Braun–Emmet–Teller; DMEM, Dulbecco’s modified Eagle’s medium; SEM, scanning electron microscopy; TEM, transmission electron microscopy; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; LDH, lactate dehydrogenase; ROS, reactive oxygen species; carboxy-H2DCFDA, 5-(and-6)carboxy-20 ,70 -dichlorodihydrofluorescein diacetate. ⇑ Corresponding author. Address: Tissue Engineering, Biomaterials & Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Ankara University Stem Cell Institute, Ankara 06100, Turkey. Tel.: +90 (312) 212 6720; fax: +90 (312) 223 2395. E-mail address: [email protected] (Y.M. Elçin). http://dx.doi.org/10.1016/j.tiv.2014.06.016 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.
electronic and optical (Lia et al., 2010) sensor systems. As the use of nanomaterials increase worldwide, the risk on human health and environmental nanomaterials increases. It is, therefore, necessary to assess the potential adverse effects of exposure, on human health and the environment. Metal oxide nanoparticles (NPs) have been widely used in nanotechnology-based applications, such as catalysis, sensors, environmental remediation and in consumer products. In particular, zinc oxide (ZnO), gold (Au), platinum (Pt) and titanium dioxide (TiO2) NPs find application in personal care products, especially sunscreens and toothpastes (Fröhlich and Roblegg, 2012), in coatings and paints, due to their UV absorption efficiency and transparency to visible light (Franklin et al., 2007). Despite many advantages in the wide applications of NPs, nanomaterials may cause serious environmental and health problems. Ever increasing use of the nanotechnology products requires their elaborate toxicological evaluations. Many types of NPs have proven to be toxic to human tissue and cell cultures. In particular, the respiratory and intestinal tracts and skin are in direct contact with the environment. Adding to these, NPs can translocate from these routes via different pathways and mechanisms. It is very important to characterize
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NPs throughout their interaction with the biological system and to accurately determine nanoparticle uptake site and concentration (Maurer-Jones et al., 2010). Although studies regarding the environmental toxicity and potential harm on human health have increased during the last course of the decade, there is still a significant lack of knowledge related to the impact of nanomaterials on human health and the environment (Böhmert et al., 2012). Nanotoxicology studies performed directly on model animals or cultured cells provide significant information on the effects it might have on human and other species (Suh et al., 2009). In particular, in vitro methods are generally utilised for investigating NP interactions with the biological systems (Arora et al., 2012; Hillegass et al., 2010). For example, cytotoxic responses such as changes in cell morphology, cell adhesion, cell–cell interactions and cell proliferation can be monitored in real-time by using the electrical impedance-based high-throughput method without using any label (McGuigan and Li, 2014). Toxic properties of nanomaterials depend on a variety of parameters such as surface charge, chemical composition, quantity, resolution, shape and surface area. Besides, impurities subsided during NP production may be involved in material toxicity. Different biological systems may show distinct sensitivities to nanomaterials (Oberdörster et al., 2004). ZnO is one of the most widely used semi-conducting oxide (wide direct band gap of 3.37 eV) in electrical (Gulino and Fragala, 2002), electrochemical (Yumak et al., 2011), magnetic (Norton et al., 2003), and optical (Fujihara et al., 2001) applications. ZnO NPs have received considerable attention for years due to their wide range of possible technological applications such as solar cells (Jiang et al., 2007), photovoltaic devices (Ravirajan et al., 2006), varistors (Pillai et al., 2004) and sensors (Lin et al., 1998; Muti et al., 2010). Various methods (chemical precipitation, microwave technique, solvothermal, sol–gel, hydrothermal and solid state) have been used to synthesize NPs. In this work, hydrothermal method was chosen to synthesize ZnO NPs owing to particular advantages such as, allowing superior compositional and morphological control, and neither requiring calcination nor milling steps. In this study, the physicochemical properties of hydrothermally synthesized ZnO NPs were characterized through zeta potential, particle size distribution via dynamic light scattering (DLS) and Braun–Emmet–Teller (BET) analyses. Biological responses of periodontal ligament fibroblasts (cells of the supportive tissue of the teeth) and dermal fibroblasts (cells of the skin tissue) against ZnO NPs were determined by transmission electron microscopy (TEM). Degree of intracellular reactive oxygen species (ROS) generation, cell impedance, mitochondrial activity and membrane leakage were also measured. Our findings demonstrated that ZnO NPs at 50–100 lg/mL were significantly cytotoxic to primary hPDLF and mDF cell cultures. 2. Materials and methods 2.1. Synthesis and characterization of NPs All chemicals used for NP synthesis were of analytical grade. Triton X-100, zinc acetate and ethanol were purchased from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification. We synthesized ZnO NPs by using the hydrothermal method as previously described (Sinag et al., 2011). Detailed characterization including the XRD analysis of the ZnO NPs is reported elsewhere (Sinag et al., 2011). 2.2. Atomic force microscopy (AFM) AFM was used to investigate further the morphology and size distribution of the nanosized ZnO, coated onto polished gold
surface. Study was performed on an NI–AFM model atomic force microscope (Nanomagnetics Inst., Ankara, Turkey), operating in dynamic mode in air (Seker et al., 2010). A Tap300A1 model cantilever (Budget Sensors, Innovative Solutions, Sofia, Bulgaria) with a resonance frequency of 320 kHz was used. The coated surfaces were scanned with 40 N/m force constant and imaged at a scan area of 2 lm 2 lm. The NP solution was prepared (100 lg/mL) in dichloromethane (Sigma) at 25 °C and dispersed using a Fisher FB15060 model sonicator (Fisher Scientific, Schwerte, Germany) for an hour. For AFM evaluation, thin films on polished gold surfaces were prepared at 3000 rpm by a Primus SB15 model spin coater (Singen, Germany), then the samples were dried in air at room temperature. 2.3. BET analysis The Braun–Emmet–Teller (BET) surface area of the ZnO NPs was determined using a NOVA 2200e volumetric gas adsorption instrument (Quantachrome Instruments, Boynton Beach, FL, USA), according to the procedures described elsewhere (Yagmur et al., 2008). 2.4. hPDLF cell isolation and culture Periodontal ligament fibroblast cells were isolated from periodontal ligament tissue from the root surfaces of premolar teeth, following review board approval and informed consent before extraction (Albandar, 2005; Inanc et al., 2007). Briefly, periodontal ligament fibroblast tissue from the middle third of the tooth roots were scraped with sterile blades, minced and transferred to culture flasks containing DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin, 1% nonessential amino acid stock solution, and 2 mM L-glutamine (all from Sigma). The explants were cultured at 37 °C, 5% CO2 and 95% humidity until the fibroblast cells became confluent. hPDLF cells between passages 8–10 were used in the experiments. 