Study of Biological Activities of Magnetic Iron Oxide Nanoparticles Prepared by Co-Precipitation Method

*Corresponding Author: Rania Hasan Hussein as.19.14@grad.uotechnology.edu.iq Abstract Iron oxides are the most common types of substances available in nature in various forms such as oxides, hydroxides and oxide-hydroxides, which obtained from various aqueous reactions. In the present study, iron oxide (Fe3O4) nanoparticles (NPs) had been synthesized using co precipitation method and determined some biological activities. Iron oxide NPs had been characterized by spectrophotometry, XRay Diffraction (XRD) analysis and Field Emission Scanning Electron Microscopic (FE-SEM). The XRD analysis confirmed the purity and crystalline nature of fabricated NPs. In contrast, four biological activities of iron oxide NPs were determined. High removal of methylene blue pollutant dye (37.62 %) was observed when iron oxide NPs were used at 400 μg/ml within 48 hrs. Furthermore, iron oxide NPs revealed high inhibition zones of 27.5 and 30 mm, at 1000 μg/ml, toward Staphylococcus aureus and Escherichia coli respectively. The hemolysis activity of fabricated NPs is increases whenever concentrations increased. The lower hemolysis percentage of iron oxide NPs was (69.76%) when the NPs concentration was 250 μg/ml. Finally, cytotoxic activity was estimated against MCF7 cell line and normal cell line WRL68 by MTT assay. Significant and decrease in viability of (MCF-7 to 65.1%) when 400 μg/ml of iron oxide NPs was examined, while WRL68 viability was (75.03%).


Introduction
Metal oxide nanoparticles have exceptional properties that lead to great interest in some biological activities. Iron (Fe) is a transitional mineral and the backbone of the earth's crust appeared in different forms and magnetic properties, either as hematite (α-Fe2O3), magnetite (Fe3O4) or maghemite (ɣ-Fe2O3) [1,2]. Magnetite is the most common types of substances available in nature in various forms such as oxides, hydroxides and oxide-hydroxides, are obtained from various aqueous reactions (oxidation and reduction) [3]. Iron oxide NPs revealed more stable, superparamagnetic, biocompatible, and low toxicity compounds. They also have high stability in water when exposed to difficult conditions such as high temperature and high pH, and therefore their conjugation with biomolecules is very easy [4]. Moreover, synthesis methods of iron oxide NPs are extensively developed, like solgel, co-precipitation, thermal decomposition, and micro emulsion method [5]. On the other hand, iron oxide NPs have a high surface area to volume ratio, which gives the particles high binding ability and good dispersion in solution. Iron oxide nanoparticles have amazing magnetic properties and high biocompatibility that have attracted widespread interest in the biomedical field [1,6]. In the present study, iron oxide NPs synthesized using coprecipitation process and then characterized by (UV-V) is spectroscopy, XRD and FE-SEM. The aim of this study is determining their compatibility, the ability to remove pollutant dye, hemolysis, antibacterial and antitumor activities had been investigated.

Chemicals and Cells
Iron (II) chloride tetrahydrate (FeCl2.4H2O2), Iron (III) chloride (FeCl3), and Ammonium hydroxide (NH4OH) were bought from Merck (India). Each solution was prepared to utilize deionized water. Bacterial isolates of Staph, aureus and E. coli were purchased from medical lab technique, Al Esraa university, Baghdad, Iraq, while MCF-7 and WRL68 cell lines were supplied by Sterllixir Biotech, industrial area, Bangalore, India.

Synthesis of Magnetic Iron Oxide NPs
The co-precipitation method was used for Fe 3+ ions and Fe 2+ ions to synthesize magnetic Fe3O4 nanoparticles in the presence of ammonium hydroxide solution (NH4OH). The synthesis process was accomplished by mixing 4.58g of FeCl2.4H2O with 8.93g FeCl3.6H2O and dissolving them in an aqueous medium (80 mL). The solution was then heated at 80 °C with continues stirring. After heating, 10 mL of NH4OH solution is added to the reaction solution. Crystal growth continuous for 30 minutes without changing the reaction conditions. Then the reaction solution is purified by washing it several times with distilled water drying in an oven at 50 °C [7].

