Colloids and Surfaces B: Biointerfaces 122 (2014) 209–215
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Synthesis, characterization and toxicological evaluation of iron oxide nanoparticles in human lung alveolar epithelial cells Sourabh Dwivedi a,b , Maqsood A. Siddiqui a,b,∗ , Nida N. Farshori c , Maqusood Ahamed d , Javed Musarrat a,b , Abdulaziz A. Al-Khedhairy a,b a
Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Al-Jeraisy Chair for DNA Research, King Saud University, Riyadh, Saudi Arabia c Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia d King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, Saudi Arabia b
a r t i c l e
i n f o
Article history: Received 13 March 2014 Received in revised form 9 June 2014 Accepted 30 June 2014 Available online 7 July 2014 Keywords: A-549 cells Iron oxide nanoparticles Cytotoxicity Oxidative stress
a b s t r a c t The present investigation was aimed to characterize the synthesized iron oxide nanoparticles (Fe3 O4 -NPs) and to assess their cytotoxicity and oxidative stress in human lung alveolar epithelial cells (A-549). Fe3 O4 NPs were characterized by X-ray diffraction, transmission electron microscopy, dynamic light scattering, and atomic force microscopy. The morphology of the Fe3 O4 -NPs was found to be variable with a size range of 36 nm. A-549 cells were exposed to Fe3 O4 -NPs (10–50 g/ml concentrations) for 24 h. Post exposure, cytotoxicity assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT; neutral red uptake, NRU; and cellular morphology) and oxidative stress (lipid peroxidation, LPO and glutathione, GSH) were evaluated. Further, intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were also studied. MTT and NRU assays revealed a concentration-dependent decrease in the cell viability of A-549 cells. Fe3 O4 -NPs exposed cells also altered the normal morphology of the cells. Furthermore, the cells showed significant induction of oxidative stress. This was confirmed by the increase in LPO and ROS generation, and the decrease in the GSH level and MMP. Our results demonstrated that Fe3 O4 -NPs induced cytotoxicity is likely to be mediated through the oxidative stress and ROS generation in A-549 cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Metal oxide nanoparticles are widely being used in many industrial products, i.e., catalysts, pigments, food additives, sun screens and cosmetics [1,2]. The applications and productions of these nanoparticles at large number have brought attention to their risk factors. It is well known that nanoparticles are being released during particle synthesis and handling of dry powders and liquid suspensions [3]. Toxicity of various metal oxide nanoparticles has also been reported in vitro [4–9] as well as in vivo [10–12] setups. Numerous studies indicate that metal oxide nanoparticles have the ability to generate reactive oxygen species (ROS) [13,14] and they are involved in the cytotoxicity due to their small size
∗ Corresponding author at: Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Tel.: +966 14699532; fax: +966 542967835. E-mail addresses: [email protected], [email protected] (M.A. Siddiqui). http://dx.doi.org/10.1016/j.colsurfb.2014.06.064 0927-7765/© 2014 Elsevier B.V. All rights reserved.
and large surface area [15]. Experimental evidences also showed that nanoparticles released by sprays and powders can potentially deposit in the respiratory system [16,17]. Iron oxide (Fe3 O4 ) nanoparticles have been applied broadly to bioscience and clinical research for various purposes; the most common includes magnetic cell labeling [18,19], separation and tracking [20], for therapeutic purposes in hyperthermia [21], in drug delivery [22], and for diagnostic purposes, e.g., as contrast agents for magnetic resonance imaging (MRI) [23]. Although the cytotoxic effects of iron oxide nanoparticles are known [24–26], the mechanism(s) of their induced cytotoxicity is not clearly understood. Since, human exposure to iron oxide may occur through the exposure routes of inhalation and ingestion at occupational settings. Therefore, the present investigation was aimed to understand the mechanism(s) of cell death induced by iron oxide nanoparticles in human lung alveolar epithelial cells (A-549) under in vitro conditions. These in vitro systems are cost-effective, rapid and reproducible with low or no ethical dubious [27,28]. In this study, firstly we have focused on the detailed synthesis and physicochemical characteristics of iron oxide nanoparticles; secondly, the effect
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of iron oxide nanoparticles on (i) cell viability, (ii) morphological alterations, (iii) oxidative stress markers, (iv) ROS generation, and (v) mitochondrial membrane potential (MMP) in A-549 cells. 2. Materials and methods 2.1. Cell culture A-549 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.2% sodium bicarbonate, and antibiotic/antimycotic solution (100×, 1 ml/100 ml of medium, Invitrogen, Life Technologies, USA). The cells were maintained in 5% CO2 , 95% atmosphere at 37 ◦ C. Cells were assessed for cell viability by trypan blue dye exclusion assay as described earlier [29] and batches showing more than 98% cell viability were used in the experiments. 2.2. Reagents and consumables All other specified chemicals, culture mediums, reagents, and diagnostic kits were procured from Sigma Chemical Company Pvt. Ltd., St. Louis, MO, USA unless otherwise stated. Culture wares and other plastic consumables used in the study were procured from Nunc, Denmark. 2.3. Experimental design A-549 cells were treated with various concentrations of Fe3 O4 NPs (10–50 g/ml) for 24 h. Cells were studied for the cytotoxicity by MTT and NRU assays and morphological alterations. Further, oxidative stress markers, i.e. lipid peroxidation (LPO), glutathione (GSH), reactive oxygen species (ROS) generation, and MMP were studied. 