2.5. mDF cell isolation and culture Dermal fibroblast cells were isolated from the tail skin of 8–10 weeks old adult mice according to the standards of international regulations, using the method described elsewhere (Lichti et al., 2008). Briefly, mouse was anesthetized with intraperitoneal injection of avertin and under anesthesia, the tail was disinfected with ethanol 70%. Tails of ten to fifteen animals were cut at the base and the skin was sectioned and removed. After cutting into square pieces along with strong antisepsis and washing, the fragments were transferred to a petri dish (60 15 mm) containing 20 ml of 0.5% trypsin solution and stored overnight at 4 °C to separate the epidermis from the dermis. Then, the dermal explants were cultured and grown in fibroblast medium [DMEM highglucose medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicilin, 100 lg/mL streptomycin and 1% non-essential amino acids (all from Sigma, St. Louis, MO)] at 37 °C, humidified atmosphere with 5% CO2. After 48 h, the non-adherent cells were removed and the adherent cells were cultured. Culture medium was changed twice a week. 2.6. Preparation of ZnO NP dispersion for cell culture The nano sized ZnO was sterilized using UV irradiation (254 nm) for 30 min before use. The stock solution of ZnO NPs was prepared in cell culture medium and dispersed by using a sonicator for 30 min. Then, the stock solution was diluted to different concentrations in cell culture medium completed by 10% fetal
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bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin and 1% non-essential amino acids. 2.7. Determination of particle size distribution Zeta potential and particle size distribution measurements of the ZnO NPs were determined using Malvern’s NanoZetasizer-ZS (Worcestershire, UK). The ZnO NPs at concentration of 100 lg/mL were suspended in cell culture medium. Prior to measurements, the particle suspension was sonicated in an ultrasonic bath (FB15060, Fisher Scientific, Hampton, NH, USA) for 5 min to ensure homogenous dispersion in the medium. Sample–was placed in clean disposable cuvettes, and thirteen sequential measurements were performed. All the measurements were taken at ambient temperature of 25 °C. 2.8. Treatment with nanoparticles For in vitro cytotoxicity studies, the cells were cultured with various concentrations (0.1, 1, 10, 50 and 100 lg/mL) of nanosized ZnO for 6 h, 24 h and 48 h. In brief, cells at 80–85% confluence were trypsinized with 0.25% trypsin–EDTA solution (Sigma) and subsequently seeded onto 96-well plates at a density of 1 104 cells per well. Cells were cultured in a humidified incubator at 37 °C and 5% CO2 atmosphere for 2 days before the assays. Then, the medium was replaced with the NP-dispersed culture medium and the cells were treated with varying concentrations of NPs for 6 h, 24 h and 48 h. In control cultures, the cells were seeded at the same cell density and cultured without the ZnO NPs. At the end of the exposure period, toxicity assays (MTT, LDH, impedance measurements and ROS) were evaluated in control and NP-exposed cells.
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collected by centrifugation at 1100 rpm for 5 min. Samples were prepared for TEM using the method described elsewhere (Elcin et al., 2003). Briefly, after centrifugation, cells were collected, prefixed with 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in a graded alcohol series, embedded in epoxy resin and sectioned using a Leica ultramicrotome (Ultracut UCT, Vienna, Austria). Thin sections were examined under a JEOL 100 CX TEM (Tokyo, Japan) after post-staining with 4% uranyl acetate in methanol and Reynolds lead citrate. 2.12. MTT assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma) was performed to evaluate the effect of ZnO NPs on the mitochondrial activity of fibroblast cells. At the end of the exposure, the cell culture medium was discarded cautiously and the cells were washed with serum-free medium three times to remove excess nanoparticles. 20 lL of fresh MTT solution (5 mg/mL) and 180 lL of DMEM (without serum) was added into each well, and the cells were incubated at 37 °C and 5% CO2 for 4 h. After incubation, the medium was aspirated with care; then 200 ll MTT solvent (0.1 N HCl in anhydrous isopropanol) was added to each well and mixed to dissolve the blue formazan crystals. The 96-well plate was centrifuged for 5 min at 250 g at 25 °C, supernatants were transferred to new 96-well plates to remove remaining NPs. Formation of formazan was measured at 570 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The increasing concentration of formazan product as measured by the amount of absorbance [A] is directly proportional to the number of living cells in culture. The relative cell viability (%) was calculated from [A]sample/[A]control 100, where [A]sample is the absorbance of the test sample and [A]control is the absorbance of the control sample.
2.9. Cell morphology 2.13. Lactate dehydrogenase (LDH) release assay In order to document morphological changes in fibroblast cells in response to NPs, after 48 h of incubation, the phase contrast images of the cells exposed to all concentrations of ZnO NPs were obtained using a Primo Vert digital inverted microscope (Zeiss, Jena, Germany). 2.10. Scanning electron microscopy (SEM) analyses SEM was used to investigate the surface morphology of exposed cells. Briefly, the cells were seeded onto coverslips and allowed for 2 days to cover the surface. The cell cultures were treated with nanosized ZnO (50 lg/mL) for 48 h upon reaching 80% confluency. Following exposure, the samples were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Then, samples were dehydrated in graded ethanol series, mounted on aluminum supports and were sputter-coated with gold, using a Sputter Coater (Desk II, Denton Vacuum, Cherry Hill, NJ, USA) (Emin et al., 2008). A JEOL 100S (Tokyo, Japan) SEM was utilized at a voltage of 20 kV. 2.11. Subcellular localization of nanoparticles TEM was used to visualize the morphological changes and subcellular localization of ZnO NPs within cells. Briefly, the cells were seeded into 6-well tissue culture-treated plates at a density of 0.3 106 cells per well in 5 mL culture medium and allowed to attach for 2 days. Upon reaching 80% confluency, the cell cultures were treated with nanosized ZnO (50 lg/mL) for 48 h. The untreated cells were used as the control. After 48 h exposure to NPs, cells were washed three times with the corresponding medium and removed from the plate through trypsinization and
The activity of cytoplasmic LDH released into the culture medium was determined using an LDH Toxicology Assay kit (Sigma). The positive control was prepared by adding 10 lL of lysis solution (per well) to the control cells at 45 min prior to centrifugation (Chang et al., 2011), and set as 100% LDH leakage. After treatment, cell material from 96-well plates was centrifuged under room temperature for 5 min at 250 g, to remove excess particles. The resulting supernatants were transferred to new 96-well plates to perform the assay. LDH assay mixture was prepared fresh for each experiment. Briefly, equal volumes of LDH assay substrate, LDH assay dye, and 1 LDH assay cofactor were added together. Fifty lL of cell medium and 100 lL of the substrate/dye/cofactor solution were mixed for LDH analysis, light-protected and incubated at room temperature for 30 min. The reaction was terminated by the addition of 15 lL of 1 N HCl to each well. The absorption was measured using the SpectraMax M5 microplate reader at 490 nm. Absorbance at a wavelength of 490 nm was subtracted by the absorbance readings at a wavelength of 690 nm. All samples were read in triplicate. The LDH leakage (% of positive control) is expressed as the percentage of (ODsample ODnegative)/(ODpositive ODnegative) 100, where ODsample is the optical density of cells exposed to NPs, ODpositive is the optical density of the positive control cells, and ODnegative is the optical density of the cell culture medium. 2.14. Detection of intracellular ROS The production of intracellular reactive oxygen species was measured using 5-(and-6)-carboxy-20 ,70 -dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). The positive controls were