Characterization of Iron Oxide NPs
(UV-Vis) is absorption spectra of iron oxide NPs were determined on UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The crystal phase and particles size of the prepared sample was identified by X-ray technology (XRD-6000, Shimadzu) with Cu Kα source (wavelength of 1.54056 ˚A) at diffraction angle (2θ) from 20° to 80°. Furthermore, the morphology of the prepared iron oxide NPs can be identified by field emission scanning electron microscopy (Mira 3-TESCAN) and quickening voltage (20-30 kV).

Biological Activities Determination of Fe3O4 NPs 2.4.1. Removal of Methylene Blue
The adsorption activity of methylene blue dye has been studied. Various concentrations (5-20 mg) of the prepared iron oxide NPs with methylene blue dye (50 mL, 40 mg L -1 ) had been mixed. The reaction solution was mixed over a hot plate stirrer at different times 2-72 hr. and the process was repeated three times at room temperature. Finally, the removal percentage of methylene blue dye was measured using UV-Vis spectroscopy at wavelength 668 nm. Kinetics of adsorption was studied by analyzing adsorptive uptake of the dye color from the aqueous solution at different time intervals [8].

Antibacterial Activity
The well-diffusion technique was utilized in [9] with some modification. Fifty microliter of E. coli, and S. aureus cultures of 18-24 h (10 6 / CFU/ ml) growth were spread on the surface of sterilized Mueller-Hinton agar (Oxoid, England) poured in Petri plates. Wells were made and each well was packed with different concentrations of iron oxide NPs ranging from 250, 500, 750, and 1000 µg/ml DMSO. The plates were incubated at 37 O C for 24. DMSO was used as control. The antibacterial activity of (DMSO) used as control. The average of inhibition zones of duplicate treatment was determined in millimeters [9].

Hemolytic Activity Assay
Hemolytic activity of Fe3O4 NPs was established according to the procedure mentioned in [10] with some modification. Briefly, 200 μL of the separated blood was mixed with 1600 μL of normal saline. Then, 200 μL of different concentrations of NPs (250, 500, 750, and 1000 µg / mL) were added to the diluted blood. The samples were incubated at 37°C for 1 h and centrifuged at 700 rpm for 5 min. Positive and negative control includes Distilled water and normal saline, respectively, were used for comparison. Sample absorbance was measured spectrophotometry at 541 nm and hemolysis percentage was estimated by utilizing the following equation 1 [10]: Where, A: is the absorbance. The absorbance of positive and negative controls was found to be 1.764 and 0.048, respectively and A (-): is Negative control includes distilled water and A (+): is Positive control includes distilled water with normal saline.

Anticancer Activity
MCF-7 and WRL 68 cell lines were grown in RPMI-1640 medium (Sigma-Aldrich, USA) at 37 o C with 5% CO2 in 96 -well flat bottom culture plates at density 1×10 4 cells /ml for 48 hrs. The cells were treated, in duplicate, with the concentrations of 20 -400 μg/ml of NPs and incubated for 24 hrs. to determine the toxicity against examined cell lines. 10 μl of MTT solution was added to each well, and the plates were incubated for 4 hrs. at 37 o C. The media were then removed, and the remaining formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm using an ELISA microplate reader to determine the toxicity, once with a tumor cell line and another with normal cells. The cytotoxicity percentage was calculated by the equation 2 [11]: A: an optical density of the control, B: an optical density of the treated sample.

