Formulation and Characterization of Poly (Ethylene Glycol)-Coated Core-Shell Methionine Magnetic Nanoparticles as a Carrier for Naproxen Delivery: Growth Inhibition of Cancer Cells

Simple Summary Naproxen was loaded onto a magnetic nanoparticle coated with polyethylene glycol. Magnetic nanoparticles (MNPs) were used in this study to develop a smart naproxen delivery system. One of the most potent COX-1 and COX-2 inhibitors is naproxen, which belongs to the NSAID family of drugs. Although this drug has a short half-life, it has considerable toxicities and side effects on gastrointestinal tissues. The significant potential of our proposed nanocarrier for biomedical applications has been widely recognized; we modified MNPs to attach to this drug via disulfide bonds, promote the selective release of naproxen in inflammatory cells, and prevent adverse effects on the digestive system. It was found that the cytotoxicity of the drug was lowered by this change, which prevented unspecific protein binding. Abstract An efficient and selective drug delivery vehicle for cancer cells can remarkably improve therapeutic approaches. In this study, we focused on the synthesis and characterization of magnetic Ni1−xCoxFe2O4 nanoparticles (NPs) coated with two layers of methionine and polyethylene glycol to increase the loading capacity and lower toxicity to serve as an efficient drug carrier. Ni1−xCoxFe2O4@Methionine@PEG NPs were synthesized by a reflux method then characterized by FTIR, XRD, FESEM, TEM, and VSM. Naproxen was used as a model drug and its loading and release in the vehicles were evaluated. The results for loading efficiency showed 1 mg of Ni1−xCoxFe2O4@Methionine@PEG NPs could load 0.51 mg of the naproxen. Interestingly, Ni1−xCoxFe2O4@Methionine@PEG showed a gradual release of the drug, achieving a time-release up to 5 days, and demonstrated that a pH 5 release of the drug was about 20% higher than Ni1−xCoxFe2O4@Methionine NPs, which could enhance the intracellular drug release following endocytosis. At pH 7.4, the release of the drug was slower than Ni1−xCoxFe2O4@Methionine NPs; demonstrating the potential to minimize the adverse effects of anticancer drugs on normal tissues. Moreover, naproxen loaded onto the Ni1−xCoxFe2O4@Methionine@PEG NPs for breast cancer cell lines MDA-MB-231 and MCF-7 showed more significant cell death than the free drug, which was measured by an MTT assay. When comparing both cancer cells, we demonstrated that naproxen loaded onto the Ni1−xCoxFe2O4@Methionine@PEG NPs exhibited greater cell death effects on the MCF-7 cells compared with the MDA-MB-231 cells. The results of the hemolysis test also showed good hemocompatibility. The results indicated that the prepared magnetic nanocarrier could be suitable for controlled anticancer drug delivery.