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prepared by culturing the normal cells in culture medium containing 200 mM H2O2. The cells were incubated with different NP concentrations for 30 min. At the end of exposure, the cells were washed three times with PBS to remove particles and then incubated with 20 lM carboxy-H2DCFDA (Invitrogen) for 30 min at 37 °C. After the incubation, the fluorescence intensity was immediately determined at 485-nm excitation and 520-nm emission using a microplate reader (SpectraMax M5). As a negative control, cells were treated with normal cell culture medium. The ROS level is expressed as the ratio of (Fsample)/(Fcontrol), where Fsample is the fluorescence intensity of the cells exposed to NPs; Fcontrol is the fluorescence intensity of the control cells. 2.15. Real-time cell impedance measurements Cellular response to ZnO NPs was continuously monitored using the xCELLigence Real-Time Cell Analyzer (RTCA) DP (Roche Applied Science, Penzberg, Germany) that measures electrical impedance of cells grown on microelectrodes placed at the bottom of plate wells. The hPDLF and mDF cells were seeded on electrode-plates (E-plates, Roche Diagnostics, Basel, Switzerland) at a density of 6.25 103 and 12.50 103 cells/well, respectively and cultured inside the incubator at 37 °C and 5% CO2 until reaching the log phase (10 h for hPDLFs and 15 h for mDFs). Then, NP suspensions at different concentrations were added into each well (final concentration of 0.1, 1, 10, 50 and 100 lg/mL). The cells were incubated with the NP suspensions or cell culture medium (negative control). The cell impedances were continuously recorded at 15 min intervals for 90 h. In order to determine possible interference of NPs, the impedance measurement was performed with cell culture medium (no cell) containing ZnO NPs at the highest concentrations used. The half maximal inhibitory concentrations (IC50) were calculated using the xCELLigence Software. 2.16. Statistical analysis Data from all groups were obtained from three independent experiments, except the MTT assay was performed with six independent experiments. Data was expressed as the mean ± SD, and analyzed by using the one-way analysis of variance and post hoc tests. A value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. AFM analyses The topography of the surfaces coated with ZnO NPs was characterized by AFM. The AFM images of the coated surface are
shown in Fig. 1. The results demonstrate that the metal oxide film was uniformly coated onto gold surface and exhibited homogenous distribution of ZnO NPs (Fig. 1A). The cross section measurements made for the entire scan area of multiple samples (n = 3) demonstrated a coating thickness of around 2–8 nm with a rootmean-square (rms) roughness of 7.5. 3.2. BET analyses BET analysis results are shown in Table 1. Surface analysis of the ZnO nanoparticles determined from the BET measurements showed that ZnO NPs had high surface area (208.481 m2/g) for the nitrogen to be adsorbed. 3.3. Zeta potential and particle size distribution using DLS DLS measurement determined the average particle size distribution, zeta (f) potential and polydispersity index (PDI) of the ZnO NP suspensions in cell culture medium. The average hydrodynamic diameter according to NanoZetasizer-ZS measurements is 154.5 ± 6.5 nm (Fig. 2 and Table 1). 3.4. Cell morphology Inverted phase-contrast micrographs of hPDLF and mDF cells, exposed for 48 h duration to NPs and the control cultures are shown in Fig. 3. The cells cultured under standard conditions had evenly spread on culture dishes and showed characteristic morphology. The cells exposed to ZnO NPs gradually lost their characteristic phenotype in a concentration dependent manner. As seen in Fig. 3, dramatic morphology changes occurred at 50 lg/mL NP concentration; cells started to shrink and obtained irregular shapes. Compared with control cells, ZnO-treated cells showed a significant difference in their adhesion to the culture dish at 50 lg/mL concentration. At higher concentration of ZnO NPs, the cells became completely necrotic and detached from the culture plates. The phase contrast images indicated that the damage on cell morphology was dependent on ZnO concentration. 3.5. SEM for cell morphology SEM images provided information on cell morphology after nanoparticle exposure. ZnO NPs seemed to be phagocytosed by the cells. The lumps and bumps recognized on the fibroblast surfaces indicated the possibility of NP penetration into the vesicles of the fibroblasts (Fig. 4). In addition, NPs were strongly adsorbed on the cell surfaces on account of their high specific surface area and high surface energy (Fig. 4).
Fig. 1. Representative three-dimensional and cross-sectional AFM images (A, B) and cross section profiles of ZnO NPs (C).
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Table 1 Characterization of ZnO NPs. Crystalline sizes in XRD (nm) BET results (m2/g) Average diameter in DLS (nm) Zeta potential (mV) Polydispersity index
8 208.481 154.5 ± 6.5 28.2 ± 6.03 0.229 ± 0.0067
Fig. 2. Particle size distribution of the ZnO NPs obtained by DLS.
3.6. TEM for visualization of cellular uptake Fig. 5 represents TEM results obtained after exposure of hPDLFs (5A, 5C) and mDFs (5B, 5D) to ZnO NPs. TEM images of NP-treated cells indicated that nanoparticles were often present within the cytoplasmic vesicles and nucleus (arrows), while localization in organelles was not observed. Fig. 5 also demonstrates that, NPs appear as aggregates within the cells. Compared to the control hPDLFs (5B) and control mDFs (5D), NP-treated cells were considerably rich in cytoplasmic vesicles. Also, the ZnO NP exposure caused significantly distorted nucleus shape. Control cells had normal cell morphology, their vesicles appeared to be uniform and free of any particles. The larger nuclei indicate the beginning of cell division. 3.7. Mitochondrial function The cell viability of hPDLF and mDF cultures was measured by the MTT assay after culturing for 6 h, 24 h and 48 h in the presence of ZnO nanoparticles. Exposure to NPs resulted in a concentrationdependent decrease of mitochondrial activity in cells, as given in Fig. 6. The MTT assay for mDF cells showed that ZnO NPs had no significant effect on the mitochondrial activity at concentrations tested for 6 h, while hPDLFs were affected by the NPs at this time point. For the 24 and 48 h exposure time points, the viability of mDF cells treated with ZnO NPs was lower at concentrations of 50–100 lg/ml. However, ZnO NPs exhibited a significant reduction in hPDLF cell viability at 50–100 lg/mL for 6, 24 and 48 h. The results demonstrated that ZnO NPs caused a significant reduction in mDF and hPDLF viability at 50–100 lg/ml. 3.8. LDH measurements To investigate the impact of ZnO NPs on cell membrane integrity, lactate dehydrogenase leakage was assessed under NP-exposed conditions. The results indicated that exposure to ZnO NPs resulted in a concentration- and time-dependent increase in LDH leakage. In mDF cells, ZnO NPs caused a sharp increase in LDH leakage at 50–100 lg/ml for 6, 24 and 48 h time points. In hPDLF cells, ZnO NPs had significant effect on membrane integrity at the 50–100 lg/mL concentration range (Fig. 7).
Fig. 3. Representative light micrographs demonstrating hPDLF and mDF cultures following exposure to ZnO NPs at different concentrations. Scale bars: 100 lm.
