In vitro toxicity evaluation of ultra-small MFe₂O₄ (M = Fe, Mn, Co) nanoparticles using A549 cells

Abstract

As ferrite nanoparticles (MFe₂O₄) have been widely used in biomedical field, their safety evaluation has been paid great attention both in vitro and in vivo. In this paper, the ultra-small MFe₂O₄ (M = Fe, Mn, Co) nanoparticles with the average size less than 5 nm were prepared by thermal decomposition method and characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), high-resolution transmission electron microscopy (HRTEM), and vibrating sample magnetometry (VSM). The toxic effect on human lung epithelial A549 cells treated with MFe₂O₄ nanoparticles at the different concentrations was evaluated in vitro. Mitochondrial function (MTT assay), cellular morphology, reactive oxygen species (ROS), superoxide dismutase (SOD), membrane lipid peroxidation (LPO) and glutathione (GSH) were assessed as toxicity end points. The results showed that the cytotoxicity of ultra-small MFe₂O₄ nanoparticles was in a dose- and time-dependent manner. Moreover, ultra-small Fe₃O₄ nanoparticles were found to be nearly non-toxic in A549 cells, while MnFe₂O₄ nanoparticles exhibited cytotoxic effects, and CoFe₂O₄ nanoparticles exerted higher cytotoxic effects among the three studied particles at the same concentration.

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1 Introduction

Magnetic iron oxide nanoparticles have attracted great interest from scientists and technologists due to their favorable magnetic and electrical properties. Spinel ferrites (MFe₂O₄, M = Fe, Mn, Co, Zn, or Ni) nanoparticles are the most attractive iron oxide nanoparticle and have been commonly and widely used in biomedical research,^1–3 such as magnetic resonance imaging (MRI),^4–6 in clinical diagnosis and the treatment of diseases,^7,8 magnetic carriers for drug targeting and catalysis,^9–11 immune assays^12,13 and targeting photodynamic therapy.¹⁴ And their applications are affected by the composition, shape, particle size, size monodispersity, surface chemistry, magnetization, stability, non-toxicity, biocompatibility and short blood half-life of nanoparticles.¹⁵ Especially, the size control is one of the most important factors.¹⁶ The large nanoparticles are easily removed from the circulation by the reticuloendothelial system (RES).^17,18 Therefore, the study on ultra-small nanoparticles (<20 nm) is significant due to the longer blood half-time and a wider biodistribution.^19–21

Moreover, it is well known that the biocompatibility and cytotoxicity of nano-sized materials, expected to be used in biomedical field, should be taken into consideration. Owing to high particle reactivity, the particles with small size have ability to cross cell and tissue barriers, and resistance to biodegradation. Some studies have revealed the nano-sized materials are toxic to cells, tissues, organs, and organisms compared with the bulk-sized particles with the same composition.^22,23 The key paradigms of nanotoxicity consist of activation of oxidative stress and genotoxicity, which had been reported by many researches.^24,25 The mitochondrial function (MTT assay) and the cell morphologic characteristics could be used to evaluate cell viability through the mitochondrial dysfunction and changes of cell shape, respectively. Increased reactive oxygen species (ROS) levels, depleted antioxidant glutathione (GSH) levels, decreased superoxide dismutase (SOD) levels and elevated thiobarbituric acid reactive substance (TBARS) levels are applied to manifest nanoparticles-induced oxidative stress. For example, Han et al. used MTT and lactate dehydrogenase (LDH) assays examined the potential toxicity of organophyllosilicates on cells from different organs and indicated that organoclays had little cytotoxicity at concentrations as high as 500 μg mL⁻¹ in A549, HT-29 (colon epithelial cancer), MRC-5 (lung fibroblast) and CCD-986sk (skin fibroblast) cells tested.²⁶ Moreover, Könczöl et al. reported that ROS formation played an important role in the genotoxicity of magnetite in A549 cells. The enhanced ROS production induced slight cytotoxicity after the A549 cells exposed to different size fractions of magnetite particles after 24 h which demonstrated that magnetite particles were able to induce concentration-dependent and a slight size-dependent genotoxicity.²⁷