Introduction
Cancer includes a variety of diseases in which the growth of malignant cells has the potential to spread to other parts of the body [1][2][3]. One of the most common cancers is breast cancer in women; most deaths from breast cancer are due to its drug resistance and the potential for metastasis to distant organs [4][5][6]. There are several pathways involved in the modulation of breast cancer. One of these pathways is the use of nanoparticles (NPs) carrying anticancer agents that can be delivered to the targeted tumor and the treatment of breast cancer [7,8].
Drug delivery is a major topic of research in biomedicine that controls drug delivery and therapeutic drugs to target cells [9][10][11]. However, nanotechnology creates products that exhibit novel properties at nanoscale levels, which offer new tools for biologists [12][13][14]. Recently, the use of various organic-inorganic hybrid NPs because of advances in nanotechnology has drawn significant attention [15]. Studies have shown that modified magnetic NPs can be used in vivo as they increase the efficacy of drug delivery [16].
Magnetic NPs (MNPs), due to their potential in medical applications, have received extensive attention in biomedical communities [17][18][19]. They have a spinel structure with unique properties such as a considerable specific surface area, a small size, high saturation magnetization, and controlled magnetic behavior [20][21][22][23]. Amongst the magnetic NPs (MNP), cobalt ferrite (CoFe 2 O 4 ) has drawn significant attention because the change of metals in its composition can substantially affect its physical and chemical characteristics as well as its biocompatibility to serve as a drug delivery system [22,24,25]. Spinel ferrites (SFs) can be used in medical diagnostics and therapy such as magnetic resonance imaging, controlled drug delivery, gene delivery, bioseparation, and therapeutic hyperthermia [26,27].
Biomedical applications must modify the surface of magnetic NPs to make them more stable before they are used for drug delivery to improve the limitations of these NPs, which include their destabilization effect and non-specific uptake by the RES; the size of magnetic NPs is also limited by the main immunogenic region [28][29][30][31]. Surface modification is required for biomedical applications to begin coating them with stimuli-responsive magnetic NPs that respond when exposed to external stimuli and can generate physicochemical changes in the structure that favor controlled drug release or release at a specific place [32][33][34]. Recently, the use of various organic-inorganic hybrid nanoparticles because of advances in nanotechnology has drawn significant attention. Methionine is one of the most crucial and primary biocompatible amino acids in the human body and it has been specialized for physiological purposes in vivo. Methionine has two active functional groups (-COOH and -NH 2 ) that can be used for metal atom conjugation [35][36][37]. The conjugation of amino acids with polyethylene glycol (PEG) shows important therapeutic benefits. The use of NPs with PEG coatings inside biological samples has several advantages. For in vivo applications, PEG coatings can increase the particle circulation time by reducing protein and cell adsorption on the particles and decreasing the clearance rate of "PEGylated" materials. Herein, we report the magnetic nanoformulations in which Ni 1−x Co x Fe 2 O 4 NPs were coated with methionine as an inner layer and polyethylene glycol as an outer layer. NSAIDs (non-steroidal anti-inflammatory drugs) such as naproxen, which are potent COX-1 and COX-2 inhibitors [38], have been studied; drug loading and release behavior using naproxen as a drug model on Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs have not been previously studied. MTT assays with different doses and incubation times were used to determine cytotoxicity in vitro using this as a carrier.

Preparation of Methionine-Coated Magnetite (Ni 1−x Co x Fe 2 O 4 @Methionine)
In a typical experiment to synthesize an Ni 1−x Co x Fe 2 O 4 NP coated with methionine using the reflux method, briefly 1.621 g FeCl 3 , 0.713 g CoCl 2 , and 0.713 g NiCl 2 were stirred for 15 min in 90 mL of deionized water under an N 2 atmosphere. The mixture was stirred followed by the addition of 25 mL of NaOH (1.5 M) until the mixture reached a pH of around 12. When the precipitate was obtained, magnetic stirring was used to dissolve 0.5 g of methionine in deionized water. A 70-80 • C heat was applied to the mixture and it was allowed to reflux for two hours. Magnetic separation was used to collect the final products and they were rinsed several times with a solution of ethanol and deionized water. The same experimental technique was used to produce bare Ni 1−x Co x Fe 2 O 4 NPs without methionine.

Preparation of PEG-Coated Magnetite (Ni 1−x Co x Fe 2 O 4 @Methionine) NPs
A polyethylene glycol solution with a suitable concentration was prepared by dissolving PEG-6000 powder in a phosphate-buffered saline (PBS) solution. The mixture of the PBS solution and magnetic (Ni 1−x Co x Fe 2 O 4 @Methionine) NPs was vigorously stirred for 30 min and then centrifuged at 6000 rpm to obtain the final NPs [29].

Characterization
An X-ray diffraction (XRD) analysis of the samples was recorded by an A X' Pert Pro MPD, Panalytical (Amsterdam, Netherlands) with Cu Kα radiation (λ = 1.54060 Å) and with a 2θ range of 10-80 • at room temperature. The emissions from the field of the NPs were sized and morphologically characterized using scanning electron microscopy (FESEM) and a transmission electron microscope (Zeiss EM900 Transmission Electron Microscope, Cologne, Germany). It was possible to obtain infrared spectra in the 4000-400 cm −1 range by using a BRUKER spectrometer (VERTEX 70). As a function of the soaking time, the amount of adsorbed and released medication was measured with a UV-Vis spectrophotometer (Shimadzu, UV-1700 Pharma spec, Tokyo, Japan). A Quantum Design MPMS-XL-7 superconducting quantum interference device (SQUID) with an external magnetic field assessed the magnetic characteristics of the NPs produced at an ambient temperature.