3.9. Intracellular ROS measurements In order to determine the oxidative stress induced by NP exposure, ROS formation in hPDLF and mDF cells was assessed using carboxy-H2DCFDA (Fig. 8). It was found that, the concentration-dependent effect of the NPs on the oxidation of 5-(and-6)carboxy-20 ,70 -dichlorodihydrofluorescein to 5-(and-6)-carboxy-20 , 70 -dichlorofluorescein was minimal. To sum up, NP exposure resulted in oxidative stress, but there was no significant increase of ROS generation in cells in a concentration-dependent manner for the levels in question. 3.10. Electrical impedance measurements The changes in cell index (CI) caused by ZnO NPs are presented in Fig. 9. The CI values of hPDLF cells treated at 50–100 lg/mL NP concentration started to decrease at 6 h. After 14 h of exposure to
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Fig. 4. SEM images of control and ZnO NP-exposed (48 h) fibroblast cells.
Fig. 5. TEM micrographs of hPDLF (A, C) and mDF (B, D) cells: ZnO NP-treated hPDLFs (C); ZnO NP-treated mDFs (D); control PDLFs (A), control mDFs (B). Arrows point NP agglomerates. Abbreviations: PM: plasma membrane, N: nucleus, M: mitochondria, er: endoplasmic reticulum, v: vesicle, bar = 1 lm.
NPs, CI was decreased by 55% in the 50 lg/mL ZnO NPs group and remained almost stable. At the 100 lg/mL ZnO NP concentration, CI had significantly decreased (near zero level). Changes in the CI of mDF cells are presented in Fig. 9B. As seen in Fig. 9, ZnO NPs exhibited cytotoxic effect at doses of 50–100 lg/ mL at 4 h after exposure. At 50 lg/mL NP concentration after 8 h of exposure, the CI decreased by 75%; after 12 h the CI decrease slowed down. The highest NP concentration resulted in near zero cell index during the experiments. On the other hand, during exposure below 50 lg/mL concentration, the CI of both types of fibroblastic cells was quite similar to that of the controls. IC50 values (lg/mL) of NP exposure was determined for both cell types using the xCELLigence RTCA software. While the calculated IC50 value for hPDLFs was 49.1 lg/mL, this was 47.5 lg/mL for mDF cells, indicating quite similar cytotoxic responses.
4. Discussion Despite well established benefits of nanotechnology, a wide range of studies indicate the potential hazards of certain metal oxide NPs on human health and the environment due to their small size and unique properties (Li et al., 2011). Therefore, it is important to assess the risk/benefit ratio of using NPs for particular applications (Medina et al., 2007). This study confirms that the cytotoxicity responses of periodontal ligament fibroblast cells (the teeth’s supportive tissue) and dermal fibroblast cells (skin tissue) to ZnO NPs depended on, concentration, and exposure time. Given the risk of oral exposure to these NPs, or their potential utility in sealers and root-end filling materials, it seems very important to examine the possible toxic effects of this material on periodontal ligament fibroblast cells.
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Fig. 6. Effect of NPs on the mitochondrial activity of hPDLF and mDF cells. Cells were treated with 0.1–100 lg/ml of ZnO NPs for 6, 24, 48 h. Statistically significant differences (p < 0.05) between doses of 10 lg/mL and 50 lg/mL doses, and between doses of 10 lg/mL and 100 lg/mL were found at 24 h and 48 h for both cell types.
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Skin is the largest organ in the body and directly comes into contact with many toxic agents. Dermal exposure to NPs is usual and is clearly an important issue, since the range of different NPs in creams are likely to increase (Pinnell et al., 2000). ZnO NPs included in sunscreens is the primary source of dermal exposure, since most of the population has been using sunscreen products for protection against the hazardous effects of the sun. Therefore, in vitro toxicology studies were carried out to examine the exposure effects on skin cells. In vitro cytotoxicity assays have the potential to reduce the number of animal research for preliminary testing of nanomaterials to assess their potential toxic effects (Kroll et al., 2011). Three
toxicity end-points (MTT, LDH and ROS) that were selected in the current study represent biological activities of two types of fibroblast cells. Since many NPs can interfere with reagents used in classical cell-based assays, these methods may not provide reliable data (Kroll et al., 2012). In order to minimize particle interference in vitro, metal oxide NPs were thoroughly removed by washing and centrifugation, prior to incubation. It is advantageous to remove NPs via centrifugation before MTT and LDH assays in order to discard pellets containing NPs and the cell debris. This step reduces interference effect of the particles (Stone et al., 2009). Electrical impedance measurements of the cells grown on electrodes were performed as a function of time. The results obtained
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Concentration (μg/mL) Fig. 8. Effect of NPs on the ROS formation in hPDLF and mDF cells. Cells were treated with 1–100 lg/ml of ZnO NPs for 30 min. PC: Positive control; cells were exposed to H2O2 (200 mM). Statistically significant differences (p < 0.05) between doses of 10 lg/mL and 100 lg/mL were found for both cell types.
with the xCELLigence System were in line with the findings from the conventional assays (MTT, and LDH). Our data from the cellimpedance measurements, MTT and LDH assays indicated that ZnO NPs at concentrations of 50–100 lg/mL showed toxic effect in terms of CI decrease, for both cell types. It is important to note that measuring the electrical impedance is more accurate and a reliable alternative to traditional cytotoxicity assays, due to advantages such as, real-time and label-free detection of cell responses (Chuang et al., 2013). The CI values of cells incubated with different concentrations of ZnO NPs did not indicate explicit difference at concentrations up to 50 lg/mL; thus, the cell growth curve was considerably similar to that of the control cells, especially the mDF cells. Exposure to NP dose of 50 lg/mL resulted in deceleration of cell growth. Another finding was that, this dose was better tolerated by the mDF cells, as compared with the hPDLF cells. At the dose of 100 lg/mL, both types of cells showed significant
decrease in viability over time, as indicated by the significant decrease in CI values. CI measurements also denoted that cellular response of mDF to ZnO NPs occured in a shorter time span, compared to that of the hPDLF cells. We note that, changes in cell morphology, harmful effects on cell viability and membrane integrity were induced by the nano ZnO, in general. Microscopy revealed that fibroblasts exposed to ZnO NPs demonstrated signs of necrosis and cell death, in terms of morphological changes, cellular shrinkage or detachment from the culture substrates at doses higher than 50 lg/mL (Fig. 3). The hydrodynamic size, which indicates the degree of particle aggregation in solution, were measured inside the cell medium instead of water, since the DLS measurement in water is not representative of the actual behavior (Gualtieri et al., 2012). It is important to determine the hydrodynamic size and surface charges of NP dispersion to evaluate the toxic responses of cells to NPs (Jiang et al., 2009). The DLS observation showed that the NPs formed agglomerates in the solution. ZnO NPs were typically 19 times larger in solution than their size in dry form, estimated by XRD analysis. The polydispersity index of ZnO NP suspensions (
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells Sß ükran Sß eker a,b, A. Eser Elçin a,b, Tug˘rul Yumak b, Ali Sınag˘ b, Y. Murat Elçin a,b,⇑ a b
Ankara University Stem Cell Institute, Ankara 06100, Turkey Ankara University Faculty of Science, Ankara 06100, Turkey
a r t i c l e
i n f o
Article history: Received 31 January 2014 Accepted 25 June 2014 Available online 9 July 2014 Keywords: Nanoparticles Toxicity Zinc oxide Impedance measurement Periodontal ligament fibroblasts Dermal fibroblasts
a b s t r a c t The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7–8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun–Emmet–Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 lg/mL for 6, 24 and 48 h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of 50–100 lg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The unique size-dependent properties of nanomaterials make them very attractive for potential applications within the biomedical, commercial and environmental sectors (Aili and Stevens, 2010; Harishchandra et al., 2010). Due to their superior electrical, thermal, mechanical, and imaging properties, engineered nanomaterials have found applications such as in cell imaging (Zhong et al., 2012), drug delivery (Panyam and Labhasetwar, 2012), cancer therapy (Xiao et al., 2012) and piezoelectric (Seker et al., 2010), Abbreviations: NP, nanoparticle; ZnO, zinc oxide; hPDLF, human periodontal ligament fibroblast; mDF, mouse dermal fibroblast; AFM, atomic force microscopy; BET, Braun–Emmet–Teller; DMEM, Dulbecco’s modified Eagle’s medium; SEM, scanning electron microscopy; TEM, transmission electron microscopy; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; LDH, lactate dehydrogenase; ROS, reactive oxygen species; carboxy-H2DCFDA, 5-(and-6)carboxy-20 ,70 -dichlorodihydrofluorescein diacetate. ⇑ Corresponding author. Address: Tissue Engineering, Biomaterials & Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Ankara University Stem Cell Institute, Ankara 06100, Turkey. Tel.: +90 (312) 212 6720; fax: +90 (312) 223 2395. E-mail address: [email protected] (Y.M. Elçin). http://dx.doi.org/10.1016/j.tiv.2014.06.016 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.
