Enhancing the Cytocompatibility of Cobalt‐Iron Ferrite Nanoparticles Through Chemical Substitution and Surface Modification

Due to their potential in numerous interdisciplinary fields, such as drug delivery, hyperthermia, and magnetic resonance imaging, magnetic nanoparticles have attracted widespread attention. In this study, cobalt ferrite nanoparticles are synthesized with varying amounts of iron and cobalt ions, and their cytocompatibility is evaluated before and after surface modification with amino‐silane (AEPTMS). Characterization of the synthesized nanoparticles is performed using scanning electron microscopy, energy‐dispersive X‐ray spectroscopy, X‐ray diffraction, and Fourier transform infrared spectroscopy. In addition, it is observed that a slight reduction in the saturation magnetization of the coated materials. The cytotoxicity of these materials is evaluated using the MTT assay on fibroblast cells. This study focuses on the importance of the interface between the surface of magnetic nanoparticles, their coating, and their interaction with cells. This study presents an innovative approach to enhancing the cytocompatibility of cobalt ferrite nanoparticles through chemical substitution and surface modification with AEPTMS. The key findings demonstrate that the iron and cobalt ion ratio significantly impacts cytotoxicity, with the AEPTMS coating found to increase cell viability. These findings demonstrate the potential of these materials for biomedical applications, highlighting the importance of both the composition and surface coating of magnetic nanoparticles for biomedical applications.


Introduction
Nanoparticles have attracted a lot of interest in the biomedical field due to their distinctive characteristics. They have DOI: 10.1002/admi.202300206 special physical and chemical properties that can be taken advantage of for a variety of interdisciplinary applications, including imaging, [1,2] drug delivery, [3,4] cancer therapy [5][6][7] as hyperthermia mediators [8][9][10][11] because of their small size and high surface areato-volume ratio. One of the key factors that influences the properties of nanoparticles is their composition, [12,13] which can be controlled through various synthesis methods such as coprecipitation. [14,15] This can have a significant impact on the physicochemical properties of the nanoparticles, as well as their interactions with biological environments. The addition of coatings to nanoparticles can also significantly alter their properties, enabling them to be more biocompatible or to better interact with their surroundings. [16,17] The interface between cells and nanoparticles is of critical importance for understanding the behavior and potential applications of nanomaterials in biomedicine. The surface properties of nanoparticles play a key role in determining their interaction with cells, including their uptake, toxicity, and ability to trigger cellular responses.
Moreover, magnetic fluids, or ferrofluids, are colloidal suspensions of small magnetic nanoparticles (10-20 nm) in a carrier fluid. They exhibit unique physicochemical properties that are triggered by a magnetic field and have been widely studied. These colloidal materials, consisting of fine magnetic particles suspended in a liquid medium, have been widely investigated in various areas of science and engineering due to their ability to be used in a variety of technological and biological applications. Ferrofluids have shown to be highly effective in fields such as heat transfer, detection of different parameters, cancer therapy, and removal of contaminants, among others. [18] Iron oxide nanoparticles have been extensively investigated for their potential biomedical applications due to their magnetic properties, low toxicity, and FDA approval. [19] However, some limitations such as the desire to control the saturation magnetization, coercivity as well as cytocompatibility, have motivated researchers to explore alternative materials. One such material is cobalt-iron ferrite, which can potentially overcome the limitations of iron oxide nanoparticles. Co x Fe (1-x) Fe 2 O 4 is a type of ferrite material with the formula MFe 2 O 4 , where M is a divalent metal such as iron (Fe) or cobalt (Co). The exact arrangement of the atoms or ions within the structure has a significant impact on the magnetic properties of these materials. [20,21] In this study, we will be examining the effects of substituting divalent iron ions with divalent cobalt ions on the structure and properties of Co x Fe (1-x) Fe 2 O 4 nanoparticles. This exchange is expected to affect the magnetic behavior of the nanoparticles, as well as other properties such as cell viability. Cell lines' viability can be used to measure the potential biological impacts of nanoparticles. In vitro studies using these cell lines can provide important insights into the potential effectiveness and safety of nanoparticles for use in biomedical applications. [22] Recent studies have shown that the surface chemistry, size, shape, and charge of nanoparticles can all influence their cellular interactions, highlighting the need for a better understanding of the mechanisms underlying these interactions. [23][24][25][26] Amino silane is a commonly used coating for nanoparticles due to its biocompatibility and ability to improve the stability of the particles in biological environments. [26][27][28][29][30] In the literature, various modifications have been proposed for magnetite or cobalt ferrite magnetic nanoparticles, including the use of other materials such as silica, [31] chitosan, [32] or polyethylene glycol (PEG), [33] to improve their properties for biomedical applications. These modifications have shown promising results in terms of increased stability, biocompatibility, and reduced toxicity of the nanoparticles. In addition, the current literature on the subject has primarily focused on the synthesis routes and the characterization of magnetic nanoparticles [34][35][36][37] However, to the best of our knowledge, the synthesis and characterization of cobalt-iron ferrite nanoparticles with varying concentrations of iron and cobalt ions and coated with 3-(2-aminoethyl amino) propyltrimethoxysilane (AEPTMS) have not been extensively studied in the literature. Our work fills this gap by presenting a systematic study of the synthesis and characterization of these nanoparticles and their cytotoxicity in vitro. In this study, we used AEPTMS coatings on the Co x Fe (1-x) Fe 2 O 4 nanoparticles. The novelty of our work lies in the combination of these two strategies to enhance the biocompatibility and stability of the nanoparticles, as well as their potential for biomedical applications.