UV-Vis Spectrophotometry Analysis
UV-Vis Spectroscopy is an analytical technique used to identify the absorbance of colloidal solutions and the optical properties of prepared compounds in a range of wavelengths (200-800) nm. Figure 1a, b shows the absorption spectrum of Fe3O4 NPs. The spectrum of Fe3O4 NPs shows continuous absorption along wavelengths and no strong absorption peaks observed. The results are similar to previous literature by Nagaraj et al., [12] and Yen et al., [13]. Moreover, the band gap energy spectrum of Fe3O4 NPs had been determined, as exhibited in Figure (1 b). The optical band gap energy can be calculated from the Tauc's equation 3 [14]: Where, Eg is the band gap of the sample, h is blank constant, C is a constant, and α is the absorption coefficient. The gap energy is estimated by drawing (αhυ) 2 against the photon energy (hυ). It was found that the gap energy value of the Fe3O4 NPs up to 5.22 eV.

X-Ray Diffraction Analysis
XRD is an analytical technique utilized to identify the crystalline nature and particle size of prepared crystalline nanomaterials. Figure 2 Whereas D represents crystal size, θ is diffraction angle (deg.), λ is the X-ray wavelength (0.15056nm), k is a dimensionless shape factor (0.9), FWHM (β) is the full width at half maximum of the peak. The results are similar to previous literature [17,18].

FE-SEM Images
FE-SEM is an analysis that provides information about surface morphology and particle size of the prepared samples. Figure 3 displays the FE-SEM analysis of iron oxide NPs. The microscopic images reveal the presence of spherical particles of different sizes and distribution. Also, an increase in the ratio of particle agglomeration was noticed due to the electrostatic interaction between the layers of nanoparticles. The average particle size is 27.12 nm, close to the crystal size calculated from the XRD diffraction analysis. The results show the formation of iron oxide nanoparticles, and this is in agreement with was previously published research [19]. On the other hand, EDS a diagnostic tool used to identify the elementary components of the chemical composition of the prepared sample. Figure 4 represents to EDS spectrum of iron oxide NPs with strong peak for the oxygen element and weak peaks for the Fe element were observed, indicating the formation of Fe3O4 compound. The results are identical to previous studies prepared by Saddam et al,. [9].

Removal of Methylene Blue (MB) Dye
Methylene blue had been used as pollutant dye model. The removal percentage of the dye had been estimated in the existence of iron oxide NPs in liquid phase. Absorbance of removed dye was measured in the existence of various concentrations of iron oxide NPs by spectrophotometer at 668 nm, within 48 hrs. at room temperature. High and significant removal of dye (37.62 %) was observed when iron oxide NPs was used at 400 µg/ml within 48 hr as shown in Figure 5. Most destruction and disruption of water ecosystems caused by untreated dye came from different industrial products, like textile, paper, and food processes. Further than their toxicity, most dyes are mutagenic and carcinogenic agents. Synthetic methylene blue is a cationic and aromatic dye used wildly in textile industry that cause skin damage and eyes allergy and other defects when ingested [20,21]. Different remedying methods have been applied to remove methylene blue from water environments. The rapid and specific technique is the method of choice to isolate and concentrate the specific pollutant from water system. Magnetic nanoparticles represent easily and cheap prepared adsorbent material to remove contaminations [22]. On the other hand, although the reported advantages of nanoparticles, the toxicity of the iron oxide NPs need more to explore. Despite whether the applications were in vivo or in vitro, iron oxide NPs should be biocompatible and non-toxic. Moreover, surface area to volume of nanoparticles, size, shape, concentration of NPs, concentration of methylene blue, reaction temperature, type of coat, and pH effect dye removal [23]. Therefore, the results obtained in the study appeared low removal of methylene blue in contrast to results obtained by other studies [24].