electronic and optical (Lia et al., 2010) sensor systems. As the use of nanomaterials increase worldwide, the risk on human health and environmental nanomaterials increases. It is, therefore, necessary to assess the potential adverse effects of exposure, on human health and the environment. Metal oxide nanoparticles (NPs) have been widely used in nanotechnology-based applications, such as catalysis, sensors, environmental remediation and in consumer products. In particular, zinc oxide (ZnO), gold (Au), platinum (Pt) and titanium dioxide (TiO2) NPs find application in personal care products, especially sunscreens and toothpastes (Fröhlich and Roblegg, 2012), in coatings and paints, due to their UV absorption efficiency and transparency to visible light (Franklin et al., 2007). Despite many advantages in the wide applications of NPs, nanomaterials may cause serious environmental and health problems. Ever increasing use of the nanotechnology products requires their elaborate toxicological evaluations. Many types of NPs have proven to be toxic to human tissue and cell cultures. In particular, the respiratory and intestinal tracts and skin are in direct contact with the environment. Adding to these, NPs can translocate from these routes via different pathways and mechanisms. It is very important to characterize
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NPs throughout their interaction with the biological system and to accurately determine nanoparticle uptake site and concentration (Maurer-Jones et al., 2010). Although studies regarding the environmental toxicity and potential harm on human health have increased during the last course of the decade, there is still a significant lack of knowledge related to the impact of nanomaterials on human health and the environment (Böhmert et al., 2012). Nanotoxicology studies performed directly on model animals or cultured cells provide significant information on the effects it might have on human and other species (Suh et al., 2009). In particular, in vitro methods are generally utilised for investigating NP interactions with the biological systems (Arora et al., 2012; Hillegass et al., 2010). For example, cytotoxic responses such as changes in cell morphology, cell adhesion, cell–cell interactions and cell proliferation can be monitored in real-time by using the electrical impedance-based high-throughput method without using any label (McGuigan and Li, 2014). Toxic properties of nanomaterials depend on a variety of parameters such as surface charge, chemical composition, quantity, resolution, shape and surface area. Besides, impurities subsided during NP production may be involved in material toxicity. Different biological systems may show distinct sensitivities to nanomaterials (Oberdörster et al., 2004). ZnO is one of the most widely used semi-conducting oxide (wide direct band gap of 3.37 eV) in electrical (Gulino and Fragala, 2002), electrochemical (Yumak et al., 2011), magnetic (Norton et al., 2003), and optical (Fujihara et al., 2001) applications. ZnO NPs have received considerable attention for years due to their wide range of possible technological applications such as solar cells (Jiang et al., 2007), photovoltaic devices (Ravirajan et al., 2006), varistors (Pillai et al., 2004) and sensors (Lin et al., 1998; Muti et al., 2010). Various methods (chemical precipitation, microwave technique, solvothermal, sol–gel, hydrothermal and solid state) have been used to synthesize NPs. In this work, hydrothermal method was chosen to synthesize ZnO NPs owing to particular advantages such as, allowing superior compositional and morphological control, and neither requiring calcination nor milling steps. In this study, the physicochemical properties of hydrothermally synthesized ZnO NPs were characterized through zeta potential, particle size distribution via dynamic light scattering (DLS) and Braun–Emmet–Teller (BET) analyses. Biological responses of periodontal ligament fibroblasts (cells of the supportive tissue of the teeth) and dermal fibroblasts (cells of the skin tissue) against ZnO NPs were determined by transmission electron microscopy (TEM). Degree of intracellular reactive oxygen species (ROS) generation, cell impedance, mitochondrial activity and membrane leakage were also measured. Our findings demonstrated that ZnO NPs at 50–100 lg/mL were significantly cytotoxic to primary hPDLF and mDF cell cultures. 2. Materials and methods 2.1. Synthesis and characterization of NPs All chemicals used for NP synthesis were of analytical grade. Triton X-100, zinc acetate and ethanol were purchased from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification. We synthesized ZnO NPs by using the hydrothermal method as previously described (Sinag et al., 2011). Detailed characterization including the XRD analysis of the ZnO NPs is reported elsewhere (Sinag et al., 2011). 2.2. Atomic force microscopy (AFM) AFM was used to investigate further the morphology and size distribution of the nanosized ZnO, coated onto polished gold
surface. Study was performed on an NI–AFM model atomic force microscope (Nanomagnetics Inst., Ankara, Turkey), operating in dynamic mode in air (Seker et al., 2010). A Tap300A1 model cantilever (Budget Sensors, Innovative Solutions, Sofia, Bulgaria) with a resonance frequency of 320 kHz was used. The coated surfaces were scanned with 40 N/m force constant and imaged at a scan area of 2 lm 2 lm. The NP solution was prepared (100 lg/mL) in dichloromethane (Sigma) at 25 °C and dispersed using a Fisher FB15060 model sonicator (Fisher Scientific, Schwerte, Germany) for an hour. For AFM evaluation, thin films on polished gold surfaces were prepared at 3000 rpm by a Primus SB15 model spin coater (Singen, Germany), then the samples were dried in air at room temperature. 2.3. BET analysis The Braun–Emmet–Teller (BET) surface area of the ZnO NPs was determined using a NOVA 2200e volumetric gas adsorption instrument (Quantachrome Instruments, Boynton Beach, FL, USA), according to the procedures described elsewhere (Yagmur et al., 2008). 2.4. hPDLF cell isolation and culture Periodontal ligament fibroblast cells were isolated from periodontal ligament tissue from the root surfaces of premolar teeth, following review board approval and informed consent before extraction (Albandar, 2005; Inanc et al., 2007). Briefly, periodontal ligament fibroblast tissue from the middle third of the tooth roots were scraped with sterile blades, minced and transferred to culture flasks containing DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 lg/mL streptomycin, 1% nonessential amino acid stock solution, and 2 mM L-glutamine (all from Sigma). The explants were cultured at 37 °C, 5% CO2 and 95% humidity until the fibroblast cells became confluent. hPDLF cells between passages 8–10 were used in the experiments. 2.5. mDF cell isolation and culture Dermal fibroblast cells were isolated from the tail skin of 8–10 weeks old adult mice according to the standards of international regulations, using the method described elsewhere (Lichti et al., 2008). Briefly, mouse was anesthetized with intraperitoneal injection of avertin and under anesthesia, the tail was disinfected with ethanol 70%. Tails of ten to fifteen animals were cut at the base and the skin was sectioned and removed. After cutting into square pieces along with strong antisepsis and washing, the fragments were transferred to a petri dish (60 15 mm) containing 20 ml of 0.5% trypsin solution and stored overnight at 4 °C to separate the epidermis from the dermis. Then, the dermal explants were cultured and grown in fibroblast medium [DMEM highglucose medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicilin, 100 lg/mL streptomycin and 1% non-essential amino acids (all from Sigma, St. Louis, MO)] at 37 °C, humidified atmosphere with 5% CO2. After 48 h, the non-adherent cells were removed and the adherent cells were cultured. Culture medium was changed twice a week. 2.6. Preparation of ZnO NP dispersion for cell culture The nano sized ZnO was sterilized using UV irradiation (254 nm) for 30 min before use. The stock solution of ZnO NPs was prepared in cell culture medium and dispersed by using a sonicator for 30 min. Then, the stock solution was diluted to different concentrations in cell culture medium completed by 10% fetal