In Vitro Loading and Release of Naproxen
First, 0.1 g of nanoparticles (Ni 1−x Co x Fe 2 O 4 @Methionine, Ni 1−x Co x Fe 2 O 4 @Methionine @PEG) were dispersed in a PBS solution. We then prepared different contents of naproxen with 20 mL of a buffer of pH 7.4. The naproxen solution was added to the initial solution and the mixture stood for 24 h whilst being constantly agitated. After that, the dispersion was centrifuged at 6000 rpm and washed twice to collect the naproxen-loaded NPs; the supernatant was saved for estimating the drug loading concentration. UV-Vis spectroscopy was used to determine the loading capacity at a wavelength of 230 nm to assess the remaining drugs in the supernatants. The loading capacity was then computed using a standard calibration curve with various known drug concentrations. Equation (1) was used to compute the amount of loaded naproxen: where C 0 indicates the initial concentration of naproxen, C t represents the drug concentration determined by the standard naproxen curve, α is the dilution ratio, V 0 and V t are the volumes of the aqueous phase in mL, and w (mg) is the weight of the nanocarrier. Samples containing 20 mg of naproxen were placed in 20 mL of PBS at various pH values (5 and 7.4) and shaken at a constant rate (100 r/min) for a specific time in the dark at a constant temperature (37.5 • C). Each time the supernatant was removed, the volume of the new PBS with the same pH value was equally balanced. According to Equation (2), the UV-Vis method at a wavelength of 230 nm was used to calculate the proportion of naproxen that had been released: where V (mL) is the volume of buffer solution, W (mg) is the amount of drug loading, and Ce (mg/mL) is the concentration of naproxen in the supernatant. We used various kinetic models such as zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell.

In Vitro Cytotoxicity
On cancer cell lines MCF-7 and MDA-MB-231 as well as a normal cell line (MCF10A), the samples were tested for their cytotoxicity using an MTT assay, which is a commonly used technique. There were 2 × 10 4 cells per well and 37 • C humidified incubators in 96-well plates with FBS and penicillin-streptomycin to seed the cells. After a 24 h incubation period, suspensions of various samples (0-35 µg/mL) were added to the medium and incubated for another 24 h period as well as for 48 and 72 h. In the next step, the contents of the 96-well plates were replaced by the MTT solution in each well after another 4 h of incubation at 37 • C in a 5% CO 2 environment. To dissolve the purple formazan crystals, the medium was replaced and added to each well with 0.05 mL of dimethyl sulfoxide (DMSO) [39,40]. Finally, the absorbance of each well was measured using a microplate reader at 570 nm. The IC 50 concentration and the cytotoxicity rate were estimated using Equation

Hemocompatibility Test
Fresh blood samples were taken from a healthy volunteer and centrifuged for 5 min at 2000 rpm to isolate the RBC cells. A total of 400 µL of the washed RBCs was diluted with 3.6 mL of PBS to produce the clear supernatant and then the cells were washed three times with PBS. Various concentrations (2000, 1750, 1500, 1250, 1000, and 750 µg·mL −1 ) of the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG nanoformulation in PBS (0.8 mL) was mixed with the diluted RBC suspension (0.2 mL) and kept without shaking for 2 h at room temperature. The negative control was PBS. Centrifugation and UV-Vis spectroscopy were used to measure the absorbance of the supernatant at 540 nm after 2 h to determine the percentage of hemolysis in the solution of each sample [41]. The following equation (Equation (4)) was used to estimate the percentage of hemolysis in each sample: Percent Hemolysis(%) : (sample absorbance − negative control absorbance/ positive control absorbance − negative control absorbance) × 10.

