# Synthesis, Characterization, and Cytotoxicity of Iron Oxide Nanoparticles

Authors: S. Kanagesan; M. Hashim; S. Tamilselvan; N. B. Alitheen; I. Ismail; A. Hajalilou; K. Ahsanul
Journal: Advances in Materials Science and Engineering
 (2013)

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## Abstract

In order to study the response of human breast cancer cells' exposure to nanoparticle, iron oxide (α-Fe2O3) nanoparticles were synthesized by a simple low temperature combustion method using Fe(NO3)3·9H2O as raw material. X-ray diffraction studies confirmed that the resultant powders are pure α-Fe2O3. Transmission electron microscopy study revealed the spherical shape of the primary particles, and the size of the iron oxide nanoparticles is in the range of 19 nm. The magnetic hysteresis loops demonstrated that the sample exposed ferromagnetic behaviors with a relatively low coercivity. The cytotoxicity of α-Fe2O3 nanoparticle was also evaluated on human breast cancer cells to address the current deficient knowledge of cellular response to nanoparticle exposure.


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## Body

## 1. Introduction

Recently, synthesis of oxide nanoparticles with homogeneous size has attracted a great deal of attention from researchers due to its unique optical, electrical, and magnetic properties and their potential interests in a variety of biomedical applications [1]. Iron oxides are very important materials, classified into three different forms, namely, hematite (α-Fe2O3), maghemite (γ-Fe2O3), and magnetite (Fe3O4) [2]. Among them, hematite (α-Fe2O3) is a thermodynamically stable iron oxide under ambient conditions. It has the corundum structure and offers high resistance to corrosion. Its low cost and nontoxicity make it the most environmentally friendly n-type semiconductor material (band gap of 2.1 eV) [3–7].They hold bear have great scientific interest due to its various extensive technological applications such as magnetic recording media, catalysis, pigments, electrode materials, gas sensors, optical devices, electromagnetic devices, lithium ion battery electrodes, sorbents, and drug delivery [8–13].The properties ofα-Fe2O3 strongly depend on the particle size, morphology, and its structure [14]. In order to obtain nanostructures of α-Fe2O3 powder, several techniques have been designed such as thermal oxidation technique [15], hydrothermal processing [16], mechanical millings [17], microwave synthesis [18], and sol-gel [19]. Among these methods, sol-gel combustion method has simple synthesis procedure and is cost-effective. This method can result in ultrafine homogeneous powder [20]. Magnetic nanoparticles have displayed great promise for use as tools in a wide variety of biomedical applications including drug delivery in cancer chemotherapy.Nanoparticles are attractive for cancer applications as they can be engineered to have multifunctionality serving simultaneously as imaging contrast agents, therapeutic agents, and drug delivery vehicles. Meanwhile, effects of nanoparticles on health and safety have also attracted more and more attention [21, 22]. It is already known that nanoparticles of different sizes have different abilities of entering target cells. Some researches have reported that small-sized nanoparticles are more readily able to enter the cancer cells and interact with components inside the cells; hence, those nanoparticles may act as a good system for drug delivery and offer new perspectives for diagnostic and targeted therapeutic approaches for cancers [23, 24]. Furthermore, magnetic particles like Fe2O3 nanoparticles have excellent biocompatible properties and thus could be used to perform many in vitro and in vivo applications including resonance imaging [25]. Some reports have indicated that magnetic particles of iron oxide could be phagocytosed by cancer cells and interact with components inside the cells; hence, those magnetic nanoparticles may act as a good system for drug delivery and offer new perspectives for diagnostic and targeted therapeutic approaches for cancers [26, 27]. Several researchers have studied the toxic effects of metal nanoparticles and reported the changes in cellular morphology, mitochondrial function, and apoptosis [28–30]. Numerous metal nanoparticles exhibited cytotoxicity after exposure to cells, and Fe2O3 nanoparticles were found to be more potent [31–33]. It has been shown that iron oxide nanoparticle had no measurable cytotoxic effect at lower doses (10–50 μg/mL), while there was a significant effect at higher levels (100–250 μg/mL) [34, 35]. Furthermore, Veranth and coworkers [36] studied the cytokine response in BEAS-2B cells due to metal oxide nanoparticles and found that metal oxide particles have a lower toxicity than an equal mass of micron-sized particles of the same nominal composition. Despite the wide-spread application of magnetic nanoparticles, there is a serious lack of information concerning the toxicity of these nanoparticles at the cellular and molecular levels. Only a few significant studies reported the potential cytotoxicity of Fe2O3 nanoparticles [28–30]. In view of this, we have successfully synthesized α-Fe2O3 nanoparticles by the simple sol-gel combustion method and have explored the cytotoxic effect on human breast cancer MCF-7 cells.