Results and Discussion
In this study, we prepared samples of magnetite (x = 0) and cobalt ferrite (x = 1) to verify the suitability of the synthesis parameters for preparing these materials. We then applied these same parameters to the materials that had the substitution of iron ions with cobalt. Figure 1A shows the X-ray diffraction (XRD) patterns of all the samples while Figure 1B shows XRD pattern of cobalt ferrite compared with the JCPDS (Joint Committee in Powder Diffraction Standards) crystallographic charts. The comparison of the obtained patterns with the standardized patterns indicated that the crystalline structures of the prepared materials were formed, as there was a similarity in the diffraction peaks between the obtained patterns and the standard. The XRD spectra showed the presence of reflections corresponding to the spinal cubic phase, with no peaks corresponding to any secondary phases. The (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) reflections were observed for all ferrite samples. The (311) peak was found to be the most intense peak for all concentrations, indicating a preferred orientation of the crystallites. It was also noted that the diffraction peaks shifted toward higher 2 angles with an increase in cobalt concentration. We also observed that when iron ions were replaced with cobalt ions, there was no variation in the crystal structure of the material, which maintained its cubic ordering of atoms. However, the domain size of the crystalline structure, calculated with the Scherrer formula, decreased as the cobalt content increased (Figure 2). This was due to the smaller atomic radius of cobalt compared to iron, which caused the crystalline structure and nanoparticles to reduce in size. Therefore, the crystalline structure of the material will tend to reduce its size as more cobalt atoms substitute iron atoms.
The chemical composition of the uncoated ferrite nanoparticles was analysed using energy dispersive X-ray spectroscopy (EDS) to confirm the presence of iron, cobalt, and oxygen. This technique was performed using a scanning electron microscope (SEM) and involves detecting the characteristic X-ray radiation emitted by the elements present in the sample. Figure 3 displays the SEM images and EDS spectrum of bare cobalt ferrite (Co x Fe 1-x Fe 2 O 4 , x = 1.0), where peaks corresponding to cobalt, iron, and oxygen can be seen. In the EDS of the sample coated with AEPTMS, the presence of the silicon element is also visible. The morphology of all samples was semi-spherical in shape, with a high degree of agglomeration due to the large surface energy of the nanoparticles. The high surface area to volume ratio of these small particles leads to an increase in surface energy and a tendency to form aggregates in order to reduce energy.
The Fourier transform infrared (FTIR) spectra of all the samples (Figure 4) were analysed to investigate the chemical bonding and functional groups present in the samples. In all the spectra, a vibration band ≈650 cm −1 was observed, which is attributed to the bonding between the metals and oxygen. This indicates the presence of metal-oxygen complexes in the octahedral sites, which is a characteristic of ferrite materials. In the spectra of the AEPTMS-coated nanoparticles, additional bands were observed. These bands were assigned to the vibrations of the functional groups present in the AEPTMS coating. The N-H stretching, and wagging vibrations are represented by bands at 3667 and 812 cm −1 , whereas the C-H stretching and scissoring vibrations   Figure 5. The Co 2+ content is found to have a significant effect on the magnetic properties of the nanoparticles, while the AEPTMS coating has a minor effect on the saturation magnetization. In particular, the saturation magnetization decreases with increasing Co 2+ concentration. The results confirm that the magnetic properties of Co x Fe (1-x) Fe 2 O 4 nanoparticles can be tailored by controlling the Co concentration as previously reported. [38,39] The observed differences in magnetic properties are attributed to the different moments of the Fe +2 and Co +2 ions. The moment of Fe +2 is greater than those of Co +2 due to its four unpaired electrons in the 3d orbital, which combine to form a resultant magnetic moment. The magnetization values obtained for the nanoparticles synthesized in this study were found to be comparable to or higher than those reported in previous studies for similar concentrations. It should be noted that the similar concentration of 0.05 M, in our case for x = 1.0, yields a magnetization of 36.5 emu g −1 , whereas in the article cited by the reviewer, this concentration gives a magnetization of ≈37.5 emu g −1 but with a notable coercivity. [40] Our study found that the magnetic properties of the materials remained superparamagnetic even after coating. However, the saturation magnetization of the coated materials was slightly lower than that of the uncoated ones, with a reduction from 55.84 to 52.78 emu g −1 for x = 1.0. This decrease can be attributed to the presence of a silica layer surrounding the particles, which contains diamagnetic silicon. Diamagnetic materials tend to oppose the direction of the applied magnetic field, resulting in a slight decline in the saturation magnetization of the material.