XRD Analysis
The XRD patterns of the as-prepared magnetite Ni 1−x Co x Fe 2 O 4 NPs, methioninecoated samples, and PEG-coated magnetite (Ni 1−x Co x Fe 2 O 4 @Methionine) NPs are shown in Figure 1 [29,37]. After the PEGylation of the core-shell of the Ni 1−x Co x Fe 2 O 4 @Methionine NP with decreasing 2theta values, polyethylene glycol entered into the network cavities and the network space increased. However, the peaks were broad after capping with PEG.
The negative control was PBS. Centrifugation and UV-Vis spectroscopy were measure the absorbance of the supernatant at 540 nm after 2 h to determine the per of hemolysis in the solution of each sample [41]. The following equation (Equa was used to estimate the percentage of hemolysis in each sample:

XRD Analysis
The XRD patterns of the as-prepared magnetite Ni1−xCoxFe2O4 NPs, meth coated samples, and PEG-coated magnetite (Ni1−xCoxFe2O4@Methionine) NPs are in Figure [29,37]. A PEGylation of the core-shell of the Ni1−xCoxFe2O4@Methionine NP with decreasin values, polyethylene glycol entered into the network cavities and the network s creased. However, the peaks were broad after capping with PEG.

FTIR Analysis
The FTIR spectra of all samples were recorded using an FTIR spectrophotometer in the range of 400 to 4000 cm −1 . As shown in Figure 2, methionine showed peaks at 1517 cm −1 (symmetric N-H bending), 1630 cm −1 (asymmetric N-H bending), 1419 cm −1 , 1600 cm −1 (symmetric and asymmetrical stretching of COO), and 1232-1330 cm −1 (C-O band). The peaks of the tetrahedral and octahedral metal-oxygen complexes of Ni 1−x Co x Fe 2 O 4 , which are mainly dependent on Fe-O distances, were located at 400 and 600 cm −1 , respectively. gion of 1481-1604 cm −1 was caused by aromatic C=C stretching and the peaks of 1682 cm −1 and 1728 cm −1 referred to the C=O H bonds and non-H bonds stretching, respectively.
In the curve, the O-H stretch band was approximately presented at 3404 cm −1 , the C-H stretch band at 2835 cm −1 , and the C-O stretch band at 1058 cm −1 . These were are all from the PEG-coated Ni1−xCoxFe2O4@Methionine NP, demonstrating the existence of a residual PEG in the final sample. As seen in Figure 2 in curve e, hydrogen bonds were thought to be present between naproxen and the analyzed NPs. Given the C-H stretching groups in polyethylene glycol and the presence of oxygen in the naproxen, this possibility was reasonable.  The absorption bands in the pure naproxen spectrum around 1028 cm −1 and 1264 cm −1 were due to symmetric Aryl-O stretching and asymmetric Aryl-O stretching, respectively. The spectra around 1090 cm −1 were related to C-O stretching. The peak in the region of 1481-1604 cm −1 was caused by aromatic C=C stretching and the peaks of 1682 cm −1 and 1728 cm −1 referred to the C=O H bonds and non-H bonds stretching, respectively.
In the curve, the O-H stretch band was approximately presented at 3404 cm −1 , the C-H stretch band at 2835 cm −1 , and the C-O stretch band at 1058 cm −1 . These were are all from the PEG-coated Ni 1−x Co x Fe 2 O 4 @Methionine NP, demonstrating the existence of a residual PEG in the final sample. As seen in Figure 2 in curve e, hydrogen bonds were thought to be present between naproxen and the analyzed NPs. Given the C-H stretching groups in polyethylene glycol and the presence of oxygen in the naproxen, this possibility was reasonable.

Magnetic Properties
As can be seen in Figure 3, the magnetic properties of the synthesized Ni 1−x Co x Fe 2 O 4 @Methionine NPs, Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs, and bare Ni 1−x Co x Fe 2 O 4 NPs were measured at room temperature, indicating that the Ni 1−x Co x Fe 2 O 4 NPs exhibited a superparamagnetic behavior. Figure 3a shows that the saturation magnetization of the bare Ni 1−x Co x Fe 2 O 4 NPs was estimated to be 46.09 emu/g. Similar behavior for the Ni 1−x Co x Fe 2 O 4 @Methionine NPs was observed but with less magnetization of 21.86 emu/g (Figure 3b), and even less saturation magnetization for the Ni 1−x Co x Fe 2 O 4 @Methionine@ PEG NPs, which was estimated to be 16.26 emu/g.