2.4. Synthesis of Fe3 O4 -NPs Fe3 O4 -NPs were prepared by chemical co-precipitation of Fe2+ and Fe3+ ions in an alkaline solution followed by a treatment under hydrothermal conditions [30]. Briefly, FeSO4 ·7H2 O (2.7 g) and FeCl3 (5.7 g) were dissolved in 10 ml Mili-Q water. Then, these two solutions were thoroughly mixed and added to double volume of 10 M ammonium hydroxide with constant stirring at 28 ◦ C. After that the dark black slurry of Fe3 O4 particles was heated to 80 ◦ C in a water bath for 30 min. Ion impurities such as chlorides and sulphates were removed by washing the particles several times with Mili-Q water. The particles were then dispersed in 10 ml Mili-Q water and sonicated for 20 min. 2.5. Characterization of Fe3 O4 -NPs 2.5.1. X-ray diffraction analysis of Fe3 O4 -NPs Finely powdered sample of Fe3 O4 -NPs was analyzed using X’pert PRO analytical diffractometer (Almelo, The Netherlands) ˚ in the range of 20◦ ≤ 2 ≤ 80◦ using CuK␣ radiation ( = 1.54056 A) at 40 keV. In order to calculate the particle size (D) of Fe3 O4 -NPs, the Scherrer’s equation (D = 0.9/ˇ cos ) has been used [31], where is the wavelength of X-ray, ˇ is the broadening of diffraction line measured as half of its maximum intensity in radians and is the Bragg’s diffraction angle. The average particle size of Fe3 O4 -NPs was estimated from the line width of the XRD peak. 2.5.2. Analysis of Fe3 O4 -NPs by transmission electron microscopy (TEM) TEM analysis of Fe3 O4 -NPs was performed on a transmission electron microscope (Hitachi, H-7500, Japan) at an accelerating
voltage of 90 kV. Samples were prepared by drop-coating Fe3 O4 NPs solutions (1% Fe3 O4 -NPS) onto carbon-coated gold TEM grids. Film of Fe3 O4 -NPs sample on TEM grid was allowed to stand for 2 min. The extra solution was removed using a blotting paper and the grid was allowed to dry prior to measurement. 2.5.3. Atomic force microscopy (AFM) of Fe3 O4 -NPs Fe3 O4 -NPs were examined using AFM (Veeco Instruments, USA). Analysis was performed by running the machine in non-contact tapping mode [32]. Characterization of Fe3 O4 -NPs was done by observing the patterns appeared on the surface topography and analyzing the AFM data. Tapping mode imaging was implemented in ambient air by oscillating the cantilever assembly at or near the cantilever’s resonant frequency using a piezoelectric crystal. The topographical images were obtained in tapping mode at a resonance frequency of 218 kHz. Data were analyzed through WSXM software. 2.5.4. Dynamic light scattering Fe3 O4 -NPs powder was suspended in deionized ultrapure water to obtain a concentration of 50 g/ml, and was sonicated at 40 W for 15 min. Hydrodynamic particle size and Zeta potential () of Fe3 O4 NPs in an aqueous suspension were determined by measuring the dynamic light scattering using of a ZetaSizer-HT (Malvern, UK). 2.6. Cytotoxicity of Fe3 O4 -NPs 2.6.1. MTT assay Percent cell viability was assessed using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described by Siddiqui et al. [33]. Briefly, cells (1 × 104 ) were allowed to adhere for 24 h in CO2 incubator at 37 ◦ C in 96 well culture plates. After the exposure, MTT (5 mg/ml of stock in phosphate buffer saline, PBS) was added (10 l/well in 100 l of cell suspension), and plates were incubated for 4 h. The supernatant was discarded and 200 l of DMSO were added to each well and mixed gently. The developed color was read at 550 nm. Untreated sets were also run under identical conditions and served as control. 2.6.2. Neutral red uptake (NRU) assay NRU assay was carried out following the protocol described [34]. After the exposure, the medium was aspirated and cells were washed twice with PBS, and incubated for 3 h in a medium supplemented with neutral red (50 g/ml). The medium was then washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. Cells were further incubated for 20 min at 37 ◦ C in a mixture of acetic acid (1%) and ethanol (50%) to extract the dye. The plates were read at 550 nm. The values were compared with the control sets. 2.6.3. Morphological analysis by phase contrast microscope Morphological changes in A-549 cells treated with Fe3 O4 -NPs were observed to determine the alterations induced by Fe3 O4 -NPs. The cell images were taken using an inverted phase contrast microscope at 20× magnification. 2.7. Lipid peroxidation (LPO) Lipid peroxidation was performed using thiobarbituric acidreactive substances (TBARS) protocol [35]. Briefly, after the exposure, A-549 cells were collected by centrifugation and sonicated in ice cold potassium chloride (1.15%) and were centrifuged for 10 min at 3000 × g. Resulting supernatant (1 ml) was added to 2 ml of thiobarbituric acid (TBA) reagent (15% TCA, 0.7% TBA and 0.25 N HCl) and was heated at 100 ◦ C for 15 min in a boiling water
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ROS generation was assessed using 2,7-dichlorodihydrofluorescein diacetate dye (DCFH-DA; Sigma–Aldrich, USA) as a fluorescence agent following the protocol earlier described [7]. Cells were washed with PBS and incubated for 60 min in DCFH-DA (20 M) containing incomplete culture medium in dark at 37 ◦ C. Then, the cells were analyzed for intracellular fluorescence using fluorescence microscope.
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2 theta Fig. 1. XRD pattern of the iron oxide nanoparticles (Fe3 O4 -NPs). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
MMP was measured following the protocol of Zhang et al. [37]. Briefly, control and treated cells were washed twice with PBS. Then the cells were further treated with 10 g/ml of Rhodamine-123 fluorescent dye for 1 h at 37 ◦ C in dark. Cells were then washed twice with PBS. The fluorescence intensity of Rhodamine-123 was measured using fluorescence microscope by grabbing the images at 20× magnification. 2.11. Statistical analysis
bath. Samples were then placed in cold ice and were centrifuged at 1000 × g for 10 min. The absorbance of the supernatant was measured at 550 nm. 2.8. Glutathione (GSH) content Intracellular GSH content was estimated as described [36]. Briefly, cells were collected by centrifugation and the cellular protein was precipitated by incubating 1 ml sonicated cell suspension with 1 ml TCA (10%) on ice for 1 h, followed by 10 min centrifugation at 3000 rpm. The supernatant was added to 2 ml of 0.4 M Tris buffer (pH 8.9) containing 0.02 M EDTA, followed by an addition of 0.01 M 5,5 -dithionitrobenzoic acid (DTNB) to a final volume of 3 ml. The tubes were incubated for 10 min at 37 ◦ C in shaking water bath. The absorbance of the yellow color developed was read at 412 nm.