Although, a lot of studies have been conducted to evaluate the cytotoxicity of MFe₂O₄ nanoparticles,^28–31 only a few significant studies have reported potential cytotoxicity of different ultra-small MFe₂O₄ nanoparticles with average size less than 5 nm. In the present study, the ultra-small MFe₂O₄ (M = Fe, Mn, Co) nanoparticles, with the average size less than 5 nm, were prepared and characterized. The cytotoxicity of MFe₂O₄ nanoparticles was further evaluated by using A549 cells types of MFe₂O₄ (M = Fe, Mn, Co) nanoparticles exerted different cytotoxic effects through MTT assay, cellular morphology, ROS, SOD, MDA and GSH to assess the end points of toxicity.

2 Experimental

2.1 Materials

2.1.1 Reagents and materials. Ferric chloride hexahydrate (FeCl₃·6H₂O), ferrous sulfate heptahydrate (FeSO₄·7H₂O), 2,2-azobisisobutyronitrile (AIBN), and ammonium hydroxide (25%, aqueous solution) were obtained from Sinopharm Chemical Reagent Co. Ltd, and used without further purification. Methacrylicacid (MAA) was obtained from Shanghai Lingfeng Chemical Reagent Co. Ltd 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (USA). Dulbecco's modified Eagle's medium (DMEM) cell culture medium, penicillin/streptomycin, trypsin and fetal bovine serum (FBS) were bought from Gibco Invitrogen (USA). All other chemicals and reagents were of analytical grade and used as received.

2.2 Methods

2.2.1 Synthesis and characterization of ultra-small magnetite MFe₂O₄ nanoparticles. MFe₂O₄ (M = Fe, Mn, Co) nanoparticles were synthesized following the thermal decomposition method as described elsewhere.^7,32,33 In brief, thiol-functionalized poly(methacrylic acid) (PMAA-PTMP) (number-molecular-weight (Mn) = 6901 g mol⁻¹, weight-molecular-weight (M_w) 7041 g mol⁻¹) was dissolved in 50 mL Milli-Q water (0.768 mM) and purged with nitrogen to remove oxygen. The polymer solution was heated to reflux. Then, the diluted HCl solution containing FeCl₃·6H₂O (0.54 mmol) and FeSO₄·7H₂O (0.27 mmol) was quickly injected into the hot solution, followed by concentrated ammonia solution (28%). The solution kept refluxing for 2 h before cooled down. The solution was dialyzed against Milli-Q water (the molecular weight cut-off was 8000–10 000 g mol⁻¹) for 72 h to remove impurities. Then the dialyzed solution was free-dried. The other MFe₂O₄ nanoparticles were prepared by the similar procedure except the use of a mixture of Fe³⁺- and Fe²⁺-precursors.

The ultra-small MFe₂O₄ nanoparticles were characterized by means of powder X-ray diffraction (XRD), X-ray photoelectron spectrometer (XPS), high-resolution transmission electron microscopy (HRTEM) and vibrating sample magnetometer (VSM). The particle size and shape of products were measured with HRTEM with a JEOL model JEM 2100 electron microscope operating at an accelerating voltage of 200 kV. The crystal structure of the ultra-small MFe₂O₄ nanoparticles was recorded on a Rigaku corporation D/MAX 2550 VB/PC Multi-Purpose X-ray diffractometer with Cu Kα radiation (λ = 0.1542 nm). The surface composition of the as-prepared hybrid was detected by XPS (ESCALAB 250Xi, Thermo Fisher). Magnetic properties were recorded using a VSM (Lake Shore, USA).

2.2.2 Cell culture. A549 cells were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in a full DMEM containing FBS (10%), penicillin/streptomycin (1%), and incubated under 5% CO₂ in atmosphere at 37 °C.