The cell viability of the proposed materials was evaluated using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results of the cytotoxicity of bare ferrite are shown in Figure 6A, where it can be observed that according to the statistical analysis, there are clear significant differences between the materials at all concentrations of NPs tested. Cobalt ferrite was found to have a more toxic effect in its pure state (x = 1.0), but the substitution of the Co +2 ion for the Fe +2 ion reduced its cytotoxicity. In a study by Marmorato et al., a dependence was found between the uptake of cobalt ferrite NPs by fibroblasts and their concentration, observing that these cells can even endocytose aggregates of NPs, and it was found that cobalt accumulation in the nuclear region was greater than that of iron. [41] On the other hand, Figure 6A also shows that pure magnetite (x = 0) tends to be more toxic than the substituted ferrites, which could be due to the oxidative stress caused by the Fe +2 ion. [42] However, further testing is needed to determine the causes of cell death.
In general, ferrites with different stoichiometric ratios (x = 0.2, 0.4, 0.6, and 0.8) were able to maintain cell viability to a greater extent than pure magnetite and cobalt ferrite, indicating that the variation of the Fe +2 /Co +2 ratio in the ferrites has a positive effect on their cytocompatibility. Iron ions can be metabolized by cells since it is an essential element for various catalytic redox reactions as it is part of the structure of proteins involved in important metabolic processes (e.g., hemoproteins). In the second case, cobalt is an essential trace element that is required for the proper functioning of certain enzymes. For example, cobalt is a component of cobalamin, also known as vitamin B12, which is essential for the synthesis of red blood cells and the maintenance of the nervous system. Cobalt is also a component of other enzymes, such as superoxide dismutase, which plays a role in protecting against oxidative stress. However, excessive exposure to cobalt ions has been linked to genetic damage and oxidative stress. Therefore, the potential cytotoxic effects of these materials should be carefully considered in their application.
The influence of coating with AEPTMS on cellular viability was evaluated using the MTT assay. Results shown in Figure 6B indicate that there were still significant differences between the materials. The cytotoxic behavior of the coated materials showed the same trend as the uncoated materials, with substituted ferrites still presenting lower toxicity than pure magnetite and cobalt ferrite, indicating that the proportion stoichiometry factor has a greater impact on cytocompatibility than the coating. It is important to note that the percentage of viability obtained with the coated nanoparticles increased. AEPTMS is a silica coating with an organic modification, and it has been demonstrated that in physiological environments, silica provides greater stability to nanoparticles and greater resistance to degradation, which may be related to a lower release of Co +2 and Fe +2 ions and, therefore, to the reduction of the toxic potential of the nanoparticles.

Conclusions
In this study, we evaluated the cytotoxicity of bare and AEPTMS coated cobalt-iron ferrite (Co x Fe 1-x Fe 2 O 4 ) nanoparticles and investigated the potential of substituting cobalt ions with iron ions to improve their cytocompatibility for biomedical applications. Our findings show that the substitution of Co +2 ions with Fe +2 ions had a positive effect on the cytocompatibility of the materials. Additionally, the AEPTMS coating did not significantly affect the cytotoxicity of the materials but increased the viability of the cells. These results suggest that the combination of cobaltiron ferrite nanoparticles with the AEPTMS coating may be a promising strategy for biomedical applications such as hyperthermia therapy. The presence of the coating may also contribute to the stability and degradation resistance of the nanoparticles. Our structural and chemical characterization using SEM, EDS, XRD, and FTIR confirmed the presence of the coating and the ferrite structure of the nanoparticles. The novelty of this research lies in the evaluation of the cytocompatibility of cobalt-iron ferrite nanoparticles with the substitution of cobalt ions with iron ions and the coating with AEPTMS. Our study provides new insights into the cytotoxicity of these materials and offers a potential strategy for improving their cytocompatibility. Our findings contribute to the ongoing efforts to develop safer and more effective nanomaterials for biomedical applications.