Results were expressed as mean ± standard error of three experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Post hoc Dunnett’s test was applied to compare values between control and treated groups. The values depicting p < 0.05 were considered as statistically significant. 3. Results 3.1. Characterization of iron oxide nanoparticles (Fe3 O4 -NPs) Fig. 1 shows that the XRD pattern of Fe3 O4 -NPs clearly exhibiting the crystalline nature of this material. Based on the comparison of their XRD patterns with the standard pattern of Fe3 O4 (JCPDS 750033). The diffraction peaks corresponding to (2 2 0), (3 1 1), (4 0 0),
Fig. 2. Panel (A) representative transmission electron micrograph recorded from a drop-coated film of the aqueous solution of Fe3 O4 -NPs; (B) represents the 3D topography of Fe3 O4 -NPs in top view by atomic force microscopic analysis; and (C) average hydrodynamic size of Fe3 O4 -NPs.
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Fig. 3. Cytotoxicity assessments by MTT assay in A-549 cells following the exposure of various concentrations of iron oxide (Fe3 O4 ) nanoparticles for 24 h. Values are mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 vs control).
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Fig. 4. Cytotoxicity assessments by NRU assay in A-549 cells following the exposure of various concentrations of seed extract of iron oxide (Fe3 O4 ) nanoparticles for 24 h. Values are mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 vs control).
(5 1 1), and (4 4 0) are quite identical to the characteristic peaks of the Fe3 O4 crystal structure. The crystallite size has been estimated from the XRD pattern using the Scherrer’s equation [31]. The average crystallite size of Fe3 O4 -NPs was also found to be 36 nm. Fig. 2A shows the typical TEM image of Fe3 O4 -NPs. The average diameter was calculated by measuring over 100 particles in random fields of TEM view. The average TEM diameter of Fe3 O4 -NPs was 22 nm which is supporting the XRD data. The morphology and size of the Fe3 O4 -NPs were further validated by AFM analysis. Fig. 2B, shows the AFM image of Fe3 O4 -NPs obtained on scanning probe microscope in tapping mode, under ambient conditions. The average size of the nanoparticles and surface roughness (Ra ) were determined to be 31 and 22 nm, respectively using WSXM and SPIP softwares. The results of hydrodynamic size of analysis obtained with dynamic light scattering are shown in Fig. 2C. The distribution curves show the Fe3 O4 -NPs aggregates of 174 nm in Milli-Q water.
Depletion in the glutathione level in cultured A-549 cells exposed to 10–50 g/ml concentrations of Fe3 O4 -NPs are summarized in Fig. 6B. The results indicate that Fe3 O4 -NPs decreased the GSH levels in a concentration-dependent manner. A significant decrease in GSH level was observed as 15%, 38%, and 54% at 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively as compared to the control.
3.2. Cytotoxicity assessment by MTT assay
3.7. ROS generation
The results of cytotoxicity assessment of Fe3 O4 -NPs in A-549 cells by MTT assay are summarized in Fig. 3. A concentrationdependent statistically significant (p < 0.001) decrease in percentage cell viability of A-549 cells was recorded after 24 h of exposure of Fe3 O4 -NPs. Cell viability at 10, 25, and 50 g/ml of Fe3 O4 -NPs was recorded as 84%, 72%, and 56%, respectively.
Statistically significant (p < 0.001) ROS generation was observed in A-549 cells exposed to Fe3 O4 -NPs at 10, 25, and 50 g/ml concentrations for 24 h (Fig. 7A and B). An increase in ROS generation was observed in a concentration-dependent manner i.e., 23%, 46%, and 82% of untreated control following the exposure of 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively.
3.3. Cytotoxicity by NRU assay
3.8. Mitochondrial membrane potential (MMP)
Cytotoxicity assessment of Fe3 O4 -NPs by NRU assay is summarized in Fig. 4. A concentration-dependent statistically significant (p < 0.001) decrease in cell viability of A-549 cells was also recorded following the exposure of Fe3 O4 -NPs for 24 h. Cell viability was recorded as 87%, 79%, and 65% at 10, 25, and 50 g/ml, respectively.
The effect of Fe3 O4 -NPs exposure on MMP in A-549 cells was evaluated. A concentration-dependent statistically significant (p < 0.001) decrease in the level of MMP was also observed in A-549 cells after the exposure of Fe3 O4 -NPs for 24 h. The decrease in MMP was observed to be 21%, 44%, and 62% at 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively as compared to untreated control (Fig. 8A and B).
3.4. Morphological changes The morphological changes observed in A-549 cells exposed to Fe3 O4 -NPs for 24 h are shown in Fig. 5. Alterations in the morphology of A-549 cells were observed under phase contrast inverted microscope. The cells indicate the most prominent effects after the exposure of Fe3 O4 -NPs, the changes in their morphology were found in a concentration- and time-dependent manner. A-549 cells exposed to 10, 25, and 50 g/ml concentrations reduced the normal morphology of the cells and cell adhesion capacity in compared to the control.
3.5. Lipid peroxidation Lipid peroxidation level induced by Fe3 O4 -NPs is summarized in Fig. 6A. A concentration-dependent statistically significant increase in lipid peroxidation was also observed. An increase of 15%, 32%, and 91% was observed at 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively. 3.6. Glutathione depletion
4. Discussion In spite of their many advantages, nanoparticles may have hazardous effects because of their unique physicochemical properties. Iron oxide nanoparticles (Fe3 O4 -NPs) have attracted considerable attention in terms of their potential role in biomedical and clinical research for various purposes, including magnetic cell labeling, separation and tracking, drug delivery, therapeutics, and diagnosis. The physicochemical characterization of nanoscale materials is
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Fig. 5. Morphological changes in A-549 cells exposed to various concentrations of iron oxide (Fe3 O4 ) nanoparticles for 24 h. Images were taken using an inverted phase contrast microscope (OLYMPUS CKX 41) at 20× magnification.