2.2.3 Cytotoxicity assay. The in vitro cytotoxicity was assessed by using the MTT assay in A549 cells. Cells (1 × 10⁵ per well) were inoculated into a 96-well cell-culture plate and then incubated for 24 h. All incubations were performed at 37 °C in a 5% CO₂-humidified incubator. 200 μL of MFe₂O₄ nanoparticles with different concentrations (0, 25, 50, 100, 200, 400, and 800 μg mL⁻¹, DMEM) were added to the wells, separately. After incubation for 24 h and 48 h the supernatant was removed. Subsequently, MTT (20 μL, 5 mg mL⁻¹) solved in DMEM (200 μL) were added and the plates were incubated for another 4 h. Then supernatant was removed before DMSO was added to each well to dissolve the formazan. The absorbance at 492 nm and 630 nm was detected with spectrophotometric microplate reader (THERMO Multiskan MK3 spectrometer). Each data point was collected by averaging that of three wells, and the untreated cells were used as controls.

2.2.4 Qualitative observation of external cellular morphology. A549 cells were plated in 96-well plates with different concentrations (0, 100, 500, 1000 μg mL⁻¹, DMEM) of MFe₂O₄ nanoparticles and incubated for 48 h. Cells cultured in the medium without adding nanoparticles were taken as the control. After completion of the exposure period, cells (controlled and exposed) were observed by an inverted microscopy.

2.2.5 Intraceller ROS measurement. The generation of intracellular ROS was measured using peroxide-sensitive fluorescent probe, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, A549 cells were seeded into 96-well plate with a density of 1 × 10⁵ cells per well and then incubated for 24 h. 200 μL of MFe₂O₄ nanoparticles with different concentrations (0, 25, 50, 100 μg mL⁻¹, DMEM) were added into the wells, respectively. After 24 h, the mixture was washed twice with PBS and then incubated in working solution of 10 μM DCFH-DA (the Beyotime Institute of Biotechnology, Jiangsu, China) at 37 °C for 30 min. Fluorescence was then determined at 488 nm excitation and 525 nm emission.

2.2.6 Lipid peroxidation assay. The MDA content, an index of lipid peroxidation, was carried out by the Lipid Peroxidation MDA Assay Kit (the Beyotime Institute of Biotechnology, Jiangsu, China). A549 cells were plated into a 6-well plate at a density of 4.0 × 10⁵ cells per well and exposed to MFe₂O₄ nanoparticles at the concentrations of 0, 25, 50 and 100 μg mL⁻¹ for 24 h. The cells were washed twice with ice-cold PBS and then were lysed. Lysed cells were centrifuged to remove debris. The supernatant was used to measure MDA levels and the protein content. The absorbance of the supernatant was measured at 532 nm using the UV-Visible spectrophotometer (Evolution 220, Japan). The concentrations of TBARS were calculated using tetraethoxypropane as a reference standard. Protein content was determined for the same cell homogenate. The content of MDA levels were presented as the percentage of MDA production over the control.

2.2.7 Quantification of intracellular SOD levels. The A549 cells (1 × 10⁵ per well) were incubated into a 6-well cell-culture plate and then incubated at 37 °C in a 5% CO₂-humidified incubator for 24 h. 200 μL of MFe₂O₄ nanoparticles with different concentrations (0, 25, 50, 100 μg mL⁻¹, DMEM) were separately added into the wells. After incubation for 24 h, the cells were washed twice with ice-cold PBS and lysed. The cell homogenate was centrifuged at 4 °C for 10 min. The assay was performed on centrifugation supernatants according to SOD Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). The absorbance of cell supernatant was read at 450 nm using a UV-Visible spectrophotometer.

