Structural, optical, magnetic, photocatalytic activity and related biological effects of CoFe2O4 ferrite nanoparticles

The synthesis of magnetic nano-sized spinel ferrites has become an important area of research, due to their several potential applications. In this work, CoFe2O4 nanoparticles were synthesized by the co-precipitation method. Structural, magnetic, and photocatalytic properties of cobalt ferrites were analyzed based on their chemical composition considering their biological properties. Structural and morphological properties were investigated by X-ray diffraction analysis (XRD) and scanning electron microscope (SEM), respectively. Lattice parameters and cell volumes were calculated from XRD data. SEM images revealed uniform surface morphology and spherical shape of nanoparticles. Magnetization measurements were measured by using Lake Shore 7304 model Vibrating Sample Magnetometer (VSM). In hemolytic activity tests, formation of a precipitate with a characteristic black color provided an explicit evidence to the formation of heme–iron complexes. Undesirable hemolytic effect of CoFe2O4 nanoparticles on human erythrocytes at both concentrations was attributed to the comparatively high amount of reactive oxygen species formed by CoFe2O4 nanoparticles. The theoretical concentration Co (theory) obtained by second-order model (0.82 mg/L) fit with the experimental value of Co (experimental) (0.95 mg/L) well in photocatalytic activity tests.


Introduction
In recent years, nano-sized metal oxide particles have been widely investigated due to their huge contact area between materials, good mechanical, and electrical properties arising from the adjustment of dimensions.
Especially, nano-sized iron oxides have gained extreme attention due to their unique material properties, such as high surface/volume ratio, excellent photostability, and high quantum yield [1]. They become very popular in the fields of optics, electronics, chemical sensors, bio-imaging applications, and medicine. The ability to control the behavior of the nanomaterials using an external magnetic field leads to potential applications, including magnetic resonance imaging, controlled drug delivery, and magnetic hyperthermia [2,3]. Nanoparticles, which are planning to be used for such applications, should have a low-toxic nature, high stability, significant selectivity of accumulation in targeted area, and superparamagnetic properties at room temperature [4].
For instance, superparamagnetic particles were found positively responsive toward erythrocytes [5,6] and some cancer cells, such as lung and breast cancer even without immunospecific coatings [7,8]. NiCo 2 O 4 [9,10], CuCo 2 O 4 [11], and MnFe 2 O 4 [12,13] nanoparticles were also investigated for their morphological, electrical, magnetic, and photocatalytic properties, which are directly related to their biomedical application performance. Besides iron oxide, binary iron oxides and other iron containing nanomaterials also have magnetic properties which are essential for biomedical applications. Especially, the spinel ferrites have gained considerable scientific and technological interest due to their unique physical and chemical properties as well as their technological applications.
Spinel ferrites can be represented by the formula of MFe 2 O 4 with a face-centered cubic structure where M is a divalent cation, such as Co, Cu, Ni, Mn, or Fe, and commonly synthesized by sol-gel method [14]. Among spinel ferrites, copper ferrite (CuFe 2 O 4 ) and cobalt ferrite (CoFe 2 O 4 ) are of great interest from physics, chemistry, and biomedical aspects because of their excellent chemical and mechanical stability, high magnetocrystalline anisotropy, and high coercivity [15][16][17].
Improved mechanical hardness and stability of CoFe 2 O 4 under physiological conditions and tunable superparamagnetic behavior by changing particle size [18] make these nanomaterials a tough competitor over other magnetic materials, such as traditional iron oxides, Fe 3 O 4 and Fe 2 O 3 . Moreover, undesirable interactions of red blood cells (RBCs) and traditional iron oxides could be prevented by ferrites providing better hemocompatibility [19]. A scientific method to adjust the toxicity of nano-sized materials is to apply biocompatible coatings, such as polysiloxane, alginate, and citric acid, to the surface [20,21] [24]. Manohar et al. [25,26] investigated magnetic, dielectric, and photocatalytic properties of both manganese-and zinc-doped Fe 2 O 4 nanoparticles. They claimed both nanomaterials synthesized by solvothermal method as promising in the field of biomedical applications.
It appears from our literature research that spinel ferrites were used in various areas, including catalysis and water treatment. Ferrites were also introduced as promising materials for especially biomedical applications. However, the interactions between these nano-sized materials and tissues and blood components are still an uncompleted issue. Thus, it is still an open question whether these nanomaterials are suitable to be used in vivo applications or cause any undesired interactions between the cells of living tissues. This research reports the synthesis and material properties of CoFe 2 O 4 nanoparticles together with their blood compatibilities. In this study, we were also concerned about the photodegradation mechanism of crystal violet (CV) by CoFe 2 O 4 nanoparticles which has not been studied sufficiently. This study aims to investigate and clarify basic biological effects of these nanoparticles for their potential use in biomedical area. Concurrently, we aimed to assess whether CoFe 2 O 4 nanoparticles could be used as an effective photocatalyst in water treatment.