## 2. Experimental Techniques

### 2.1. Nanopowder Synthesis

In the typical procedure, 1 mol of Fe(NO3)3·9H2O solution was added with 100 mL of ethylene glycol, and the mixture was continuously stirred at room temperature to get a clear brown-coloured solution. This solution was continuously stirred for 8 h at a temperature of 90°C. Viscosity of the solution increased constantly with time. The reddish brown gel was calcinated at 250°C in a hot air oven for 24 h. The resultant powder was subsequently washed several times with distilled water and ethanol. The structure, morphology, and magnetic properties of the as-prepared sample were characterized by powder X-ray diffraction (XRD) and transmission electron microscopy (TEM). The magnetic characteristics of the specimen were measured at room temperature using a vibrating sample magnetometer (VSM) (LDJ9600).


### 2.2. Cell Proliferation Assays

MCF-7 cells, human breast cancer cells, were obtained from American Type Culture Collection, and the cells were grown in DMEM at 37°C under 5% CO2 in a humidified incubator. Cells were harvested, counted, and transferred to 96-well plates and incubated for 24 h prior to the addition of the nanoparticle. The iron oxide nanoparticles were processed and applied in various concentrations, and the treated cells were incubated for 24 h. MCF-7 cells were plated (1×104 cells/well) in triplicate in 96-well sterile microtiter plates and allowed to grow for 24 h for proper cell adherence. Cells were then treated with different concentrations of iron oxide nanoparticles for 24 h. Cells incubated with 25 μL of PBS were used as a control. After incubation, the media were discarded and replaced with 175 μL of fresh medium and 25 μL of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution then incubated at 37°C under 5% CO2 for 4 hours. The medium was removed, and 100 μL of dimethyl sulfoxide was added to solubilize the formazan crystals. Optical density was then taken at 575 nm. The results were expressed as the percentage proliferation with respect to vehicle-treated cells.


### 2.3. Trypan Blue Exclusion Test

The trypan blue exclusion assay was performed to analyze the cell viability afterα-Fe2O3 nanoparticle treatment. Equal amounts of MCF-7 cell suspension and trypan blue (0.4%) were mixed for 1-2 min and 10 μL of the mixture was placed on hemocytometer and observed under light microscope, and the cells were counted in all four fields.


### 2.4. Acridine Orange/Propidium Iodine (AO/PI) Staining

MCF-7 cells were treated with various concentrations ofα-Fe2O3 nanoparticles and stained with AO/PI (Sigma-Aldrich) dual dye to detect apoptotic or necrotic nuclei. The cells were seeded in 6-well tissue culture plates for 24 h followed by the α-Fe2O3 nanoparticle treatment at desired concentrations. After the incubation period, the media were removed, and the cells were washed twice with PBS. Fresh media were added in each well, and the cells were stained by adding 10 μL of AO/PI mix (10 mg mL−1 AO and 10 mg mL−1 PI in PBS) then incubated for 10 min. The cells were washed with PBS before visualizing under a fluorescence microscope (Nikon Eclipse, TS100, Tokyo) with an excitation filter of 480/30 nm.


### 2.5. Annexin V-PI Staining

The FITC annexin V staining was carried out to examine the induction of apoptosis inα-Fe2O3 nanoparticles treated MCF-7 cells. In order to differentiate the necrotic cells from apoptotic ones, the iron oxide nanoparticle treated MCF-7 cells were simultaneously stained with PI. Cells were grown in 6-well plates (1×105 cells/well) and then incubated for 24 h in the presence of various doses of iron oxide nanoparticles. At the required time point, the cells were harvested and then stained with FITC annexin V-PI as per manufacturer’s instructions (FITC Annexin V Apoptosis Detection Kit, BD Pharmingen, NJ) and analyzed in a FACS Calibur (BD Biosciences, NJ) flow cytometer. The results were analysed using CellQuest Pro software.