Antibacterial activity
Various concentrations of Fe3O4 NPs had been examined against E. coli (Gram negative) and S. aureus (positive bacteria. The well diffusion method was used to detected the antibacterial activity of Fe3O4 NPs on Muller Hinton medium within 24 hrs. at 37 ○ C. Results were expressed as mean ± standard error (SE) and values of p<0.05 were considered significant. The obtained results demonstrated that iron oxide NPs have antibacterial activity against both E. coli and S. aureus and the activity increases with an increase in concentration of nanoparticles. The oxide NPs, at 1000 µg/ml, revealed Highest and significant inhibition zones of 27.5 and 30 mm in diameter against S. aureus and E. coli, as shown in Figure 6. the other hand, investigation in many research had been done to explain the mechanism of iron oxide NPs antibacterial activity. One of the mechanisms referred to the interaction between opposite charge between the negative charge of bacterial surface and the potential positive charge of nanoparticles [25]. Due to the presence of positive charge on the surface of prepared iron oxide NPs [10,5], we think the attracted electrostatic led to an increase in the interaction between prepared NPs and the examined bacteria. Furthermore, in the present study iron oxide NPs could be the source that produces reactive oxygen species (ROS) leading to the inhibition of bacterial growth.

Hemolysis Activity
The absorbance of human red blood cells corpuscles (RBC) lysis had been determined by spectrophotometer at 541 nm within one hour at 37 ○ C. According to the standard ISO 10993-4 to evaluate materials and devices that compatible for blood cells, the hemolysis percentage should be less than 5 [27]. Different concentrations of iron oxide NPs were used to estimate their hemolysis activity, as shown in Figure 7. The hemolysis activity of iron oxide NPs is increasing whenever concentrations increased. Lower hemolysis percentages of iron oxide NPs (69.76%) were observed at concentration of 250 µg/ml. Iron oxide NPs have wildly investigated and developed for biological and medical applications, but a few studies discussed the biocompatibility and less on their hemocompatibility. The concentration of iron oxide NPs and incubation time are other factors that affect hemolysis estimation in vitro. Hemolysis rates were found to be lower than 2 % for a low content 17.4 µg/ml and the highest 34.8 µg/ml of iron oxide NPs [28], while another study revealed maximum hemolysis (5.11%) was obtained at 125 µg/ml of iron oxide NPs [29]. On contrast study, the results indicate that the iron oxide NPs had non-hemolytic reaction at all examined concentrations up to 3.0 mg/ml [30].

Antitumor activity
Antitumor activity of different concentrations of iron oxide NPs were measured against MCF-7, as tumor cell line, and WRL68, as normal cell line. The MTT assay was used to determine cell viability after incubation of 25, 50 ,100, 200 and 400 µg/ml of NPs at 37 ○ C for 24 hr. in the presence of 5% CO2. Significant and decreasing MCF-7 viability reach 65.1% when 400 µg/ml of iron oxide NPs were examined, while the viability of WRL68 was 75.03%, as shown in Figure 8. Tumor can be treated either physically by radiation and hyperthermia or chemically by intracellular entry of nanoparticles to induce ROS and most importantly, the apoptotic, programmed cell death, and necrotic, direct cell damage, in tumor cells populations. The apoptotic and necrotic populations in MCF-7 cells increased by 61% in 200 g/mL of iron oxide NPs [31]. Moreover, treatment of DU145 and PC-3 cell lines with 100 µg/mL iron oxide NPs for 72 h resulted in significant inhibition of cell viability [32]. On the other hand, iron oxide NPs with 39 nm in size appeared a remarkable antitumor activity against renal tumor at concentration of 400 µg/ml [33][34][35].

Conclusions
The synthesis of iron oxide NPs by co -precipitation method revealed purity and crystalline nature by XRD. The FE-SEM images of iron oxide NPs reveal a homogeneous structure with spherical shapes closely related to each other. Iron oxide NPs can remove methylene, as a pollutant dye, but it needs more optimization. Moreover, the hemolysis activity of fabricated NPs is increasing whenever concentrations increased. On the other hand, it seems to be E. coli was more sensitive to iron oxide NPs than S. aureus. Finally, the iron oxide NPs revealed low toxicity against both MCF-7 and WRL 68, as tumor and natural cell lines, respectively.