Structural analysis
The structure of the CoFe 2 O 4 nanoparticles was characterized by XRD measurements using a PANalytical X'pert Powder 3 model X-ray diffractometer device with CuKa (k = 1.5418) radiation at room temperature in the scan range of 2h = 10°-90°with a scan speed of 3°/min and a step increment of 0.02°. The surface morphologies of precipitated powders were examined by using a SEM of the Zeiss EVO MA model. Optical properties of the CoFe2O4 nanoparticles were measured by using Shimadzu 2600 UV-Spectrometer with an integrating sphere in 300-1000 nm wavelength range. Magnetic hysteresis experiments were carried out in a Lake Shore model 7304 Vibrating Sample Magnetometer (VSM), operating within the 15-300 K temperature range.

Photocatalytic activity measurements
The photocatalytic activity of CoFe 2 O 4 nanoparticles was investigated by photodegradation of CV under 254 nm irradiation. The concentration values of nanoparticle dispersion and CV solution were 1.0 mg/mL and 2.5 9 10 -6 M, respectively. Distilled deionized water (DDW) obtained from Human Zeneer Power1 water purification system was used in the experiments. CV solutions containing the appropriate amount of nanoparticles were kept under magnetic stirring for 30 min (in darkness) to establish an adsorption/desorption equilibrium of CV molecules on the nanoparticle surface. After 30 min, 254 nm irradiation of CV-nanoparticle suspensions was started under continuous magnetic stirring. Aliquot parts were taken at appropriate time intervals and centrifuged at 4000 rpm to precipitate suspended nanoparticles. The absorption maxima of each supernatant was verified using a UV/Vis spectrophotometer (Shimadzu UV mini 1240) at a wavelength of 591 nm corresponding to the absorption maxima of CV. Distilled water was used as reference.
Zero order: First order: Second order: where C o and C represent the initial concentration of CV at t = 0 and the concentration of CV after a certain irradiation time (t), respectively. k, k 1, and k 2 were the rate constants for photocatalytic degradation.

Blood compatibility tests
Hemolytic activity of CoFe 2 O 4 ferrites against human erythrocyte membrane was investigated. 0.108 mM aqueous solution of trisodium citrate was used as stabilizing agent to prevent the coagulation of whole blood samples collected from healthy volunteers. Blood:anticoagulant ratio was adjusted as 9:1. Phosphate buffer solution (PBS) having a pH of 7.35 was prepared using Na 2 HPO 4 Á2H 2 O (1.78 g/L), KH 2 PO 4 (0.24 g/L), KCl (0.2 g/L), and NaCl (8 g/L), then autoclave sterilized at 1 atm and 121°C for 15 min. Ca-and Mg-free PBS was used to dilute the anticoagulated whole blood samples. RBCs were separated from plasma by centrifugation at 4000 rpm for 5 min. Precipitated RBCs were diluted up to 50 mL by adding PBS. 1.0 mg/mL and 5.0 mg/mL concentrations of CoFe 2 O 4 suspensions were prepared and mixed with 0.8 mL of RBC stock solution. DDW and PBS were used in positive and negative control tests, respectively. In positive control test DDW causes complete (100%) hemolysis of all erythrocytes and negative control test corresponds to 0% hemolysis. RBCs were incubated in the presence of varying nanoparticle concentrations under magnetic stirring at 37°C for 3 h. Each test was performed twice. At the end of the incubation period, the samples were centrifuged at 3000 rpm for 5 min and the absorbance (ABS) value of supernatant was used to quantify the degree of hemoglobin release into the medium following cell lysis. Percent hemolysis values were calculated using the ABS value at 540 nm using the equation given below [30]: 3 Results and discussions