state. This finding is supported by our previous investigations [40–42]. To evaluate the cytotoxicity of the synthesized Fe3 O4 -NPs on human lung alveolar epithelial cells (A-549), we have employed MTT and NRU assays. The viability of A-549 cells was significantly decreased with the increasing concentrations of Fe3 O4 -NPs. The cell mortality data obtained from this study allowed us to predict their potential cytotoxic effect. Our data are in well agreement with the previous reports of Fe3 O4 -NPs induced cytotoxicity in various cell types in vitro, including; ARPE-19 cells [43], human bronchial and alveolar epithelial cells [44], human lymphoblastoid TK6 cells, primary human blood cells [45], and in vivo rat model [46]. Our results also support a concentration-dependent response of Fe3 O4 -NPs as observed by Mendes et al. [47], who showed that carbon coated Fe3 O4 -NPs induce cytotoxicity in a concentration-dependent
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essential in biomedical research for better interpretation of results [7,38]. Parameters including shape, size, crystal structure, purity, hydrodynamic size, and agglomeration have been suggested for NPs characterization [39]. We have utilized the XRD, TEM, AFM and DLS techniques to characterize the NPs. Our XRD results confirm the crystalline nature of Fe3 O4 -NPs. TEM showed that NPs were fairly distributed with a smooth surface of an average diameter of 22 nm. Agglomeration and stability of NPs is also a major concern in nanomedicine research. The average hydrodynamic size of the present Fe3 O4 NPs in water determined by DLS was 174 nm. DLS is widely used to determine the size of brownian NPs in colloidal suspensions in the submicron and nano ranges. The higher size of nanoparticles in aqueous suspension as compared to TEM might be due to the tendency of particles to agglomerate in aqueous
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Fig. 6. Iron oxide (Fe3 O4 ) nanoparticles induced oxidative stress in A-549 cells exposed for 24 h. (A) Lipid peroxidation; (B) glutathione depletion. Values are mean ± SE of three independent experiments (*p < 0.01, **p < 0.001 vs control). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. (A) Iron oxide (Fe3 O4 ) nanoparticles induced ROS generation in A-549 cells. ROS generation was studied using dichlorofluorescindiacetate (DCFH-DA) dye after the exposure of iron oxide (Fe3 O4 ) Nanoparticles for 24 h. (B) Percent induction of ROS generation in A-549 cells following the exposure of various concentrations of iron oxide (Fe3 O4 ) nanoparticles for 24 h (*p < 0.01, **p < 0.001 vs control).
manner in HeLa Kyoto, human osteosarcoma (U2 OS), mouse fibroblasts (NIH 3T3), and mouse macrophages (J7442) cells. The cytotoxic effects of Fe3 O4 -NPs in A-549 cells may be due to the active physicochemical interaction of iron atoms with the functional groups of the intracellular proteins, which interact with the proteins and form a protein coat, which is believed to cause the toxicity [48]. The morphological alterations in A-549 cells exposed to Fe3 O4 NPs as observed by phase contrast inverted microscope showed the detachment with increasing concentrations. Our data support that cellular stress induced by nanoparticles can result in the alteration of the cellular morphology, when cells were treated with higher concentration of nanoparticles [49] resulting in a decrease in the total cell areas and a deviation of the cells from typical morphology [26]. Although, excessive oxidative stress can be harmful, however it is involved in normal cellular processes of cell signaling. Oxidative stress is a prominent feature of the response to metal oxide nanoparticles. Many studies have shown that exposure to nanoparticles promotes cellular oxidative stress [50–52]. These include
evidences of increased ROS production, depletion of antioxidants or elevation in oxidative products such as LPO, in a variety of cell types, including microglia [53], fibroblast [54], lung epithelial cells [50], and in human liver cells HepG2 [8]. Results from the present study also showed that Fe3 O4 -NPs induce oxidative stress in A549 cells in a concentration-dependent manner with an increase in the level of lipid peroxidation and a decrease in the antioxidant enzyme GSH. These findings suggest that oxidative stress may be the primary mechanism for toxicity of Fe3 O4 -NPs in A-549 cells. Our results are also supported by the previous findings suggesting that the cytotoxicity of NPs is mediated through the oxidative stress. Several studies demonstrated metal oxide nanoparticles induced oxidative stress [7,55,56]. In general, one of the most common toxic effects for nanoparticles is the induction of reactive oxygen species (ROS) generation [57]. Similar to oxidative stress, a significant increase in intracellular ROS generation was also found in a concentration-dependent manner in A-549 cells exposed to Fe3 O4 -NPs for 24 h. Our results were also in agreement with previous studies [58,59], where they showed maximum ROS induction at 24 h of IONPs exposure.
Fig. 8. (A) Iron oxide (Fe3 O4 ) nanoparticles induced reduction in the intensity of mitochondrial membrane potential (MMP) in A-549 cells exposed for 24 h. MMP was studied using Rh123 fluorescent dye. (B) Percent induction of MMP in A-549 cells following the exposure of various concentrations of iron oxide (Fe3 O4 ) for 24 h (*p < 0.01, **p < 0.001 vs control).