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bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin and 1% non-essential amino acids. 2.7. Determination of particle size distribution Zeta potential and particle size distribution measurements of the ZnO NPs were determined using Malvern’s NanoZetasizer-ZS (Worcestershire, UK). The ZnO NPs at concentration of 100 lg/mL were suspended in cell culture medium. Prior to measurements, the particle suspension was sonicated in an ultrasonic bath (FB15060, Fisher Scientific, Hampton, NH, USA) for 5 min to ensure homogenous dispersion in the medium. Sample–was placed in clean disposable cuvettes, and thirteen sequential measurements were performed. All the measurements were taken at ambient temperature of 25 °C. 2.8. Treatment with nanoparticles For in vitro cytotoxicity studies, the cells were cultured with various concentrations (0.1, 1, 10, 50 and 100 lg/mL) of nanosized ZnO for 6 h, 24 h and 48 h. In brief, cells at 80–85% confluence were trypsinized with 0.25% trypsin–EDTA solution (Sigma) and subsequently seeded onto 96-well plates at a density of 1 104 cells per well. Cells were cultured in a humidified incubator at 37 °C and 5% CO2 atmosphere for 2 days before the assays. Then, the medium was replaced with the NP-dispersed culture medium and the cells were treated with varying concentrations of NPs for 6 h, 24 h and 48 h. In control cultures, the cells were seeded at the same cell density and cultured without the ZnO NPs. At the end of the exposure period, toxicity assays (MTT, LDH, impedance measurements and ROS) were evaluated in control and NP-exposed cells.
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collected by centrifugation at 1100 rpm for 5 min. Samples were prepared for TEM using the method described elsewhere (Elcin et al., 2003). Briefly, after centrifugation, cells were collected, prefixed with 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in a graded alcohol series, embedded in epoxy resin and sectioned using a Leica ultramicrotome (Ultracut UCT, Vienna, Austria). Thin sections were examined under a JEOL 100 CX TEM (Tokyo, Japan) after post-staining with 4% uranyl acetate in methanol and Reynolds lead citrate. 2.12. MTT assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma) was performed to evaluate the effect of ZnO NPs on the mitochondrial activity of fibroblast cells. At the end of the exposure, the cell culture medium was discarded cautiously and the cells were washed with serum-free medium three times to remove excess nanoparticles. 20 lL of fresh MTT solution (5 mg/mL) and 180 lL of DMEM (without serum) was added into each well, and the cells were incubated at 37 °C and 5% CO2 for 4 h. After incubation, the medium was aspirated with care; then 200 ll MTT solvent (0.1 N HCl in anhydrous isopropanol) was added to each well and mixed to dissolve the blue formazan crystals. The 96-well plate was centrifuged for 5 min at 250 g at 25 °C, supernatants were transferred to new 96-well plates to remove remaining NPs. Formation of formazan was measured at 570 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The increasing concentration of formazan product as measured by the amount of absorbance [A] is directly proportional to the number of living cells in culture. The relative cell viability (%) was calculated from [A]sample/[A]control 100, where [A]sample is the absorbance of the test sample and [A]control is the absorbance of the control sample.
2.9. Cell morphology 2.13. Lactate dehydrogenase (LDH) release assay In order to document morphological changes in fibroblast cells in response to NPs, after 48 h of incubation, the phase contrast images of the cells exposed to all concentrations of ZnO NPs were obtained using a Primo Vert digital inverted microscope (Zeiss, Jena, Germany). 2.10. Scanning electron microscopy (SEM) analyses SEM was used to investigate the surface morphology of exposed cells. Briefly, the cells were seeded onto coverslips and allowed for 2 days to cover the surface. The cell cultures were treated with nanosized ZnO (50 lg/mL) for 48 h upon reaching 80% confluency. Following exposure, the samples were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Then, samples were dehydrated in graded ethanol series, mounted on aluminum supports and were sputter-coated with gold, using a Sputter Coater (Desk II, Denton Vacuum, Cherry Hill, NJ, USA) (Emin et al., 2008). A JEOL 100S (Tokyo, Japan) SEM was utilized at a voltage of 20 kV. 2.11. Subcellular localization of nanoparticles TEM was used to visualize the morphological changes and subcellular localization of ZnO NPs within cells. Briefly, the cells were seeded into 6-well tissue culture-treated plates at a density of 0.3 106 cells per well in 5 mL culture medium and allowed to attach for 2 days. Upon reaching 80% confluency, the cell cultures were treated with nanosized ZnO (50 lg/mL) for 48 h. The untreated cells were used as the control. After 48 h exposure to NPs, cells were washed three times with the corresponding medium and removed from the plate through trypsinization and
The activity of cytoplasmic LDH released into the culture medium was determined using an LDH Toxicology Assay kit (Sigma). The positive control was prepared by adding 10 lL of lysis solution (per well) to the control cells at 45 min prior to centrifugation (Chang et al., 2011), and set as 100% LDH leakage. After treatment, cell material from 96-well plates was centrifuged under room temperature for 5 min at 250 g, to remove excess particles. The resulting supernatants were transferred to new 96-well plates to perform the assay. LDH assay mixture was prepared fresh for each experiment. Briefly, equal volumes of LDH assay substrate, LDH assay dye, and 1 LDH assay cofactor were added together. Fifty lL of cell medium and 100 lL of the substrate/dye/cofactor solution were mixed for LDH analysis, light-protected and incubated at room temperature for 30 min. The reaction was terminated by the addition of 15 lL of 1 N HCl to each well. The absorption was measured using the SpectraMax M5 microplate reader at 490 nm. Absorbance at a wavelength of 490 nm was subtracted by the absorbance readings at a wavelength of 690 nm. All samples were read in triplicate. The LDH leakage (% of positive control) is expressed as the percentage of (ODsample ODnegative)/(ODpositive ODnegative) 100, where ODsample is the optical density of cells exposed to NPs, ODpositive is the optical density of the positive control cells, and ODnegative is the optical density of the cell culture medium. 2.14. Detection of intracellular ROS The production of intracellular reactive oxygen species was measured using 5-(and-6)-carboxy-20 ,70 -dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). The positive controls were