XRD analysis
The X-ray diffraction (XRD) was used to investigate the crystalline phases of CoFe 2 O 4 nanoparticles. The XRD of the CoFe 2 O 4 ferrite nanoparticles is shown in Fig. 1. As shown in Fig. 1, it can be observed that CoFe 2 O 4 nanoparticles are spinel cubic structure of space group Fd-3m without secondary phases, which corresponds to PDF Card No.: 98-019-1044. The lattice parameter, a, was calculated from the diffraction pattern by using Eq. 5: The crystallite size of the CoFe 2 O 4 nanoparticles was calculated from the full width at half maximum (FWHM) of the most intense peak (113) using the Debye-Scherrer equation: where k is the X-ray wavelength of CuKa, b is the FWHM of the diffraction peaks, and h B is the angle of Bragg diffraction. The calculated crystallite size (D) and lattice parameter (a) of CoFe 2 O 4 nanoparticles for (113) peak were 39.2601 nm and 8.40 Å , respectively.

SEM analysis
The morphology of CoFe 2 O 4 nanoparticles was studied using Scanning Electron Microscope (SEM) as shown in Fig. 2. As can be observed from Fig. 2ad, the particles do not have a complete shape because they are agglomerated. As the resolution decreases, the structure appears dense and molten (Fig. 2a, b).
On the other hand, as the resolution increases, the structure looks like snowflakes (Fig. 2d)

Band gap calculation
The reflectance spectra of the CoFe 2 O 4 nanoparticles, obtained by UV-Vis diffuse reflectance Notice that the graph inset in Fig. 4 has an absorption edge close to 140 nm. The Kubelka-Munk function was used to calculate the reflection ratio F(R), which is proportional to the absorption coefficient (a) [30]: The following equation can be used to determine the optical band gap E g for the photon energy (hm) and the absorption coefficient (a):  In Eq. (8), E g and k are the optical band gap and energy-independent constants, respectively. F(R a ) is proportional to a and n is a constant that depends on the band gap type 1/2 and 2 for direct and indirect band gaps, respectively. Thus, for directly allowed transitions, n is taken as 2. Equation (8) can be transformed to In other words, F R a ð Þht ð The slope of the graphs of F R a ð Þht ð Þ 2 was approximated by using a linear fit y ht ð Þ ¼ A Â ht þ B in the leastsquares sense. To accomplish this, the error formula given in Eq. (10) was minimized for A and B: where N is the number of data points. Table 1 displays A, B band gap energies E g, and relative error value. The direct and indirect band gap energies E g , as shown in Table 1 and Fig. 4, were calculated by the linear approximation of the slope of the graph of F R a ð Þ ht ð Þ 2 to the photon energy axis where F(R a ) = 0, namely, E g ¼ ht ¼ ÀB=A, as plotted in Fig. 4. In other words, the intersection between the linear fit and the photon energy axis gave the value to E g . The direct gap energies of the Coi-ferrite nanoparticles samples were observed as 2.1 eV as shown in Fig. 4 which was accurate within three decimal digits. The values of E g depended on several factors, including lattice strain, carrier concentration, crystallite size, and the size effect of the dopant metals in Co-ferrite lattice.

Magnetic behavior
The interest in spinel ferrite CoFe 2 O 4 NPs is due to their key properties, such as mechanical hardness, chemical stability, visible light absorption capacity, low band gap energy, and high saturation magnetization (M s ) values. Vibrating Sample magnetometer (VSM) was used to characterize the magnetic nature of the CoFe 2 O 4 nanoparticles. All measurements were taken in the range of ± 1 T at room temperature. The field dependence of magnetization is shown in Fig. 5. Moreover, the magnetic properties of spinel ferrites, such as M s , remnant magnetization (M r ) and coercivity (H c ), are shown in Fig. 5. As shown in Fig. 5, the magnetization curve exhibits a narrow hysteresis. The M s and coercivity field (H c ) values are depicted in Fig. 5, for the nanoparticles. Using the formula  the field dependence of the magnetization (M) close to the saturation value is calculated [31], where M s is the saturation magnetization, b is a parameter related with the magnetocrystalline anisotropy, and H is the applied magnetic field. The magnetization versus 1/H 2 plots is shown in Fig. 6. b and M s values of the CoFe 2 O 4 nanoparticles determined from the slope of the linear fitting and the interception with the y-axis, respectively. The obtained values are depicted in Figs. 5 and 6. Once the b value is determined, the magnetic anisotropy constant (K a ) may be conveniently determined using Eq. 12 [31]: The derived K a value at 300 K is 2.26 9 10 5 erg/g. The M s value of CoFe 2 O 4 nanoparticles obtained within the framework of this study is 60.39 emu/g, which is compatible with the studies in the literature [32]. Therefore, one can suggest that our CoFe 2 O 4 NPs may be used in both biomedical and industrial fields.