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Iron oxide nanoparticles induced human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling is also reported [24]. In our study, we also found that Fe3 O4 -NPs decrease MMP in A-549 cells. It is well known that high levels of ROS can lead to cellular damage by resulting in mitochondrial damage, which can then induce cell death [60,61]. The decrease in the MMP, based on cationic fluorescent probe Rh123, indicated the role of oxidative stress and ROS generation in the toxicity of Fe3 O4 -NPs in A-549 cells due to the generation of free radicals during the mitochondrial respiration. In conclusion, this study exhibited that the synthesized Fe3 O4 NPs have a crystalline structure with smooth surface. The crystalline Fe3 O4 -NPs showed concentration-dependent cytotoxicity in human lung alveolar epithelial cells through the oxidative stress. Significant induction in intracellular ROS generation and reduction in MMP also suggests the role of Fe3 O4 -NPs in cell death. The findings from this study provide new understandings on Fe3 O4 NPs and their effects on A-549 cells. Further experiments are in process to understand the mechanism(s) of action involved in the Fe3 O4 -NPs induced cell death in A-549 cells. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This project was supported by NSTIP Strategic Technologies Programs (number 12-MED2491-02) in the Kingdom of Saudi Arabia. JM is grateful to the visiting professor program (VVP) of the King Saud University, Riyadh, Saudi Arabia, for all the help and support in conducting this collaborative research work. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Synthesis, characterization and toxicological evaluation of iron oxide nanoparticles in human lung alveolar epithelial cells Sourabh Dwivedi a,b , Maqsood A. Siddiqui a,b,∗ , Nida N. Farshori c , Maqusood Ahamed d , Javed Musarrat a,b , Abdulaziz A. Al-Khedhairy a,b a
Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Al-Jeraisy Chair for DNA Research, King Saud University, Riyadh, Saudi Arabia c Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia d King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, Saudi Arabia b
a r t i c l e
i n f o
Article history: Received 13 March 2014 Received in revised form 9 June 2014 Accepted 30 June 2014 Available online 7 July 2014 Keywords: A-549 cells Iron oxide nanoparticles Cytotoxicity Oxidative stress
a b s t r a c t The present investigation was aimed to characterize the synthesized iron oxide nanoparticles (Fe3 O4 -NPs) and to assess their cytotoxicity and oxidative stress in human lung alveolar epithelial cells (A-549). Fe3 O4 NPs were characterized by X-ray diffraction, transmission electron microscopy, dynamic light scattering, and atomic force microscopy. The morphology of the Fe3 O4 -NPs was found to be variable with a size range of 36 nm. A-549 cells were exposed to Fe3 O4 -NPs (10–50 g/ml concentrations) for 24 h. Post exposure, cytotoxicity assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT; neutral red uptake, NRU; and cellular morphology) and oxidative stress (lipid peroxidation, LPO and glutathione, GSH) were evaluated. Further, intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were also studied. MTT and NRU assays revealed a concentration-dependent decrease in the cell viability of A-549 cells. Fe3 O4 -NPs exposed cells also altered the normal morphology of the cells. Furthermore, the cells showed significant induction of oxidative stress. This was confirmed by the increase in LPO and ROS generation, and the decrease in the GSH level and MMP. Our results demonstrated that Fe3 O4 -NPs induced cytotoxicity is likely to be mediated through the oxidative stress and ROS generation in A-549 cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Metal oxide nanoparticles are widely being used in many industrial products, i.e., catalysts, pigments, food additives, sun screens and cosmetics [1,2]. The applications and productions of these nanoparticles at large number have brought attention to their risk factors. It is well known that nanoparticles are being released during particle synthesis and handling of dry powders and liquid suspensions [3]. Toxicity of various metal oxide nanoparticles has also been reported in vitro [4–9] as well as in vivo [10–12] setups. Numerous studies indicate that metal oxide nanoparticles have the ability to generate reactive oxygen species (ROS) [13,14] and they are involved in the cytotoxicity due to their small size
∗ Corresponding author at: Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Tel.: +966 14699532; fax: +966 542967835. E-mail addresses: [email protected], [email protected] (M.A. Siddiqui). http://dx.doi.org/10.1016/j.colsurfb.2014.06.064 0927-7765/© 2014 Elsevier B.V. All rights reserved.
and large surface area [15]. Experimental evidences also showed that nanoparticles released by sprays and powders can potentially deposit in the respiratory system [16,17]. Iron oxide (Fe3 O4 ) nanoparticles have been applied broadly to bioscience and clinical research for various purposes; the most common includes magnetic cell labeling [18,19], separation and tracking [20], for therapeutic purposes in hyperthermia [21], in drug delivery [22], and for diagnostic purposes, e.g., as contrast agents for magnetic resonance imaging (MRI) [23]. Although the cytotoxic effects of iron oxide nanoparticles are known [24–26], the mechanism(s) of their induced cytotoxicity is not clearly understood. Since, human exposure to iron oxide may occur through the exposure routes of inhalation and ingestion at occupational settings. Therefore, the present investigation was aimed to understand the mechanism(s) of cell death induced by iron oxide nanoparticles in human lung alveolar epithelial cells (A-549) under in vitro conditions. These in vitro systems are cost-effective, rapid and reproducible with low or no ethical dubious [27,28]. In this study, firstly we have focused on the detailed synthesis and physicochemical characteristics of iron oxide nanoparticles; secondly, the effect