## 2.1. Nanopowder Synthesis

In the typical procedure, 1 mol of Fe(NO3)3·9H2O solution was added with 100 mL of ethylene glycol, and the mixture was continuously stirred at room temperature to get a clear brown-coloured solution. This solution was continuously stirred for 8 h at a temperature of 90°C. Viscosity of the solution increased constantly with time. The reddish brown gel was calcinated at 250°C in a hot air oven for 24 h. The resultant powder was subsequently washed several times with distilled water and ethanol. The structure, morphology, and magnetic properties of the as-prepared sample were characterized by powder X-ray diffraction (XRD) and transmission electron microscopy (TEM). The magnetic characteristics of the specimen were measured at room temperature using a vibrating sample magnetometer (VSM) (LDJ9600).


## 2.2. Cell Proliferation Assays

MCF-7 cells, human breast cancer cells, were obtained from American Type Culture Collection, and the cells were grown in DMEM at 37°C under 5% CO2 in a humidified incubator. Cells were harvested, counted, and transferred to 96-well plates and incubated for 24 h prior to the addition of the nanoparticle. The iron oxide nanoparticles were processed and applied in various concentrations, and the treated cells were incubated for 24 h. MCF-7 cells were plated (1×104 cells/well) in triplicate in 96-well sterile microtiter plates and allowed to grow for 24 h for proper cell adherence. Cells were then treated with different concentrations of iron oxide nanoparticles for 24 h. Cells incubated with 25 μL of PBS were used as a control. After incubation, the media were discarded and replaced with 175 μL of fresh medium and 25 μL of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution then incubated at 37°C under 5% CO2 for 4 hours. The medium was removed, and 100 μL of dimethyl sulfoxide was added to solubilize the formazan crystals. Optical density was then taken at 575 nm. The results were expressed as the percentage proliferation with respect to vehicle-treated cells.


## 2.3. Trypan Blue Exclusion Test

The trypan blue exclusion assay was performed to analyze the cell viability afterα-Fe2O3 nanoparticle treatment. Equal amounts of MCF-7 cell suspension and trypan blue (0.4%) were mixed for 1-2 min and 10 μL of the mixture was placed on hemocytometer and observed under light microscope, and the cells were counted in all four fields.


## 2.4. Acridine Orange/Propidium Iodine (AO/PI) Staining

MCF-7 cells were treated with various concentrations ofα-Fe2O3 nanoparticles and stained with AO/PI (Sigma-Aldrich) dual dye to detect apoptotic or necrotic nuclei. The cells were seeded in 6-well tissue culture plates for 24 h followed by the α-Fe2O3 nanoparticle treatment at desired concentrations. After the incubation period, the media were removed, and the cells were washed twice with PBS. Fresh media were added in each well, and the cells were stained by adding 10 μL of AO/PI mix (10 mg mL−1 AO and 10 mg mL−1 PI in PBS) then incubated for 10 min. The cells were washed with PBS before visualizing under a fluorescence microscope (Nikon Eclipse, TS100, Tokyo) with an excitation filter of 480/30 nm.


## 2.5. Annexin V-PI Staining

The FITC annexin V staining was carried out to examine the induction of apoptosis inα-Fe2O3 nanoparticles treated MCF-7 cells. In order to differentiate the necrotic cells from apoptotic ones, the iron oxide nanoparticle treated MCF-7 cells were simultaneously stained with PI. Cells were grown in 6-well plates (1×105 cells/well) and then incubated for 24 h in the presence of various doses of iron oxide nanoparticles. At the required time point, the cells were harvested and then stained with FITC annexin V-PI as per manufacturer’s instructions (FITC Annexin V Apoptosis Detection Kit, BD Pharmingen, NJ) and analyzed in a FACS Calibur (BD Biosciences, NJ) flow cytometer. The results were analysed using CellQuest Pro software.