2.2.8 Quantification of intracellular GSH levels. The cellular levels of reduced GSH were determined using the GSH colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute, China). This method was involves on a chemical reaction between GSH and 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) to generate glutathione disulfide (GSSG) and nitro-5-thiobenzoicacid, a yellow product. Thus GSH concentration in a sample solution can be determined by the measurement at 412 nm absorbance.³⁴ A549 cells were plated into a 6-well plate at a density of 4.0 × 10⁵ cells per well and exposed to MFe₂O₄ nanoparticles at the concentrations of 0, 25, 50 and 100 μg mL⁻¹ for 24 h. After that cells were washed twice with ice-cold PBS and then lysed. The cell homogenate was centrifuged at 4 °C for 10 min. The absorbance of the supernatant was measured using a UV-Visible spectrophotometer. Protein content was determined for the same cell homogenate. GSH level was calculated from the absorbance at 412 nm and expressed as the percentage of control.

2.2.9 Statistical analysis. The significance of difference among groups was analyzed by variance analysis, and results presented as means ± standard deviation (±x − SD). The analysis was performed using one-way analysis of variance (ANOVA) test of SPSS 19.0 for windows. A value of p < 0.05 was considered significant.

3 Results and discussion

3.1 Characterization of MFe₂O₄ nanoparticles

The prepared MFe₂O₄ nanoparticles were characterized by XRD, XPS, HRTEM and VSM, respectively. The structures of MFe₂O₄ nanoparticles were analyzed by the XRD patterns showed in Fig. 1. For Fe₃O₄ particles (curve a in the Fig. 1), the characteristic (220), (311), (400), (422), (511) and (440) peaks are corresponded to the cubic ferrite (JCPDS no. 65-3107). After the substitution of M with Mn in the MFe₂O₄, three dilated peaks appear in the (311), (400) and (440) facets. Similar to MnFe₂O₄, the synthesized CoFe₂O₄ also provides the peaks of the only (311), (400) and (440) planes. Although peaks are broad, the XRD patterns of MnFe₂O₄ and CoFe₂O₄ are fit well that in JCPDS JCPDS no. 10-0319 and JCPDS no. 22-1086, respectively.

Fig. 1 XRD patterns of MFe₂O₄ (M = Fe, Mn, Co) nanoparticles.

The XPS technique was used to further confirm the surface composition of hybrids. Fig. 2A shows the XPS spectra of Fe₃O₄ nanoparticles. The binding energies at 712.2 eV and 726.0 eV are assigned to Fe 2p_3/2 and Fe 2p_1/2, which illustrate the coexistence of Fe³⁺ and Fe²⁺ valence states from Fe₃O₄.³⁵ As shown in Fig. 2B, two peaks observed at binding energies of 643.2 eV and 656.9 eV (Fig. 2B (b)) are assigned to Mn 2p_3/2 and Mn 2p_1/2. At the same time, the binding energies of Fe 2p_3/2 and Fe 2p_1/2 from Fe³⁺ were observed at 713.1 eV and 726.9 eV (Fig. 2B (a)). These results have obviously revealed the co-existence of Mn 2p and Fe 2p.^36,37 Further quantitative analysis of the Mn 2p and Fe 2p peaks displayed that the atomic ratio of Mn/Fe is about 0.5, which indicates our successful synthesis of MnFe₂O₄.³⁸ Similar results can be also observed in CoFe₂O₄. As depicted in Fig. 2C, the peaks center at 785.3, 799.3 eV, 710.9 and 725.4 eV belong to Fe 2p_3/2, Fe 2p_1/2, Co 2p_3/2 and Co 2p_1/2, respectively, and the Co/Fe atomic ratio is about 0.5.³⁹ All the data indicate we have successfully fabricated three kind of composites based on spinel structured MFe₂O₄.

Fig. 2 XPS patterns of MFe₂O₄ nanoparticles (A) Fe₃O₄, (B) MnFe₂O₄ and (C) CoFe₂O₄.