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of iron oxide nanoparticles on (i) cell viability, (ii) morphological alterations, (iii) oxidative stress markers, (iv) ROS generation, and (v) mitochondrial membrane potential (MMP) in A-549 cells. 2. Materials and methods 2.1. Cell culture A-549 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.2% sodium bicarbonate, and antibiotic/antimycotic solution (100×, 1 ml/100 ml of medium, Invitrogen, Life Technologies, USA). The cells were maintained in 5% CO2 , 95% atmosphere at 37 ◦ C. Cells were assessed for cell viability by trypan blue dye exclusion assay as described earlier [29] and batches showing more than 98% cell viability were used in the experiments. 2.2. Reagents and consumables All other specified chemicals, culture mediums, reagents, and diagnostic kits were procured from Sigma Chemical Company Pvt. Ltd., St. Louis, MO, USA unless otherwise stated. Culture wares and other plastic consumables used in the study were procured from Nunc, Denmark. 2.3. Experimental design A-549 cells were treated with various concentrations of Fe3 O4 NPs (10–50 g/ml) for 24 h. Cells were studied for the cytotoxicity by MTT and NRU assays and morphological alterations. Further, oxidative stress markers, i.e. lipid peroxidation (LPO), glutathione (GSH), reactive oxygen species (ROS) generation, and MMP were studied. 2.4. Synthesis of Fe3 O4 -NPs Fe3 O4 -NPs were prepared by chemical co-precipitation of Fe2+ and Fe3+ ions in an alkaline solution followed by a treatment under hydrothermal conditions [30]. Briefly, FeSO4 ·7H2 O (2.7 g) and FeCl3 (5.7 g) were dissolved in 10 ml Mili-Q water. Then, these two solutions were thoroughly mixed and added to double volume of 10 M ammonium hydroxide with constant stirring at 28 ◦ C. After that the dark black slurry of Fe3 O4 particles was heated to 80 ◦ C in a water bath for 30 min. Ion impurities such as chlorides and sulphates were removed by washing the particles several times with Mili-Q water. The particles were then dispersed in 10 ml Mili-Q water and sonicated for 20 min. 2.5. Characterization of Fe3 O4 -NPs 2.5.1. X-ray diffraction analysis of Fe3 O4 -NPs Finely powdered sample of Fe3 O4 -NPs was analyzed using X’pert PRO analytical diffractometer (Almelo, The Netherlands) ˚ in the range of 20◦ ≤ 2 ≤ 80◦ using CuK␣ radiation ( = 1.54056 A) at 40 keV. In order to calculate the particle size (D) of Fe3 O4 -NPs, the Scherrer’s equation (D = 0.9/ˇ cos ) has been used [31], where is the wavelength of X-ray, ˇ is the broadening of diffraction line measured as half of its maximum intensity in radians and is the Bragg’s diffraction angle. The average particle size of Fe3 O4 -NPs was estimated from the line width of the XRD peak. 2.5.2. Analysis of Fe3 O4 -NPs by transmission electron microscopy (TEM) TEM analysis of Fe3 O4 -NPs was performed on a transmission electron microscope (Hitachi, H-7500, Japan) at an accelerating
voltage of 90 kV. Samples were prepared by drop-coating Fe3 O4 NPs solutions (1% Fe3 O4 -NPS) onto carbon-coated gold TEM grids. Film of Fe3 O4 -NPs sample on TEM grid was allowed to stand for 2 min. The extra solution was removed using a blotting paper and the grid was allowed to dry prior to measurement. 2.5.3. Atomic force microscopy (AFM) of Fe3 O4 -NPs Fe3 O4 -NPs were examined using AFM (Veeco Instruments, USA). Analysis was performed by running the machine in non-contact tapping mode [32]. Characterization of Fe3 O4 -NPs was done by observing the patterns appeared on the surface topography and analyzing the AFM data. Tapping mode imaging was implemented in ambient air by oscillating the cantilever assembly at or near the cantilever’s resonant frequency using a piezoelectric crystal. The topographical images were obtained in tapping mode at a resonance frequency of 218 kHz. Data were analyzed through WSXM software. 2.5.4. Dynamic light scattering Fe3 O4 -NPs powder was suspended in deionized ultrapure water to obtain a concentration of 50 g/ml, and was sonicated at 40 W for 15 min. Hydrodynamic particle size and Zeta potential () of Fe3 O4 NPs in an aqueous suspension were determined by measuring the dynamic light scattering using of a ZetaSizer-HT (Malvern, UK). 2.6. Cytotoxicity of Fe3 O4 -NPs 2.6.1. MTT assay Percent cell viability was assessed using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described by Siddiqui et al. [33]. Briefly, cells (1 × 104 ) were allowed to adhere for 24 h in CO2 incubator at 37 ◦ C in 96 well culture plates. After the exposure, MTT (5 mg/ml of stock in phosphate buffer saline, PBS) was added (10 l/well in 100 l of cell suspension), and plates were incubated for 4 h. The supernatant was discarded and 200 l of DMSO were added to each well and mixed gently. The developed color was read at 550 nm. Untreated sets were also run under identical conditions and served as control. 2.6.2. Neutral red uptake (NRU) assay NRU assay was carried out following the protocol described [34]. After the exposure, the medium was aspirated and cells were washed twice with PBS, and incubated for 3 h in a medium supplemented with neutral red (50 g/ml). The medium was then washed off rapidly with a solution containing 0.5% formaldehyde and 1% calcium chloride. Cells were further incubated for 20 min at 37 ◦ C in a mixture of acetic acid (1%) and ethanol (50%) to extract the dye. The plates were read at 550 nm. The values were compared with the control sets. 2.6.3. Morphological analysis by phase contrast microscope Morphological changes in A-549 cells treated with Fe3 O4 -NPs were observed to determine the alterations induced by Fe3 O4 -NPs. The cell images were taken using an inverted phase contrast microscope at 20× magnification. 2.7. Lipid peroxidation (LPO) Lipid peroxidation was performed using thiobarbituric acidreactive substances (TBARS) protocol [35]. Briefly, after the exposure, A-549 cells were collected by centrifugation and sonicated in ice cold potassium chloride (1.15%) and were centrifuged for 10 min at 3000 × g. Resulting supernatant (1 ml) was added to 2 ml of thiobarbituric acid (TBA) reagent (15% TCA, 0.7% TBA and 0.25 N HCl) and was heated at 100 ◦ C for 15 min in a boiling water
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ROS generation was assessed using 2,7-dichlorodihydrofluorescein diacetate dye (DCFH-DA; Sigma–Aldrich, USA) as a fluorescence agent following the protocol earlier described [7]. Cells were washed with PBS and incubated for 60 min in DCFH-DA (20 M) containing incomplete culture medium in dark at 37 ◦ C. Then, the cells were analyzed for intracellular fluorescence using fluorescence microscope.
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2 theta Fig. 1. XRD pattern of the iron oxide nanoparticles (Fe3 O4 -NPs). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
MMP was measured following the protocol of Zhang et al. [37]. Briefly, control and treated cells were washed twice with PBS. Then the cells were further treated with 10 g/ml of Rhodamine-123 fluorescent dye for 1 h at 37 ◦ C in dark. Cells were then washed twice with PBS. The fluorescence intensity of Rhodamine-123 was measured using fluorescence microscope by grabbing the images at 20× magnification. 2.11. Statistical analysis
bath. Samples were then placed in cold ice and were centrifuged at 1000 × g for 10 min. The absorbance of the supernatant was measured at 550 nm. 2.8. Glutathione (GSH) content Intracellular GSH content was estimated as described [36]. Briefly, cells were collected by centrifugation and the cellular protein was precipitated by incubating 1 ml sonicated cell suspension with 1 ml TCA (10%) on ice for 1 h, followed by 10 min centrifugation at 3000 rpm. The supernatant was added to 2 ml of 0.4 M Tris buffer (pH 8.9) containing 0.02 M EDTA, followed by an addition of 0.01 M 5,5 -dithionitrobenzoic acid (DTNB) to a final volume of 3 ml. The tubes were incubated for 10 min at 37 ◦ C in shaking water bath. The absorbance of the yellow color developed was read at 412 nm.