Magnetic Properties
As can be seen in Figure 3, the magnetic properties of the synthesized Ni1−xCoxFe2O4@Methionine NPs, Ni1−xCoxFe2O4@Methionine@PEG NPs, and bare Ni1−xCoxFe2O4 NPs were measured at room temperature, indicating that the Ni1−xCoxFe2O4 NPs exhibited a superparamagnetic behavior. Figure 3a shows that the saturation magnetization of the bare Ni1−xCoxFe2O4 NPs was estimated to be 46.09 emu/g. Similar behavior for the Ni1−xCoxFe2O4@Methionine NPs was observed but with less magnetization of 21.86 emu/g (Figure 3b), and even less saturation magnetization for the Ni1−xCoxFe2O4@Methionine@PEG NPs, which was estimated to be 16.26 emu/g.

Morphology Study
The SEM images of the synthesized Ni1−xCoxFe2O4, Ni1−xCoxFe2O4@Methionine, and Ni1−xCoxFe2O4@Methionine@PEG NPs are shown in Figure 4a-c, which demonstrated that the magnetite nanoparticles were an irregular shape with an average particle size of about 13.3 nm. The Ni1−xCoxFe2O4@Methionine and Ni1−xCoxFe2O4@Methionine@PEG NPs were spherical and almost uniform in size with an average particle size of about 17-18 nm and 28-29 nm, respectively. Therefore, the average size of the nanoparticles made them suitable for drug delivery applications. Figure 4d-f shows the TEM images of the Ni1−xCoxFe2O4, Ni1−xCoxFe2O4@Methionine, and Ni1−xCoxFe2O4@Methionine@PEG NPs, respectively. As shown in Figure 4d, the Ni1−xCoxFe2O4 magnetic NPs had a slight agglomeration due to strong magnetic interactions between the NPs [29]. After coating the magnetic NPs with a layer of methionine as an inner layer, a light gray shell around the Ni1−xCoxFe2O4 NPs could be seen and, in the next step, the thickness of the polymer shell of the nanoparticles could clearly be seen, which was increased by adding a polyethylene glycol solution as an outer layer.