And the size and shape of MFe₂O₄ nanoparticles were determined by the HRTEM. In Fig. 3, the regular spherical MFe₂O₄ nanoparticles with the average size of 3 nm are well dispersed. The representative hysteresis loops of prepared MFe₂O₄ nanoparticles were determined at room temperature by a VSM. The saturation magnetization of Fe₃O₄ is ∼12.84 emu g⁻¹, while that of the MnFe₂O₄ is only ∼0.39 emu g⁻¹ and CoFe₂O₄ is ∼0.52 emu g⁻¹. As the particle size decreases, the magnetization at room temperature tends to decrease.⁴⁰

Fig. 3 TEM images of MFe₂O₄ nanoparticles, (A) Fe₃O₄, (B) MnFe₂O₄, and (C) CoFe₂O₄.

3.2 Cytotoxicity of MFe₂O₄ nanoparticles

Viability assays are basic steps in toxicology that explain the cellular response to a toxicant also give information on the cells death, survival, and metabolic activities. MTT assay makes use of the mitochondrial dysfunction to evaluate cell proliferation and cytotoxicity. In the present work, MTT assay was carried out to investigate the cytotoxicity of the MFe₂O₄ at the different concentrations (0, 25, 50, 100, 200, 400, and 800 μg mL⁻¹) for 24 h and 48 h on A549 cells. Cell viability after incubated with MFe₂O₄ nanoparticles at different concentrations for 24 h (A) and 48 h (B) is shown in Fig. 4. It can be seen that the cytotoxicity increases gradually with the increase of concentration and time. When the concentration is below 100 μg mL⁻¹, the viability of cells exposed to MFe₂O₄ nanoparticles is slight decreases over time, while the viability of cells is significant decreases when the concentration over 100 μg mL⁻¹. After incubated with MnFe₂O₄ sample, cell viability is only 72% and 68% at the concentrations of 400 and 800 μg mL⁻¹ at 48 h, respectively, and it is lower when exposed to CoFe₂O₄ sample (Fig. 4B). Besides, Fe₃O₄ nanoparticles show negligible toxicity towards cells, even up to a relatively high concentration of 800 μg mL⁻¹ and 48 h of incubation. These results indicate that cell viability decreased as a function of both time and dose.

Fig. 4 In vitro cytotoxicity of MFe₂O₄ nanoparticles for different concentrations to A549 cells at (A) 24 h and (B) 48 h.

3.3 Effect of MFe₂O₄ nanoparticles on cellular morphology

The morphology is an important indicator of the status of cell, which is also a most direct way to illustrate cytotoxicity through the alteration of cell shape. Together with the cytotoxicity assay, the changes of cell morphological on A549 cells after exposure to MFe₂O₄ nanoparticles were recorded. Fig. 5 illustrates the optical microscopic images of control group and cells treated with MFe₂O₄ nanoparticles with different concentrations (0, 100, 500, 1000 μg mL⁻¹). A large number of cells become rounded and shrunken, which implies a very high reduction in cell count with the increase of concentration after exposure. At the same concentration, CoFe₂O₄ nanoparticles have the highest reduction in cell count, followed by MnFe₂O₄ nanoparticles, while Fe₃O₄ nanoparticles have the least among the three MFe₂O₄ nanoparticles. In addition, there is unnoticeable difference between the Fe₃O₄ nanoparticles treated group and the control group, most cells adhere to the substrate tightly and are in normal spindle-shape under the low concentrations. The result indicates that A549 cells with Fe₃O₄ nanoparticles show more survival rate and negligible toxicity compared to cells with MnFe₂O₄ and CoFe₂O₄ nanoparticles.

Fig. 5 Morphological changes of A549 cells after 48 h exposure of Fe₃O₄, MnFe₂O₄ and CoFe₂O₄ at the concentrations of 0, 100, 500, 1000 μg mL⁻¹.