Blood compatibility tests
The synthesis of magnetic nano-sized spinel ferrites has become an important area of research, due to their several potential applications [33][34][35]. Materials planned to be used in medical area must be well tested in terms of their biocompatibility. A hemolytic activity test is a suitable and scientific way of determination of biocompatibility of a synthetic material with living systems [36].
In a related literature study, CoFe 2 O 4 nanoparticles (30-50 nm) synthesized by the conventional microemulsion technique did not recommended to be used in intravenous drug administrations due to the negative findings of complete blood count [19]. However, some studies remark the impressive physicochemical properties of CoFe 2 O 4 nanoparticles, like mechanical hardness, improved stability, and colloidal dispersibility under physiological conditions. Incompatibility between hemoglobin and iron containing materials arises from the presence of iron in the structure. These undesirable interactions could be prevented using ferrites for better tissue perfusion and hemocompatibility [37].
Nano-sized materials may change the morphology of RBCs or erythrocytes and cause hemolysis. Hemolysis is defined as the breakdown of cell membrane and lysis of cells. These undesirable interactions between nanoparticles and the blood may promote inflammatory and autoimmune disorders or leading infections as well as cancer by inducing the immune system to suppress [38]. In our study, CoFe 2 O 4 were investigated in terms of their blood compatibility. Human erythrocytes drawn from healthy volunteers were used to investigate the hemolytic potentials of CoFe 2 O 4 nanoparticles. Figure 7 shows the UV-Vis spectra of erythrocyte suspensions treated with PBS alone (control) and CoFe 2 O 4 nanoparticles with two different concentrations, which are 1.0 and 5.0 mg/mL. Hemolysis ratios corresponding to 1.0 mg/mL and 5.0 mg/mL concentrations were 5.4% and 24.7%, respectively. Hemolysis percentages lower than 5% are regarded as safe by International Standards Organization [39]. Thus, for both lower and higher concentrations of CoFe 2 O 4 , nanoparticles showed an undesirable hemolytic effect on human erythrocytes. This may be attributed to the comparatively high amount of reactive oxygen species formed by CoFe 2 O 4 nanoparticles shown in photocatalytic activity tests (Fig. 8). Besides, a sharp increase for the absorption band at 408 nm corresponds to the strong oxidation of oxyhemoglobin to methemoglobin. Moreover, in CoFe 2 O 4 test tubes, the precipitate with a characteristic black color provided an explicit evidence to indicate the formation of heme-iron complexes [40] ( Fig. 7-inset photos).
Despite the fact that cobalt is a microelement essential for living organisms as it is the cofactor of cobalamin (vitamin B12), cobalt can have toxic effects at high concentrations. Cobalt compounds are classified as class II, which means they are not extremely toxic. However, cobalt compounds cause defects in protein and carbohydrate metabolisms, anemia, and carcinogenic and mutagenic effects [41]. Cobalt is a potential inducer of oxidative stress causing reactive oxygen species generation [42]. Reactive oxygen species (ROS) cause the oxidation of hemoglobin to methemoglobin (MHb). Methemoglobin forms when the ferrous (Fe 2? ) ions in heme are oxidized to the ferric (Fe 3? ) state, and the molecule will be unable to carry oxygen to tissues. MHb can form either spontaneously or be induced to form by many substances, including chlorites, phenolic compounds, and heavy metals, such as copper, zinc, and cobalt [43][44][45]. Normally, methemoglobin is reconverted to hemoglobin spontaneously by an enzymatic reduction within the cells. Cobalt may affect this mechanism by blocking the enzymatic activity [46]. Concisely, cobalt induced oxidation of heme and increasing concentration of methemoglobin was observed with increasing CoFe 2 O 4 concentration. In conclusion, CoFe 2 O 4 nanoparticles showed undesirable hemolytic effect in both 1 mg/mL and 5 mg/mL concentrations.