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prepared by culturing the normal cells in culture medium containing 200 mM H2O2. The cells were incubated with different NP concentrations for 30 min. At the end of exposure, the cells were washed three times with PBS to remove particles and then incubated with 20 lM carboxy-H2DCFDA (Invitrogen) for 30 min at 37 °C. After the incubation, the fluorescence intensity was immediately determined at 485-nm excitation and 520-nm emission using a microplate reader (SpectraMax M5). As a negative control, cells were treated with normal cell culture medium. The ROS level is expressed as the ratio of (Fsample)/(Fcontrol), where Fsample is the fluorescence intensity of the cells exposed to NPs; Fcontrol is the fluorescence intensity of the control cells. 2.15. Real-time cell impedance measurements Cellular response to ZnO NPs was continuously monitored using the xCELLigence Real-Time Cell Analyzer (RTCA) DP (Roche Applied Science, Penzberg, Germany) that measures electrical impedance of cells grown on microelectrodes placed at the bottom of plate wells. The hPDLF and mDF cells were seeded on electrode-plates (E-plates, Roche Diagnostics, Basel, Switzerland) at a density of 6.25 103 and 12.50 103 cells/well, respectively and cultured inside the incubator at 37 °C and 5% CO2 until reaching the log phase (10 h for hPDLFs and 15 h for mDFs). Then, NP suspensions at different concentrations were added into each well (final concentration of 0.1, 1, 10, 50 and 100 lg/mL). The cells were incubated with the NP suspensions or cell culture medium (negative control). The cell impedances were continuously recorded at 15 min intervals for 90 h. In order to determine possible interference of NPs, the impedance measurement was performed with cell culture medium (no cell) containing ZnO NPs at the highest concentrations used. The half maximal inhibitory concentrations (IC50) were calculated using the xCELLigence Software. 2.16. Statistical analysis Data from all groups were obtained from three independent experiments, except the MTT assay was performed with six independent experiments. Data was expressed as the mean ± SD, and analyzed by using the one-way analysis of variance and post hoc tests. A value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. AFM analyses The topography of the surfaces coated with ZnO NPs was characterized by AFM. The AFM images of the coated surface are
shown in Fig. 1. The results demonstrate that the metal oxide film was uniformly coated onto gold surface and exhibited homogenous distribution of ZnO NPs (Fig. 1A). The cross section measurements made for the entire scan area of multiple samples (n = 3) demonstrated a coating thickness of around 2–8 nm with a rootmean-square (rms) roughness of 7.5. 3.2. BET analyses BET analysis results are shown in Table 1. Surface analysis of the ZnO nanoparticles determined from the BET measurements showed that ZnO NPs had high surface area (208.481 m2/g) for the nitrogen to be adsorbed. 3.3. Zeta potential and particle size distribution using DLS DLS measurement determined the average particle size distribution, zeta (f) potential and polydispersity index (PDI) of the ZnO NP suspensions in cell culture medium. The average hydrodynamic diameter according to NanoZetasizer-ZS measurements is 154.5 ± 6.5 nm (Fig. 2 and Table 1). 3.4. Cell morphology Inverted phase-contrast micrographs of hPDLF and mDF cells, exposed for 48 h duration to NPs and the control cultures are shown in Fig. 3. The cells cultured under standard conditions had evenly spread on culture dishes and showed characteristic morphology. The cells exposed to ZnO NPs gradually lost their characteristic phenotype in a concentration dependent manner. As seen in Fig. 3, dramatic morphology changes occurred at 50 lg/mL NP concentration; cells started to shrink and obtained irregular shapes. Compared with control cells, ZnO-treated cells showed a significant difference in their adhesion to the culture dish at 50 lg/mL concentration. At higher concentration of ZnO NPs, the cells became completely necrotic and detached from the culture plates. The phase contrast images indicated that the damage on cell morphology was dependent on ZnO concentration. 3.5. SEM for cell morphology SEM images provided information on cell morphology after nanoparticle exposure. ZnO NPs seemed to be phagocytosed by the cells. The lumps and bumps recognized on the fibroblast surfaces indicated the possibility of NP penetration into the vesicles of the fibroblasts (Fig. 4). In addition, NPs were strongly adsorbed on the cell surfaces on account of their high specific surface area and high surface energy (Fig. 4).
Fig. 1. Representative three-dimensional and cross-sectional AFM images (A, B) and cross section profiles of ZnO NPs (C).
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Table 1 Characterization of ZnO NPs. Crystalline sizes in XRD (nm) BET results (m2/g) Average diameter in DLS (nm) Zeta potential (mV) Polydispersity index
8 208.481 154.5 ± 6.5 28.2 ± 6.03 0.229 ± 0.0067
Fig. 2. Particle size distribution of the ZnO NPs obtained by DLS.
3.6. TEM for visualization of cellular uptake Fig. 5 represents TEM results obtained after exposure of hPDLFs (5A, 5C) and mDFs (5B, 5D) to ZnO NPs. TEM images of NP-treated cells indicated that nanoparticles were often present within the cytoplasmic vesicles and nucleus (arrows), while localization in organelles was not observed. Fig. 5 also demonstrates that, NPs appear as aggregates within the cells. Compared to the control hPDLFs (5B) and control mDFs (5D), NP-treated cells were considerably rich in cytoplasmic vesicles. Also, the ZnO NP exposure caused significantly distorted nucleus shape. Control cells had normal cell morphology, their vesicles appeared to be uniform and free of any particles. The larger nuclei indicate the beginning of cell division. 3.7. Mitochondrial function The cell viability of hPDLF and mDF cultures was measured by the MTT assay after culturing for 6 h, 24 h and 48 h in the presence of ZnO nanoparticles. Exposure to NPs resulted in a concentrationdependent decrease of mitochondrial activity in cells, as given in Fig. 6. The MTT assay for mDF cells showed that ZnO NPs had no significant effect on the mitochondrial activity at concentrations tested for 6 h, while hPDLFs were affected by the NPs at this time point. For the 24 and 48 h exposure time points, the viability of mDF cells treated with ZnO NPs was lower at concentrations of 50–100 lg/ml. However, ZnO NPs exhibited a significant reduction in hPDLF cell viability at 50–100 lg/mL for 6, 24 and 48 h. The results demonstrated that ZnO NPs caused a significant reduction in mDF and hPDLF viability at 50–100 lg/ml. 3.8. LDH measurements To investigate the impact of ZnO NPs on cell membrane integrity, lactate dehydrogenase leakage was assessed under NP-exposed conditions. The results indicated that exposure to ZnO NPs resulted in a concentration- and time-dependent increase in LDH leakage. In mDF cells, ZnO NPs caused a sharp increase in LDH leakage at 50–100 lg/ml for 6, 24 and 48 h time points. In hPDLF cells, ZnO NPs had significant effect on membrane integrity at the 50–100 lg/mL concentration range (Fig. 7).