3.4 Effect of MFe₂O₄ nanoparticles on ROS levels

Oxidative stress is one of nanoparticles toxic mechanisms. The generation of ROS, a very first standard of cellular toxicity cascade reactions, is one commonly proposed toxicological mechanism of nanoparticles as an early indicator for cellular responses.^27,41 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) is used to detect changes in ROS activity, which permeates the cell membrane and accumulates mostly in the cytosol following deacetylation by esterases to 2′,7′-dichlorodihydrofluorescein (DCFH). This nonfluorescent product is converted by ROS into 2′,7′-dichlorofluorescein (DCF, E_x = 488 nm, E_m = 525 nm). The potential of MFe₂O₄ nanoparticles to induce oxidative stress was assessed by measuring the ROS level in A549 cells. As shown in Fig. 6, the MFe₂O₄ nanoparticles induce the intracellular production of ROS at the concentrations of 0, 25, 50 and 100 μg mL⁻¹ for 24 h, and resulted in dose-dependent increase of the intracellular ROS levels as compared to that of the control cells. Among the three MFe₂O₄ nanoparticles samples, CoFe₂O₄ nanoparticles cause the most serious oxidative stress, while Fe₃O₄ nanoparticles cause the slightest oxidative stress under the same concentrations. For instance, at the concentration of 100 μg mL⁻¹, the ROS level for CoFe₂O₄ nanoparticles treated cells is 3.1 times higher than that of control group, while it is 2.8 for MnFe₂O₄ nanoparticles treated cells and 1.9 for Fe₃O₄ nanoparticles treated cells.

Fig. 6 Effect of Fe₃O₄, MnFe₂O₄ and CoFe₂O₄ on ROS levels in A549 cells at the concentrations of 0, 25, 50 and 100 μg mL⁻¹ for 24 h. Data were presented as mean ± SD of three independent experiments. *p < 0.05 versus control cells.

3.5 Effect of MFe₂O₄ nanoparticles on SOD levels

SOD is viewed as an important antioxidant enzyme, which catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen. It plays an important role in the balance between oxidation and antioxidation of the organism. The increase of oxidative stress usually accompanies with the decrease of SOD activity. As shown in Fig. 7, in contrast to the control group, cells exposed to MFe₂O₄ nanoparticles (25, 50 and 100 μg mL⁻¹) for 24 h show different decrease in the SOD levels, respectively. The SOD level of A549 cells exposed to CoFe₂O₄ nanoparticles has the largest reduction. At the concentration of 100 μg mL⁻¹, it has been reduced more than half (64.8 ± 3.5%) compared to the control group. However, the SOD level of cells exposed to Fe₃O₄ nanoparticles has the lowest reduction (47.0 ± 0.3%). At the concentration of 100 μg mL⁻¹, the SOD level is reduced 47.0 ± 0.3% compared to the control.

Fig. 7 Effect of Fe₃O₄, MnFe₂O₄ and CoFe₂O₄ on SOD levels in A549 cells at the concentrations of 0, 25, 50 and 100 μg mL⁻¹ for 24 h. Data were presented as mean ± SD of three independent experiments. *p < 0.05 versus control cells.

3.6 Effect of MFe₂O₄ nanoparticles on lipid peroxidation

LPO and ROS can help cells to balance out the ratio, and the extent of membrane LPO plays an important role in cell metabolism. Higher production of intracellular ROS can lead to membrane LPO, which is a major indicator of oxidative stress and has been implicated in oxidative damage of cell biomolecules.^42,43 The extent of membrane LPO was estimated by measuring the formation of MDA. MDA, one of the end products of membrane LPO, is a major indicator of oxidative stress. As shown in Fig. 8, the MDA level is increased with MFe₂O₄ nanoparticles dosage (25, 50 and 100 μg mL⁻¹) compared to the control group for 24 h. The MDA level of CoFe₂O₄ increases approximately by 3.1 folds at the concentration of 100 μg mL⁻¹, which indicates that the cytotoxicity to A549 cells is CoFe₂O₄ nanoparticles > MnFe₂O₄ nanoparticles > Fe₃O₄ nanoparticles. Fe₃O₄ nanoparticles have the highly biocompatible among the three MFe₂O₄ nanoparticles. The results are consistent with ROS activity evaluation; and free radical resulted in the production of MDA.³⁴

Fig. 8 Effect of Fe₃O₄, MnFe₂O₄ and CoFe₂O₄ on MDA levels in A549 cells at the concentrations of 0, 25, 50 and 100 μg mL⁻¹ for 24 h. Data were presented as mean ± SD of three independent experiments. *p < 0.05 versus control cells.