## 3. Results and Discussion

### 3.1. Structural Analysis

It is observed from Figure1 that all the diffraction peaks can be readily indexed to the rhombohedral phase of hematite α-Fe2O3. These diffraction patterns are in good agreement with Joint Committee on Powder Diffraction Standards (JCPDS) card number; 33-0664 with lattice parameters a=5.035Å and c=13.749Å, matching well with the reported literature [37]. A small amount of extra secondary γ-Fe2O3 phase is observed at 2θ value of 30.7°. Significant line broadening of the diffraction peaks indicates that the sample’s size is in nm range. The average crystallite size of α-Fe2O3 was estimated to be about 20.6 nm using Debye-Scherrer’s formula [38] t=0.9λ/βcosθ, where λ is the wavelength of Cu-Ka radiation source, β (in radians) is the full width at half maximum (FWHM) intensity of the diffraction peak line, and θ is the diffraction angle (Braggs angle) of the XRD spectra, calculated from the line broadening of the (104) diffraction peak.Figure 1
XRD pattern of theα-Fe2O3 nanopowder.


### 3.2. Particle Size Analysis

Figure2 shows typical TEM image of the α-Fe2O3 nanopowder calcinated at 250°C during 24 h. A good number of particles have spherical shapes, and their average size is measured to be in the range 19 nm. This is in good agreement with the particle size calculated from Debye-Scherrer formula in the X-ray diffraction method. Agglomerations observed in the TEM image might be due to the addition of small drop of iron oxide particles on microgrid during specimen preparation. The variations in particle sizes as measured by X-ray diffraction and TEM image may be due to the fact that several crystal domains build a particle [39, 40].Figure 2
TEM micrograph ofα-Fe2O3 nanopowder.


### 3.3. Magnetic Analysis

Magnetic hysteresis behaviour (Figure3) of α-Fe2O3 nanopowder was carried out at room temperature with the applied magnetic field from 250 to 20,000 gauss. It is known that the magnetic properties of the α-Fe2O3 powder were influenced by many factors such as particle size, shape, crystallinity, aspect ratio, synthesis conditions, the extent of cation substitution, and surface structure [41–46]. It can be seen from Figure 3 that the sample exhibits ferromagnetic behavior at room temperature. The saturation magnetization and coercivity values are 7.5 emu/g and 240 Oe, respectively. The inset image of Figure 3 shows a magnified view of original hysteresis loop of α-Fe2O3 nanopowder. It is known that the coercivity is mainly influenced by many potential factors such as morphologies, size, surface disorder, and structure [45]. The sample’s low coercivity value (240 Oe) may be due to high crystallinity and crystal defects [47]. Literature studies reveal that hematite exhibits antiferromagnetic behavior whereas maghemite exhibits ferromagnetic behavior at room temperature. The ferromagnetic properties of α-Fe2O3 nanopowder can be explained by the ferromagnetic contribution of γ-Fe2O3 phase along with α-Fe2O3 phase [48].Figure 3
Hysteresis loop ofα-Fe2O3 nanopowder.