Synthesis of CoxFe(1-x)Fe2O4: Co x Fe (1-x) Fe 2 O 4 (where x = 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0) nanoparticles were synthesized by chemical coprecipitation as described previously. [43] In this method, the concentration of the cations trivalent (Fe 3+ ) and divalent (M 2+ , where M = Fe, Co) remained 10 and 5 mm, respectively. In all cases, the stoichiometry of Fe 2+ and Co 2+ was adjusted to obtain the nanoparticles with a certain formula. To control the particle size and to achieve good homogeneity, the mixture of solutions was constantly stirred at 60°C till precipitate formation due to the addition of NH 4 OH. The product was washed immediately with excess of deionized water, followed by centrifugation (5 min, 3000 rpm) several times until reach pH neutral. Later, the precipitates were subjected to drying at 50°C for 12 h before surface-modification and characterization.
AEPTMS Coating: The nanoparticles with the AEPTMS coating were created using a sonochemical process. A typical reaction involved dispersing 0.10 g of ferrite nanoparticles in 150 mL of ethanol. Then, 2 mL of deionized water was added to 100 mL of AEPTMS to dissolve it. The beaker was submerged in an ultrasonic bath for two hours. The process involved collecting the finished goods using a powerful magnet and repeatedly rinsing them in absolute ethanol. The product was finally dried for 12 h at 50°C.  Characterization: Using a PANalytical X"Pert MRD PRO device with a Cu-k source ( = 1.5406 Å) operating at 40 kV and 30 mA and at a scanning rate of 0.1°2 s −1 , from 10 to 80°2 , Co x Fe (1-x) Fe 2 O 4 nanoparticles were characterized as-produced. Using Scherrer's equation and the peak with the highest intensity, samples" crystallite sizes were calculated. [44] The particle size distribution and morphology of both uncoated and AEPTMS-coated nanoparticles were examined using scanning electron microscopy (SEM) on a JEOL JSM6010LV/PLUS SEM. Using Scandium, an image processing program, 100 particles were examined to determine the mean diameter and standard deviation (SD). Comparing the mass percentage ratio of Fe/Co with the ratio discovered during the synthesis was also done using Energy Dispersive X-ray Spectroscopy (EDS) on an Inca apparatus (Oxford Instruments, Oxford, UK). A spectrometer (Nicolet 6700/Thermo Electron) was used to record Fourier transform infrared spectra (FTIR) in order to identify the functional groups that were present in the AEPTMS-modified nanoparticles. ATR infrared spectra were captured with a resolution of 4 cm −1 and a scan range of 4000-600 cm −1 .
Cell Viability Assay: An in vitro methyl thiazolyl tetrazolium (MTT) assay was carried out using 3T3 mouse embryonic fibroblast cells to check the nanoparticles' cell viability. The cells were sown in a culture medium, Eagle's Minimum Essential Medium (EMEM), which contains 200 U mL −1 of penicillin-streptomycin and 10% fetal bovine serum. 3T3 cells were seeded into a 96-well cell culture plate, and they were then kept there for 24 h at 37°C with 5% CO 2 . Then, nanoparticles dispersed in PBS were added to each well, resulting in final particle concentrations of 0, 0.05, 0.10, 0.25, 0.50, and 1.00 mg mL −1 . The cells were then kept at 37°C and exposed to 5% CO 2 for another three hours. At the conclusion of the incubation period, nonparticle-containing media was removed, wells were washed in PBS to remove the non-uptaken particles, and 90 μL of fresh medium was added. A further four hours were spent incubating the plates at 37°C with 5% CO 2 after adding 5 mg mL −1 of filter-sterilized MTT reagent in PBS to each well (100 L). 100 μL of DMSO was then added to each well to dissolve the formazan crystals after the culture medium had been incubated. A microplate reader was used to measure the absorption values of the dissolved formazan crystals in each well at 570 nm.
Statistical Analysis: The percentage difference between the absorbance of the treated cells and the untreated controls was used to define cell viability. Data are presented as the mean standard deviation (SD) and all samples were prepared in triplicate. One-way analysis of variance (ANOVA) and the Tukey's Multiple Comparison Analysis test were used to determine how different the groups were from one another. When p < 0.05, values were deemed significant.