Morphology Study
The SEM images of the synthesized Ni 1−x Co x Fe 2 O 4 , Ni 1−x Co x Fe 2 O 4 @Methionine, and Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs are shown in Figure 4a-c, which demonstrated that the magnetite nanoparticles were an irregular shape with an average particle size of about 13.3 nm. The Ni 1−x Co x Fe 2 O 4 @Methionine and Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs were spherical and almost uniform in size with an average particle size of about 17-18 nm and 28-29 nm, respectively. Therefore, the average size of the nanoparticles made them suitable for drug delivery applications. Figure 4d-f shows the TEM images of the Ni 1−x Co x Fe 2 O 4 , Ni 1−x Co x Fe 2 O 4 @Methionine, and Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs, respectively. As shown in Figure 4d, the Ni 1−x Co x Fe 2 O 4 magnetic NPs had a slight agglomeration due to strong magnetic interactions between the NPs [29]. After coating the magnetic NPs with a layer of methionine as an inner layer, a light gray shell around the Ni 1−x Co x Fe 2 O 4 NPs could be seen and, in the next step, the thickness of the polymer shell of the nanoparticles could clearly be seen, which was increased by adding a polyethylene glycol solution as an outer layer. Figure 5 shows the amount of naproxen that could be absorbed into the body. Using these findings, it was concluded that the maximum amount of naproxen adsorption of the Ni 1−x Co x Fe 2 O 4 @Methionine nanoparticles was 0.038 mg/mg when the initial naproxen concentration was 0.06 mg/mL and the maximum amount of naproxen adsorption of the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG nanoparticles was 0.051 mg/mg. As a result, 1 mg of Ni 1−x Co x Fe 2 O 4 @Methionine NPs loaded 0.38 mg and 1 mg of Ni 1−x Co x Fe 2 O 4 @Methionine @PEG NPs loaded 0.51 mg of the drug, respectively.    Figure 5 shows the amount of naproxen that could be absorbed into the body. Using these findings, it was concluded that the maximum amount of naproxen adsorption of the Ni1−xCoxFe2O4@Methionine nanoparticles was 0.038 mg/mg when the initial naproxen concentration was 0.06 mg/mL and the maximum amount of naproxen adsorption of the Ni1−xCoxFe2O4@Methionine@PEG nanoparticles was 0.051 mg/mg. As a result, 1 mg of Ni1−xCoxFe2O4@Methionine NPs loaded 0.38 mg and 1 mg of Ni1−xCoxFe2O4@Methio-nine@PEG NPs loaded 0.51 mg of the drug, respectively.  Figure 6 represents the percentage of naproxen released from the Ni1−xCoxFe2O4@Methionine and Ni1−xCoxFe2O4@Methionine@PEG NPs synthesized as described above (37 °C). For the simulated tumor environment, pH values of −5 and −7.4 were specified. In 120 h, the release rates of both carriers were lower in neutral solution settings (pH 7.4) than in acid solution conditions (pH 5). It was also found that, under the same conditions (pH 5), the drug was released about 20% faster in the Ni1−xCoxFe2O4@Methionine@PEG NPs than in the Ni1−xCoxFe2O4@Methionine NPs. The zero-order, first-order, Higuchi, and Korsmeyer-Peppas models were used in this investigation to systematically determine the release behavior of the naproxen-loaded NPs at different pH values (5 and 7.4). A higher linear regression coefficient (closer to 1) suggested the ideal sample release kinetic model, as shown in Table 1. The Higuchi model was used to calculate the release data for all samples.  In 120 h, the release rates of both carriers were lower in neutral solution settings (pH 7.4) than in acid solution conditions (pH 5). It was also found that, under the same conditions (pH 5), the drug was released about 20% faster in the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs than in the Ni 1−x Co x Fe 2 O 4 @Methionine NPs. The zero-order, first-order, Higuchi, and Korsmeyer-Peppas models were used in this investigation to systematically determine the release behavior of the naproxen-loaded NPs at different pH values (5 and 7.4). A higher linear regression coefficient (closer to 1) suggested the ideal sample release kinetic model, as shown in Table 1. The Higuchi model was used to calculate the release data for all samples.

Cytotoxicity Studies
We chose human breast cancer cells (MCF-7 and MDA-MB-231) and normal cells (MCF10A) as cell models and tested the cytotoxicity after incubation three times for 24, 48, and 72 h with naproxen loaded onto Ni 1−x Co x Fe 2 O 4 @Methionine NPs, free naproxen, and naproxen-loaded Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs, respectively. The MTT assay of the MCF-7 cells (Figure 7) showed naproxen loaded onto Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs had significant cell death, which was much higher than the naproxen loaded onto Ni1-xCoxFe 2 O 4 @Methionine; both carriers caused more cell death than free naproxen. caused higher cell death in MCF-7 than in MDA-MB-231. As shown in Figure 9, the drugloaded NPs did not cause any substantial toxicity in the normal MCF10A cells following a 72 h treatment, indicating that they were biocompatible enough to be used in drug delivery. When naproxen was loaded onto the carriers, the growth inhibition effect on the cancer cells improved their therapeutic possibilities. Table 2    According to Figure 8, the MTT experiment of the MDA-MB-231 cells showed that naproxen loaded onto Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs killed cells faster than Ni 1−x Co x Fe 2 O 4 @Methionine NPs and faster than the free drug (Figure 8 for 24, 48, and 72 h, respectively). According to the comparison of the two types of cancer cells, both carriers caused higher cell death in MCF-7 than in MDA-MB-231. As shown in Figure 9, the drug-loaded NPs did not cause any substantial toxicity in the normal MCF10A cells following a 72 h treatment, indicating that they were biocompatible enough to be used in drug delivery. When naproxen was loaded onto the carriers, the growth inhibition effect on the cancer cells improved their therapeutic possibilities. Table 2

Hemocompatibility Test
The biocompatibility of materials intended for use in the biomedical field must be appropriately evaluated. The hemolytic activity test is a reliable and scientific method for determining the biocompatibility of a synthetic material with living systems [42].