### 3.4. Cytotoxicity Analysis

Human breast epithelial MCF-7 cells were exposed toα-Fe2O3 nanoparticles at the concentrations of 25, 50, 100, 200, and 400 μg/mL for 24 h and cytotoxicity was determined using MTT assays. MTT results have shown that as the concentration of nanoparticles increased to 100, 200 and 400 μg/mL, cytotoxicity was observed in dose-dependent fashion. In MTT assay, cell viability was significantly reduced to 90%, 83%, 69%, 51% and 36% for the concentrations of 25, 50, 100, 200, and 400 μg/mL, respectively (P<0.05 for each) (Figure 4). This inhibitory growth activity of the treated cells acted in a dose-dependent manner which was also confirmed by the cell viability with the trypan blue exclusion assay. Our cytotoxicity data is in accordance with the results of Hilger et al. [31]. They investigated the cytotoxic response of iron oxide nanoparticles to adenocarcinoma cells. Results showed that iron oxide nanoparticles at low concentration did not produce cytotoxicity while the higher concentrations significantly reduced the viability of adenocarcinoma cells. As with most published work in nanotoxicology, the high concentration-induced cytotoxicity observed in this study may be difficult to translate to a realistic human exposure scenario. We believe that these data can provide a springboard for other researchers to create mechanistic pathways involved in cell death induced by iron oxide nanoparticles.Figure 4
Effect ofα-Fe2O3 on the growth of breast cancer cell lines MCF-7. Cells are seeded in 96-well plates and incubated with different concentrations of α-Fe2O3 and noted after 24 h maintaining at 37°C. Cell viabilities are determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data points are presented as means ± SD of triplicate experiments.To investigate the mode of cell death (i.e. apoptosis or necrosis) in MCF-7 cells caused by theα-Fe2O3, treated cells were stained with AO/PI dual dye and observed under the fluorescence microscope. Figure 5 shows that the cells treated with 25 μg mL−1  α-Fe2O3, like the nontreated ones, are uniformly green with normal morphology. However, extensive nuclear margination accompanied with chromatin condensation and distinctive of apoptotic cell death [49, 50] was observed in treated cells at the α-Fe2O3 concentrations of 100, 200, and 400 μg mL−1. The results of AO/PI nuclear staining indicated the induction of apoptosis in MCF-7 cells by α-Fe2O3 (Figure 5). The apoptotic cell death in MCF-7 cells induced by α-Fe2O3 was confirmed and subsequently quantified by flow cytometric analysis. Early apoptotic cells can easily be identified by green fluorescence of FITC- conjugated annexin V as annexin V has a high affinity towards phosphatidylserine (PS) residues which are externalized from inner to outer leaflet of the plasma membrane during early stages of apoptosis [51, 52]. Figure 6 shows the results of flow cytometric analysis of FITC annexin V-PI stained MCF-7 cells treated with different concentrations of α-Fe2O3. The percentages of apoptotic and necrotic populations in treated and untreated cells were calculated from the flow cytometric data and the results clearly show the dose-dependent reduction in cell viability of MCF-7 cells in presence of α-Fe2O3. Most importantly, the apoptotic and necrotic populations in MCF-7 cells increased by 34%, 61%, and 86% in presence of 100, 200, and 400 μg mL−1  α-Fe2O3, respectively. The results of flow cytometric analysis clearly establish the efficient induction of apoptotic and necrotic cell death in MCF-7 cells by α-Fe2O3 (Figure 6). While necrosis is a form of cell death resulting from direct cell damage, apoptosis is a form of programmed cell death where cells effectively commit suicide [53]. Our study demonstrates the simultaneous visualisation of viable, necrotic, and apoptotic cells in any given sample suggesting that iron oxide nanoparticles induce apoptosis and necrosis in dose-dependent manner. Similar effects have been observed in other ferrofluids which employ oleic acid [54]. In the present case, the observed cytotoxic effects are more difficult to diagnose but are possibly due to free radical generation through Fenton and/or Haber-Weiss reactions whose effects only become noticeable at higher intracellular concentrations of iron [55]. Another possible explanation for the significant cytotoxicological and morphological changes is that the phenotypic effects of iron oxide internalization may drastically impede transcriptional regulation and protein synthesis resulting in a loss of cell phenotype and possibly cell death. It has been recently demonstrated that there are many factors such as size, shape, surface charge, and solubility influencing the nature of the nanoparticle corona and playing a vital role in the interaction of the nanoparticles with proteins. In vitro and in vivo studies suggested that these interactions between nanoparticles determine the fate and impact of the particles [56, 57]. Furthermore, differences in the recent literature suggested that different cell types with different uptake mechanisms react differently to nanoparticle exposure [58, 59]. In order to evaluate these possibilities, further studies into the physicochemical basis of the observed α-Fe2O3 cytotoxicity, cellular uptake, and interaction of nanoparticles with proteins are necessary.Morphological assessment of MCF-7 cells stained with acridine orange (green) and propidium iodide (red). Cells are incubated with or withoutα-Fe2O3 at various concentrations for 24 h. Cells with intact membrane and stained green indicate viable cells; cells that are stained orange represent secondary necrotic or late apoptotic cells containing fragmented DNA (magnification 100x, scale bar 20 μm).
(a)
(b)
(c)
(d)Figure 6
Flow cytometry analysis of untreated and treated MCF-7 cells withα-Fe2O3 for 24 h stained with annexin V-FITC/propidium iodide (PI). Each bar represents the population of viable, apoptotic, and necrotic cells of three independent experiments.


## 3.1. Structural Analysis

It is observed from Figure1 that all the diffraction peaks can be readily indexed to the rhombohedral phase of hematite α-Fe2O3. These diffraction patterns are in good agreement with Joint Committee on Powder Diffraction Standards (JCPDS) card number; 33-0664 with lattice parameters a=5.035Å and c=13.749Å, matching well with the reported literature [37]. A small amount of extra secondary γ-Fe2O3 phase is observed at 2θ value of 30.7°. Significant line broadening of the diffraction peaks indicates that the sample’s size is in nm range. The average crystallite size of α-Fe2O3 was estimated to be about 20.6 nm using Debye-Scherrer’s formula [38] t=0.9λ/βcosθ, where λ is the wavelength of Cu-Ka radiation source, β (in radians) is the full width at half maximum (FWHM) intensity of the diffraction peak line, and θ is the diffraction angle (Braggs angle) of the XRD spectra, calculated from the line broadening of the (104) diffraction peak.Figure 1
XRD pattern of theα-Fe2O3 nanopowder.