Results were expressed as mean ± standard error of three experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Post hoc Dunnett’s test was applied to compare values between control and treated groups. The values depicting p < 0.05 were considered as statistically significant. 3. Results 3.1. Characterization of iron oxide nanoparticles (Fe3 O4 -NPs) Fig. 1 shows that the XRD pattern of Fe3 O4 -NPs clearly exhibiting the crystalline nature of this material. Based on the comparison of their XRD patterns with the standard pattern of Fe3 O4 (JCPDS 750033). The diffraction peaks corresponding to (2 2 0), (3 1 1), (4 0 0),
Fig. 2. Panel (A) representative transmission electron micrograph recorded from a drop-coated film of the aqueous solution of Fe3 O4 -NPs; (B) represents the 3D topography of Fe3 O4 -NPs in top view by atomic force microscopic analysis; and (C) average hydrodynamic size of Fe3 O4 -NPs.
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Fig. 3. Cytotoxicity assessments by MTT assay in A-549 cells following the exposure of various concentrations of iron oxide (Fe3 O4 ) nanoparticles for 24 h. Values are mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 vs control).
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Fig. 4. Cytotoxicity assessments by NRU assay in A-549 cells following the exposure of various concentrations of seed extract of iron oxide (Fe3 O4 ) nanoparticles for 24 h. Values are mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 vs control).
(5 1 1), and (4 4 0) are quite identical to the characteristic peaks of the Fe3 O4 crystal structure. The crystallite size has been estimated from the XRD pattern using the Scherrer’s equation [31]. The average crystallite size of Fe3 O4 -NPs was also found to be 36 nm. Fig. 2A shows the typical TEM image of Fe3 O4 -NPs. The average diameter was calculated by measuring over 100 particles in random fields of TEM view. The average TEM diameter of Fe3 O4 -NPs was 22 nm which is supporting the XRD data. The morphology and size of the Fe3 O4 -NPs were further validated by AFM analysis. Fig. 2B, shows the AFM image of Fe3 O4 -NPs obtained on scanning probe microscope in tapping mode, under ambient conditions. The average size of the nanoparticles and surface roughness (Ra ) were determined to be 31 and 22 nm, respectively using WSXM and SPIP softwares. The results of hydrodynamic size of analysis obtained with dynamic light scattering are shown in Fig. 2C. The distribution curves show the Fe3 O4 -NPs aggregates of 174 nm in Milli-Q water.
Depletion in the glutathione level in cultured A-549 cells exposed to 10–50 g/ml concentrations of Fe3 O4 -NPs are summarized in Fig. 6B. The results indicate that Fe3 O4 -NPs decreased the GSH levels in a concentration-dependent manner. A significant decrease in GSH level was observed as 15%, 38%, and 54% at 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively as compared to the control.
3.2. Cytotoxicity assessment by MTT assay
3.7. ROS generation
The results of cytotoxicity assessment of Fe3 O4 -NPs in A-549 cells by MTT assay are summarized in Fig. 3. A concentrationdependent statistically significant (p < 0.001) decrease in percentage cell viability of A-549 cells was recorded after 24 h of exposure of Fe3 O4 -NPs. Cell viability at 10, 25, and 50 g/ml of Fe3 O4 -NPs was recorded as 84%, 72%, and 56%, respectively.
Statistically significant (p < 0.001) ROS generation was observed in A-549 cells exposed to Fe3 O4 -NPs at 10, 25, and 50 g/ml concentrations for 24 h (Fig. 7A and B). An increase in ROS generation was observed in a concentration-dependent manner i.e., 23%, 46%, and 82% of untreated control following the exposure of 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively.
3.3. Cytotoxicity by NRU assay
3.8. Mitochondrial membrane potential (MMP)
Cytotoxicity assessment of Fe3 O4 -NPs by NRU assay is summarized in Fig. 4. A concentration-dependent statistically significant (p < 0.001) decrease in cell viability of A-549 cells was also recorded following the exposure of Fe3 O4 -NPs for 24 h. Cell viability was recorded as 87%, 79%, and 65% at 10, 25, and 50 g/ml, respectively.
The effect of Fe3 O4 -NPs exposure on MMP in A-549 cells was evaluated. A concentration-dependent statistically significant (p < 0.001) decrease in the level of MMP was also observed in A-549 cells after the exposure of Fe3 O4 -NPs for 24 h. The decrease in MMP was observed to be 21%, 44%, and 62% at 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively as compared to untreated control (Fig. 8A and B).
3.4. Morphological changes The morphological changes observed in A-549 cells exposed to Fe3 O4 -NPs for 24 h are shown in Fig. 5. Alterations in the morphology of A-549 cells were observed under phase contrast inverted microscope. The cells indicate the most prominent effects after the exposure of Fe3 O4 -NPs, the changes in their morphology were found in a concentration- and time-dependent manner. A-549 cells exposed to 10, 25, and 50 g/ml concentrations reduced the normal morphology of the cells and cell adhesion capacity in compared to the control.
3.5. Lipid peroxidation Lipid peroxidation level induced by Fe3 O4 -NPs is summarized in Fig. 6A. A concentration-dependent statistically significant increase in lipid peroxidation was also observed. An increase of 15%, 32%, and 91% was observed at 10, 25, and 50 g/ml of Fe3 O4 -NPs, respectively. 3.6. Glutathione depletion
4. Discussion In spite of their many advantages, nanoparticles may have hazardous effects because of their unique physicochemical properties. Iron oxide nanoparticles (Fe3 O4 -NPs) have attracted considerable attention in terms of their potential role in biomedical and clinical research for various purposes, including magnetic cell labeling, separation and tracking, drug delivery, therapeutics, and diagnosis. The physicochemical characterization of nanoscale materials is
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Fig. 5. Morphological changes in A-549 cells exposed to various concentrations of iron oxide (Fe3 O4 ) nanoparticles for 24 h. Images were taken using an inverted phase contrast microscope (OLYMPUS CKX 41) at 20× magnification.