3.7 Effect of MFe₂O₄ nanoparticles on GSH levels

GSH, an important endogenous antioxidant, is a natural antioxidant defense system. Excessive amount of ROS can lead to over much GSH, which can make cells lose normal functions causing cell death. The thiol groups of GSH can react with hydroxyl radicals, singlet oxygen, and hydrogen peroxide to protect the cell from damage. The depletion of GSH and indication of lipid peroxidation have been implicated in oxidative damage of cell biomolecules. Therefore, we further examined the GSH level in cells after 24 h treatment with 0, 25, 50 and 100 μg mL⁻¹ MFe₂O₄ nanoparticles (Fig. 9). Different MFe₂O₄ nanoparticles result in dose-dependent depletion of the intracellular GSH levels as compared to that of the control cells group. At the same concentration, CoFe₂O₄ nanoparticles have the highest reduction in GSH deletion, followed by MnFe₂O₄ nanoparticles, while Fe₃O₄ nanoparticles have the least reduction among the MFe₂O₄ nanoparticles. The results demonstrate that the toxicity of Fe₃O₄ to A549 cells is slighter than that of CoFe₂O₄ and MnFe₂O₄ nanoparticles at the same concentrations. According to Fig. 9, the intracellular GSH level of MFe₂O₄ nanoparticles is much higher than that of the control group, because MFe₂O₄ nanoparticles were coated with PMAA-PTMP containing a lot of thiol, which can increase intracellular GSH level. Furthermore, the GSH level decrease with the increase concentration of induced MFe₂O₄ nanoparticles, which in line with ROS level indicated that free radical species were generated by MFe₂O₄ nanoparticles exposure with reduction of intracellular antioxidant levels.

Fig. 9 Effect of Fe₃O₄, MnFe₂O₄ and CoFe₂O₄ on GSH levels in A549 cells at the concentrations of 0, 25, 50 and 100 μg mL⁻¹ for 24 h.

As mentioned, Fe₃O₄ nanoparticles were nearly no cytotoxicity among the three MFe₂O₄ (M = Fe, Mn, Co) nanoparticles at the same concentration. It may be explained by the core material of the MFe₂O₄ nanoparticle, Co and Mn mixed increasing the toxicity of nanoparticles. Recently, a series of works reported that Co and Mn ions induce much more cytotoxicity and apoptosis than Fe ions,^44–46 and Fe₃O₄ displayed less or no toxicity at the doses tested.⁴⁷

4 Conclusions

In summary, we prepared the ultra-small MFe₂O₄ (M = Fe, Mn, Co) nanoparticles by thermal decomposition method, and confirmed them by XRD, HRTEM, XPS and VSM. The toxicity of Fe₃O₄, MnFe₂O₄ and CoFe₂O₄ to A549 cells at different concentrations was investigated. All the results clearly indicated that the cytotoxicity of ultra-small MFe₂O₄ nanoparticles was in dose- and time-dependent manner, and cell viability appeared to be strongly relevant to ROS levels after 24 h exposure to MFe₂O₄ nanoparticles, which suggested that oxidative stress have a great effect on MFe₂O₄ nanoparticles induced cell death. It also indicated that Fe₃O₄ nanoparticles exhibited negligible cytotoxic effects in A549 cells compared with CoFe₂O₄ and MnFe₂O₄ nanoparticles. Therefore, it can be concluded that ultra-small Fe₃O₄ nanoparticles are a kind of safe material at cellular level.

Acknowledgements

This research was financially supported by the Science and Technology Commission of Shanghai Municipality (STCSM, contract No. 13ZR1412000).

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