## 3.2. Particle Size Analysis

Figure2 shows typical TEM image of the α-Fe2O3 nanopowder calcinated at 250°C during 24 h. A good number of particles have spherical shapes, and their average size is measured to be in the range 19 nm. This is in good agreement with the particle size calculated from Debye-Scherrer formula in the X-ray diffraction method. Agglomerations observed in the TEM image might be due to the addition of small drop of iron oxide particles on microgrid during specimen preparation. The variations in particle sizes as measured by X-ray diffraction and TEM image may be due to the fact that several crystal domains build a particle [39, 40].Figure 2
TEM micrograph ofα-Fe2O3 nanopowder.


## 3.3. Magnetic Analysis

Magnetic hysteresis behaviour (Figure3) of α-Fe2O3 nanopowder was carried out at room temperature with the applied magnetic field from 250 to 20,000 gauss. It is known that the magnetic properties of the α-Fe2O3 powder were influenced by many factors such as particle size, shape, crystallinity, aspect ratio, synthesis conditions, the extent of cation substitution, and surface structure [41–46]. It can be seen from Figure 3 that the sample exhibits ferromagnetic behavior at room temperature. The saturation magnetization and coercivity values are 7.5 emu/g and 240 Oe, respectively. The inset image of Figure 3 shows a magnified view of original hysteresis loop of α-Fe2O3 nanopowder. It is known that the coercivity is mainly influenced by many potential factors such as morphologies, size, surface disorder, and structure [45]. The sample’s low coercivity value (240 Oe) may be due to high crystallinity and crystal defects [47]. Literature studies reveal that hematite exhibits antiferromagnetic behavior whereas maghemite exhibits ferromagnetic behavior at room temperature. The ferromagnetic properties of α-Fe2O3 nanopowder can be explained by the ferromagnetic contribution of γ-Fe2O3 phase along with α-Fe2O3 phase [48].Figure 3
Hysteresis loop ofα-Fe2O3 nanopowder.