Hemocompatibility Test
The biocompatibility of materials intended for use in the biomedical field must be appropriately evaluated. The hemolytic activity test is a reliable and scientific method for determining the biocompatibility of a synthetic material with living systems [42].
Nanoparticle hemocompatibility is undervalued despite the fact that most nanoparticles will meet blood at some point during their path through the body. Nanoparticles can affect the morphology of red blood cells (RBCs) or erythrocytes, resulting in hemolysis. Hemolysis happens when the cell membrane is ruptured and the cells are lysed. By inciting the immune system to suppress them, these negative interactions between nanoparticles and the bloodstream may increase inflammatory and autoimmune illnesses as well as infection and malignancy [43]. The blood compatibility of Ni1−xCoxFe2O4@Methio-nine@PEG was examined in this study ( Figure 10). Hemolysis percentages lower than 5% were recorded for all concentrations of Ni1−xCoxFe2O4@Methionine@PEG (150, 125, 100, 75, 50, and 25 μg·mL −1 ), which are regarded as safe [44]. Nanoformulation has been demonstrated to be a safe method for the hemolysis of human erythrocytes. This may be due to  Nanoparticle hemocompatibility is undervalued despite the fact that most nanoparticles will meet blood at some point during their path through the body. Nanoparticles can affect the morphology of red blood cells (RBCs) or erythrocytes, resulting in hemolysis. Hemolysis happens when the cell membrane is ruptured and the cells are lysed. By inciting the immune system to suppress them, these negative interactions between nanoparticles and the bloodstream may increase inflammatory and autoimmune illnesses as well as infection and malignancy [43]. The blood compatibility of Ni 1−x Co x Fe 2 O 4 @Methionine@PEG was examined in this study ( Figure 10). Hemolysis percentages lower than 5% were recorded for all concentrations of Ni 1−x Co x Fe 2 O 4 @Methionine@PEG (150, 125, 100, 75, 50, and 25 µg·mL −1 ), which are regarded as safe [44]. Nanoformulation has been demonstrated to be a safe method for the hemolysis of human erythrocytes. This may be due to the inclusion of PEG, which facilitates NP escape from the reticuloendothelial system and prevents macrophage scavenging. This is a solution to the unfavorable hemocompatibility of CoFe 2 O 4 , which has been shown in previous studies to have a hemolytic effect on human erythrocytes [45]. the inclusion of PEG, which facilitates NP escape from the reticuloendothelial system and prevents macrophage scavenging. This is a solution to the unfavorable hemocompatibility of CoFe2O4, which has been shown in previous studies to have a hemolytic effect on human erythrocytes [45].