Fig. 3. Representative light micrographs demonstrating hPDLF and mDF cultures following exposure to ZnO NPs at different concentrations. Scale bars: 100 lm.
3.9. Intracellular ROS measurements In order to determine the oxidative stress induced by NP exposure, ROS formation in hPDLF and mDF cells was assessed using carboxy-H2DCFDA (Fig. 8). It was found that, the concentration-dependent effect of the NPs on the oxidation of 5-(and-6)carboxy-20 ,70 -dichlorodihydrofluorescein to 5-(and-6)-carboxy-20 , 70 -dichlorofluorescein was minimal. To sum up, NP exposure resulted in oxidative stress, but there was no significant increase of ROS generation in cells in a concentration-dependent manner for the levels in question. 3.10. Electrical impedance measurements The changes in cell index (CI) caused by ZnO NPs are presented in Fig. 9. The CI values of hPDLF cells treated at 50–100 lg/mL NP concentration started to decrease at 6 h. After 14 h of exposure to
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Fig. 4. SEM images of control and ZnO NP-exposed (48 h) fibroblast cells.
Fig. 5. TEM micrographs of hPDLF (A, C) and mDF (B, D) cells: ZnO NP-treated hPDLFs (C); ZnO NP-treated mDFs (D); control PDLFs (A), control mDFs (B). Arrows point NP agglomerates. Abbreviations: PM: plasma membrane, N: nucleus, M: mitochondria, er: endoplasmic reticulum, v: vesicle, bar = 1 lm.
NPs, CI was decreased by 55% in the 50 lg/mL ZnO NPs group and remained almost stable. At the 100 lg/mL ZnO NP concentration, CI had significantly decreased (near zero level). Changes in the CI of mDF cells are presented in Fig. 9B. As seen in Fig. 9, ZnO NPs exhibited cytotoxic effect at doses of 50–100 lg/ mL at 4 h after exposure. At 50 lg/mL NP concentration after 8 h of exposure, the CI decreased by 75%; after 12 h the CI decrease slowed down. The highest NP concentration resulted in near zero cell index during the experiments. On the other hand, during exposure below 50 lg/mL concentration, the CI of both types of fibroblastic cells was quite similar to that of the controls. IC50 values (lg/mL) of NP exposure was determined for both cell types using the xCELLigence RTCA software. While the calculated IC50 value for hPDLFs was 49.1 lg/mL, this was 47.5 lg/mL for mDF cells, indicating quite similar cytotoxic responses.
4. Discussion Despite well established benefits of nanotechnology, a wide range of studies indicate the potential hazards of certain metal oxide NPs on human health and the environment due to their small size and unique properties (Li et al., 2011). Therefore, it is important to assess the risk/benefit ratio of using NPs for particular applications (Medina et al., 2007). This study confirms that the cytotoxicity responses of periodontal ligament fibroblast cells (the teeth’s supportive tissue) and dermal fibroblast cells (skin tissue) to ZnO NPs depended on, concentration, and exposure time. Given the risk of oral exposure to these NPs, or their potential utility in sealers and root-end filling materials, it seems very important to examine the possible toxic effects of this material on periodontal ligament fibroblast cells.
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Concentration (μg/mL) Fig. 7. Effect of NPs on the lactate dehydrogenase leakage in hPDLF and mDF cells. Cells were treated with 0.1–100 lg/ml of ZnO NPs for 6, 24, 48 h. Statistically significant differences (p < 0.05) between doses of 10 lg/mL and 50 lg/mL, and between doses of 10 lg/mL and 100 lg/mL were found at all time points for both type of cells.
Skin is the largest organ in the body and directly comes into contact with many toxic agents. Dermal exposure to NPs is usual and is clearly an important issue, since the range of different NPs in creams are likely to increase (Pinnell et al., 2000). ZnO NPs included in sunscreens is the primary source of dermal exposure, since most of the population has been using sunscreen products for protection against the hazardous effects of the sun. Therefore, in vitro toxicology studies were carried out to examine the exposure effects on skin cells. In vitro cytotoxicity assays have the potential to reduce the number of animal research for preliminary testing of nanomaterials to assess their potential toxic effects (Kroll et al., 2011). Three
toxicity end-points (MTT, LDH and ROS) that were selected in the current study represent biological activities of two types of fibroblast cells. Since many NPs can interfere with reagents used in classical cell-based assays, these methods may not provide reliable data (Kroll et al., 2012). In order to minimize particle interference in vitro, metal oxide NPs were thoroughly removed by washing and centrifugation, prior to incubation. It is advantageous to remove NPs via centrifugation before MTT and LDH assays in order to discard pellets containing NPs and the cell debris. This step reduces interference effect of the particles (Stone et al., 2009). Electrical impedance measurements of the cells grown on electrodes were performed as a function of time. The results obtained
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Concentration (μg/mL) Fig. 8. Effect of NPs on the ROS formation in hPDLF and mDF cells. Cells were treated with 1–100 lg/ml of ZnO NPs for 30 min. PC: Positive control; cells were exposed to H2O2 (200 mM). Statistically significant differences (p < 0.05) between doses of 10 lg/mL and 100 lg/mL were found for both cell types.
with the xCELLigence System were in line with the findings from the conventional assays (MTT, and LDH). Our data from the cellimpedance measurements, MTT and LDH assays indicated that ZnO NPs at concentrations of 50–100 lg/mL showed toxic effect in terms of CI decrease, for both cell types. It is important to note that measuring the electrical impedance is more accurate and a reliable alternative to traditional cytotoxicity assays, due to advantages such as, real-time and label-free detection of cell responses (Chuang et al., 2013). The CI values of cells incubated with different concentrations of ZnO NPs did not indicate explicit difference at concentrations up to 50 lg/mL; thus, the cell growth curve was considerably similar to that of the control cells, especially the mDF cells. Exposure to NP dose of 50 lg/mL resulted in deceleration of cell growth. Another finding was that, this dose was better tolerated by the mDF cells, as compared with the hPDLF cells. At the dose of 100 lg/mL, both types of cells showed significant
decrease in viability over time, as indicated by the significant decrease in CI values. CI measurements also denoted that cellular response of mDF to ZnO NPs occured in a shorter time span, compared to that of the hPDLF cells. We note that, changes in cell morphology, harmful effects on cell viability and membrane integrity were induced by the nano ZnO, in general. Microscopy revealed that fibroblasts exposed to ZnO NPs demonstrated signs of necrosis and cell death, in terms of morphological changes, cellular shrinkage or detachment from the culture substrates at doses higher than 50 lg/mL (Fig. 3). The hydrodynamic size, which indicates the degree of particle aggregation in solution, were measured inside the cell medium instead of water, since the DLS measurement in water is not representative of the actual behavior (Gualtieri et al., 2012). It is important to determine the hydrodynamic size and surface charges of NP dispersion to evaluate the toxic responses of cells to NPs (Jiang et al., 2009). The DLS observation showed that the NPs formed agglomerates in the solution. ZnO NPs were typically 19 times larger in solution than their size in dry form, estimated by XRD analysis. The polydispersity index of ZnO NP suspensions (