Photocatalytic activity measurements
Oxidative stress mediates several pathological changes, including not only the hemolysis but also the oxidation of sulfhydryl groups on the globin moiety of hemoglobin leading to oxidative denaturation, altered endothelial cell function leading vascular disorders, such as hypertension, stroke, and heart infarction. [47,48]. Reactive oxygen species that are responsible for these undesirable pathologies can also be used to degrade organic pollutants, such as phenols, aromatic hydrocarbons, dyes, or pharmaceuticals in contaminated sources. Many nano-sized materials, which are used in medical applications and have magnetic properties, also have high electrical properties, low band gap values, and high surface area and also show catalytic effects. Combining high photocatalytic properties and biocompatibility is much more important in in vitro studies [49,50].
Changes in the UV-Vis spectra of non-photodegraded CV in the presence of CoFe 2 O 4 nanoparticles with the variation of time and second-order kinetic plot for photocatalytic degradation of CV over CoFe 2 O 4 nanoparticles are shown in Fig. 8a  In this study, when ferrite nanoparticles were subjected to the 254 nm irradiation, Co 2? sites were excited with photo-generated electrons and Co 3? ions were produced (Eq. 13). This phenomenon could be a triggering case for the oxidation of iron in hemoglobin where ferrous form turns into ferric form and induces methemoglobin generation (Eq. 14).
Electron-hole pairs generated by radiation on the valence and conduction bands of semiconductors lead to the formation 19of ROS which are assumed to be responsible for the decomposition of organic molecules [51]. Photocatalytic decomposition mechanism of organic molecules under the effect of electromagnetic radiation has been well defined as an oxidative process in which three major steps were involved. In the fist step of photocatalytic degradation, electrons in valence band were excited by the radiation having energy higher than the band gap of semiconductor. Migration of the excited electrons to an empty conduction band leaves equal number of electron holes (h ? VB ) in the valence band. Second step is the migration of excited electrons to the surface.
Finally, the photo-generated electron-hole pairs take part in redox reactions, resulting in the formation of ROS. In this last step, photo-generated electrons are trapped by the oxygen and electron holes in the valence band interact with H 2 O, resulting in the formation of superoxide (O 2 -Á ) and hydroxyl radicals (OH Á ), respectively (Eqs. 15a and 15b). This step is followed by the formation of other reactive intermediates, including H 2 O 2 and hydroperoxyl radical (HOO Á ) (Eq. 15c) [52]. Afterward, photo-generated electrons could be trapped by O 2 to form superoxide radicals (O 2 -Á ), followed by the generation of other radical species (O 2 -Á , HO Á , HO 2 Á ). Also, the Co 3? sites can react with OHand return back into Co 2? to complete the photocatalytic circle (Eq. 17) [53]. Finally, the reactive species, including O 2 -Á , HO Á , and HO 2 Á , possess sufficient energy for the photocatalytic degradation of CV (Eq. 18): Co 3þ þ Fe 2þ ferrous ð Þ!Co 2þ þ Fe 3þ ferric ð Þ; ð14Þ Co 3þ þ HO À ! Co 2þ þ HO Á ; ð17Þ Kinetic parameters of zero-order, first-order and second-order rate equations for CV degradation in the presence of CoFe 2 O 4 nanoparticles are listed in Table 2. Fitting the data of photocatalytic activity test to (C) -t and to ln(C o /C) -t showed that neither first-order nor second-order kinetic models were successful in representing the photodegradation kinetics of CV.
Second-order kinetic model was the best relationship that fits the degradation of CV by CoFe 2 O 4 nanoparticles. Additionally, the theoretical concentration C o (theory) obtained by second-order model (0.82 mg/L) fit with the experimental value of C o (experimental) (0.95 mg/L) well. Briefly, it can be stated that the second-order kinetic model was the most acceptable model to describe the experimental kinetic data of CV photodegradation.
Photocatalytic properties of CoFe 2 O 4 nanoparticles show significant CV degradation in 420 min under 254 nm irradiation and can be suitable to be used in wastewater treatment industries expurgating organic dyes. Moreover, CoFe 2 O 4 nanoparticles were also responsible for concentration-dependent hemolysis ratios as shown by hemolysis assay (Fig. 7). Results were consistent because formation of ROS is considered as one of the main reasons of oxidative stress, resulting in lysis of erythrocytes. nanoparticles significantly increased the formation of methemoglobin, but also did not cause erythrocyte destruction (hemolysis). The biological incompatibilities of traditional iron oxide nanoparticles caused by the presence of iron in their structures have been reduced in CoFe 2 O 4 nanoparticles where the cobalt is doped. It is known that spinel ferrite nanoparticles are of great importance in current techniques in both biomedical and industrial fields. It is hoped that this study will lead the way in the synthesis of nanoparticles with enhanced biocompatibility and sufficient magnetic and electrical properties with further research and modifications on CoFe 2 O 4 nanoparticles.