## 3.4. Cytotoxicity Analysis

Human breast epithelial MCF-7 cells were exposed toα-Fe2O3 nanoparticles at the concentrations of 25, 50, 100, 200, and 400 μg/mL for 24 h and cytotoxicity was determined using MTT assays. MTT results have shown that as the concentration of nanoparticles increased to 100, 200 and 400 μg/mL, cytotoxicity was observed in dose-dependent fashion. In MTT assay, cell viability was significantly reduced to 90%, 83%, 69%, 51% and 36% for the concentrations of 25, 50, 100, 200, and 400 μg/mL, respectively (P<0.05 for each) (Figure 4). This inhibitory growth activity of the treated cells acted in a dose-dependent manner which was also confirmed by the cell viability with the trypan blue exclusion assay. Our cytotoxicity data is in accordance with the results of Hilger et al. [31]. They investigated the cytotoxic response of iron oxide nanoparticles to adenocarcinoma cells. Results showed that iron oxide nanoparticles at low concentration did not produce cytotoxicity while the higher concentrations significantly reduced the viability of adenocarcinoma cells. As with most published work in nanotoxicology, the high concentration-induced cytotoxicity observed in this study may be difficult to translate to a realistic human exposure scenario. We believe that these data can provide a springboard for other researchers to create mechanistic pathways involved in cell death induced by iron oxide nanoparticles.Figure 4
Effect ofα-Fe2O3 on the growth of breast cancer cell lines MCF-7. Cells are seeded in 96-well plates and incubated with different concentrations of α-Fe2O3 and noted after 24 h maintaining at 37°C. Cell viabilities are determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data points are presented as means ± SD of triplicate experiments.To investigate the mode of cell death (i.e. apoptosis or necrosis) in MCF-7 cells caused by theα-Fe2O3, treated cells were stained with AO/PI dual dye and observed under the fluorescence microscope. Figure 5 shows that the cells treated with 25 μg mL−1  α-Fe2O3, like the nontreated ones, are uniformly green with normal morphology. However, extensive nuclear margination accompanied with chromatin condensation and distinctive of apoptotic cell death [49, 50] was observed in treated cells at the α-Fe2O3 concentrations of 100, 200, and 400 μg mL−1. The results of AO/PI nuclear staining indicated the induction of apoptosis in MCF-7 cells by α-Fe2O3 (Figure 5). The apoptotic cell death in MCF-7 cells induced by α-Fe2O3 was confirmed and subsequently quantified by flow cytometric analysis. Early apoptotic cells can easily be identified by green fluorescence of FITC- conjugated annexin V as annexin V has a high affinity towards phosphatidylserine (PS) residues which are externalized from inner to outer leaflet of the plasma membrane during early stages of apoptosis [51, 52]. Figure 6 shows the results of flow cytometric analysis of FITC annexin V-PI stained MCF-7 cells treated with different concentrations of α-Fe2O3. The percentages of apoptotic and necrotic populations in treated and untreated cells were calculated from the flow cytometric data and the results clearly show the dose-dependent reduction in cell viability of MCF-7 cells in presence of α-Fe2O3. Most importantly, the apoptotic and necrotic populations in MCF-7 cells increased by 34%, 61%, and 86% in presence of 100, 200, and 400 μg mL−1  α-Fe2O3, respectively. The results of flow cytometric analysis clearly establish the efficient induction of apoptotic and necrotic cell death in MCF-7 cells by α-Fe2O3 (Figure 6). While necrosis is a form of cell death resulting from direct cell damage, apoptosis is a form of programmed cell death where cells effectively commit suicide [53]. Our study demonstrates the simultaneous visualisation of viable, necrotic, and apoptotic cells in any given sample suggesting that iron oxide nanoparticles induce apoptosis and necrosis in dose-dependent manner. Similar effects have been observed in other ferrofluids which employ oleic acid [54]. In the present case, the observed cytotoxic effects are more difficult to diagnose but are possibly due to free radical generation through Fenton and/or Haber-Weiss reactions whose effects only become noticeable at higher intracellular concentrations of iron [55]. Another possible explanation for the significant cytotoxicological and morphological changes is that the phenotypic effects of iron oxide internalization may drastically impede transcriptional regulation and protein synthesis resulting in a loss of cell phenotype and possibly cell death. It has been recently demonstrated that there are many factors such as size, shape, surface charge, and solubility influencing the nature of the nanoparticle corona and playing a vital role in the interaction of the nanoparticles with proteins. In vitro and in vivo studies suggested that these interactions between nanoparticles determine the fate and impact of the particles [56, 57]. Furthermore, differences in the recent literature suggested that different cell types with different uptake mechanisms react differently to nanoparticle exposure [58, 59]. In order to evaluate these possibilities, further studies into the physicochemical basis of the observed α-Fe2O3 cytotoxicity, cellular uptake, and interaction of nanoparticles with proteins are necessary.Morphological assessment of MCF-7 cells stained with acridine orange (green) and propidium iodide (red). Cells are incubated with or withoutα-Fe2O3 at various concentrations for 24 h. Cells with intact membrane and stained green indicate viable cells; cells that are stained orange represent secondary necrotic or late apoptotic cells containing fragmented DNA (magnification 100x, scale bar 20 μm).
(a)
(b)
(c)
(d)Figure 6
Flow cytometry analysis of untreated and treated MCF-7 cells withα-Fe2O3 for 24 h stained with annexin V-FITC/propidium iodide (PI). Each bar represents the population of viable, apoptotic, and necrotic cells of three independent experiments.


## 4. Conclusion

In conclusion, pureα-Fe2O3 nanoparticles were synthesized by a low temperature combustion technique. The X-ray powder diffraction measurement indicates that these particles are hematite. The ultrafine α-Fe2O3 nanopowder shows a weak ferromagnetic behaviour and a significantly low coercivity performance because of their uniformity and small particle size. Cytotoxicity analysis demonstrates that the iron oxide nanoparticles lead to cellular morphological modifications and induce apoptosis and necrosis in a dose-dependent manner. This preliminary data suggest that Fe2O3 nanoparticles are likely to contribute to cytotoxicity of human breast cancer cells and can be a potential candidate for drug delivery systems.



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