state. This finding is supported by our previous investigations [40–42]. To evaluate the cytotoxicity of the synthesized Fe3 O4 -NPs on human lung alveolar epithelial cells (A-549), we have employed MTT and NRU assays. The viability of A-549 cells was significantly decreased with the increasing concentrations of Fe3 O4 -NPs. The cell mortality data obtained from this study allowed us to predict their potential cytotoxic effect. Our data are in well agreement with the previous reports of Fe3 O4 -NPs induced cytotoxicity in various cell types in vitro, including; ARPE-19 cells [43], human bronchial and alveolar epithelial cells [44], human lymphoblastoid TK6 cells, primary human blood cells [45], and in vivo rat model [46]. Our results also support a concentration-dependent response of Fe3 O4 -NPs as observed by Mendes et al. [47], who showed that carbon coated Fe3 O4 -NPs induce cytotoxicity in a concentration-dependent
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% Change in Glutathione level
% Change in Lipid peroxidation level
essential in biomedical research for better interpretation of results [7,38]. Parameters including shape, size, crystal structure, purity, hydrodynamic size, and agglomeration have been suggested for NPs characterization [39]. We have utilized the XRD, TEM, AFM and DLS techniques to characterize the NPs. Our XRD results confirm the crystalline nature of Fe3 O4 -NPs. TEM showed that NPs were fairly distributed with a smooth surface of an average diameter of 22 nm. Agglomeration and stability of NPs is also a major concern in nanomedicine research. The average hydrodynamic size of the present Fe3 O4 NPs in water determined by DLS was 174 nm. DLS is widely used to determine the size of brownian NPs in colloidal suspensions in the submicron and nano ranges. The higher size of nanoparticles in aqueous suspension as compared to TEM might be due to the tendency of particles to agglomerate in aqueous
100.0
*
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**
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**
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Fig. 6. Iron oxide (Fe3 O4 ) nanoparticles induced oxidative stress in A-549 cells exposed for 24 h. (A) Lipid peroxidation; (B) glutathione depletion. Values are mean ± SE of three independent experiments (*p < 0.01, **p < 0.001 vs control). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. (A) Iron oxide (Fe3 O4 ) nanoparticles induced ROS generation in A-549 cells. ROS generation was studied using dichlorofluorescindiacetate (DCFH-DA) dye after the exposure of iron oxide (Fe3 O4 ) Nanoparticles for 24 h. (B) Percent induction of ROS generation in A-549 cells following the exposure of various concentrations of iron oxide (Fe3 O4 ) nanoparticles for 24 h (*p < 0.01, **p < 0.001 vs control).
manner in HeLa Kyoto, human osteosarcoma (U2 OS), mouse fibroblasts (NIH 3T3), and mouse macrophages (J7442) cells. The cytotoxic effects of Fe3 O4 -NPs in A-549 cells may be due to the active physicochemical interaction of iron atoms with the functional groups of the intracellular proteins, which interact with the proteins and form a protein coat, which is believed to cause the toxicity [48]. The morphological alterations in A-549 cells exposed to Fe3 O4 NPs as observed by phase contrast inverted microscope showed the detachment with increasing concentrations. Our data support that cellular stress induced by nanoparticles can result in the alteration of the cellular morphology, when cells were treated with higher concentration of nanoparticles [49] resulting in a decrease in the total cell areas and a deviation of the cells from typical morphology [26]. Although, excessive oxidative stress can be harmful, however it is involved in normal cellular processes of cell signaling. Oxidative stress is a prominent feature of the response to metal oxide nanoparticles. Many studies have shown that exposure to nanoparticles promotes cellular oxidative stress [50–52]. These include
evidences of increased ROS production, depletion of antioxidants or elevation in oxidative products such as LPO, in a variety of cell types, including microglia [53], fibroblast [54], lung epithelial cells [50], and in human liver cells HepG2 [8]. Results from the present study also showed that Fe3 O4 -NPs induce oxidative stress in A549 cells in a concentration-dependent manner with an increase in the level of lipid peroxidation and a decrease in the antioxidant enzyme GSH. These findings suggest that oxidative stress may be the primary mechanism for toxicity of Fe3 O4 -NPs in A-549 cells. Our results are also supported by the previous findings suggesting that the cytotoxicity of NPs is mediated through the oxidative stress. Several studies demonstrated metal oxide nanoparticles induced oxidative stress [7,55,56]. In general, one of the most common toxic effects for nanoparticles is the induction of reactive oxygen species (ROS) generation [57]. Similar to oxidative stress, a significant increase in intracellular ROS generation was also found in a concentration-dependent manner in A-549 cells exposed to Fe3 O4 -NPs for 24 h. Our results were also in agreement with previous studies [58,59], where they showed maximum ROS induction at 24 h of IONPs exposure.
Fig. 8. (A) Iron oxide (Fe3 O4 ) nanoparticles induced reduction in the intensity of mitochondrial membrane potential (MMP) in A-549 cells exposed for 24 h. MMP was studied using Rh123 fluorescent dye. (B) Percent induction of MMP in A-549 cells following the exposure of various concentrations of iron oxide (Fe3 O4 ) for 24 h (*p < 0.01, **p < 0.001 vs control).
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Iron oxide nanoparticles induced human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling is also reported [24]. In our study, we also found that Fe3 O4 -NPs decrease MMP in A-549 cells. It is well known that high levels of ROS can lead to cellular damage by resulting in mitochondrial damage, which can then induce cell death [60,61]. The decrease in the MMP, based on cationic fluorescent probe Rh123, indicated the role of oxidative stress and ROS generation in the toxicity of Fe3 O4 -NPs in A-549 cells due to the generation of free radicals during the mitochondrial respiration. In conclusion, this study exhibited that the synthesized Fe3 O4 NPs have a crystalline structure with smooth surface. The crystalline Fe3 O4 -NPs showed concentration-dependent cytotoxicity in human lung alveolar epithelial cells through the oxidative stress. Significant induction in intracellular ROS generation and reduction in MMP also suggests the role of Fe3 O4 -NPs in cell death. The findings from this study provide new understandings on Fe3 O4 NPs and their effects on A-549 cells. Further experiments are in process to understand the mechanism(s) of action involved in the Fe3 O4 -NPs induced cell death in A-549 cells. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This project was supported by NSTIP Strategic Technologies Programs (number 12-MED2491-02) in the Kingdom of Saudi Arabia. JM is grateful to the visiting professor program (VVP) of the King Saud University, Riyadh, Saudi Arabia, for all the help and support in conducting this collaborative research work. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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