Discussion
In our previous experiments to produce magnetic Ni 1−x Co x Fe 2 O 4 @Methionine nanoparticles, we occasionally found aggregation among the particles that may have been due to (interparticle) hydrogen bonding between the magnetic nanoparticles and methionine. The answer to the problems posed by the aggregations among the particles in an aqueous suspension is Steric hindrances; therefore, using PEG polymers (steric stabilizers) adsorbed or grafted onto the particle surfaces, contacts among the particles were prevented by the polymer chains extending into the medium. However, with the PEGylated magnetic Ni 1−x Co x Fe 2 O 4 @Methionine nanoparticles, as shown in Figure 1c, peaks of the NPs were observed but the intensity of the peaks was significantly reduced due to the coating of amorphous PEG on the surface of the core-shell of the Ni 1−x Co x Fe 2 O 4 @Methionine NPs; this was in good agreement with Panwar et al. [46].
As can be seen in Figure 2c, the peaks of methionine that were determined by the spot chain in the spectrum were similar to the peaks of Ni 1−x Co x Fe 2 O 4 @Methionine, which clearly showed the presence of methionine on the surface of Ni 1−x Co x Fe 2 O 4 [37]. However, in magnetic Ni 1−x Co x Fe 2 O 4 NPs and methionine, van der Waals and hydrogen forces are thought to bind them. Given the amino acid groups in methionine and the presence of oxygen on the nickel structure, this possibility was reasonable.
Furthermore, before coating the Ni 1−x Co x Fe 2 O 4 @Methionine NP by PEG, peaks in the regions of 3404 cm −1 , 2835 cm −1 , and 1058 cm −1 were not observed in the spectrum of the Ni 1−x Co x Fe 2 O 4 @Methionine NP. These results clearly showed the surface modification of the Ni 1−x Co x Fe 2 O 4 @Methionine NP with PEG. On the other hand, according to the TEM image of the Ni 1−x Co x Fe 2 O 4 @Methionine NP before PEGylation, aggregation was found among the particles, which may be due to hydrogen bonding (interparticle) or van der Waals forces between the magnetic nanoparticles and methionine. After the surface coating, there was a decrease in the agglomeration, which confirmed the successful capping of Ni 1−x Co x Fe 2 O 4 @Methionine with PEG.
Naproxen-loaded Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs had C=O bands that shifted from 1728 cm −1 to 1604 cm −1 because of hydrogen bonding that formed between the surface of the carrier and the naproxen [47]. Therefore, a peak in the 808 cm −1 region of curve e related to the bending of naproxen on the plate, resulting in the successful loading of naproxen onto the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NP. Figure 3 delineates the magnetic properties with the applied magnetic field range from 15 to 15 kOe in which the magnetization curves of the nanoparticles were measured by SQUID. The curves of the magnetization samples described a superparamagnetic function. That is important for biomaterials, making them easily trackable in the magnetic field gradient whilst maintaining the advantage of a stable and homogeneous suspension during drug delivery. When methionine was coated, bare Ni 1−x Co x Fe 2 O 4 NPs were coated and the attachment of non-magnetic groups of methionine reduced the saturation magnetization. The saturation magnetization for the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs was also less than that for Ni 1−x Co x Fe 2 O 4 @Methionine. The reduction in the magnetic properties of the Ni 1−x Co x Fe 2 O 4 NPs was due to their shells. As a result, the observed values of the saturation magnetization of the Ni 1−x Co x Fe 2 O 4 NPs with different shells confirmed the magnetic properties of these nanoparticles.
As shown in Figure 5, there was an increase in naproxen adsorption when the initial concentration of naproxen was raised. These findings showed that the adsorption of naproxen benefitted from a high loading capacity. There was a strong correlation between the loading capacity of naproxen and the initial drug concentrations; this appeared to be due to the large specific surface area of the carrier and the hydrogen bonds between the naproxen and the shells of the carriers. This was demonstrated when the loading capacity of the PEGylated Ni 1−x Co x Fe 2 O 4 @Methionine NPs was raised.
As shown in Figure 6, when drugs are released into cancerous tissues, they are released into acidic intracellular lysosomes, endosomes, or diseased cells. As a result, nanoparticles could help accelerate drug release intracellularly after being internalized via endocytosis. If this is applied, anticancer drugs may have fewer side effects on healthy cells and drug losses during blood transport would be reduced. Under the same conditions, naproxen was released about 20% faster in the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs than in the Ni 1−x Co x Fe 2 O 4 @Methionine NPs because the nanoparticles could increase the intracellular drug release after being internalized by endocytosis. Under an acidic pH of 7.4, the drug release was low because a low release decreases drug loss in blood transportation; a high release at a pH of 5 facilitated the active release of anticancer drugs and the data suggested that both carriers were sensitive to the pH. The pH-dependent release was due to the strong differential in the hydrogen bonding interactions between the naproxen functional groups and the shell carrier functional groups.
The result of the MTT assay (Figure 7) showed that the cell death of naproxen loaded onto Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs was much higher than naproxen loaded onto Ni1-xCoxFe 2 O 4 @Methionine; both carriers caused more cell death than free naproxen as the method of drug absorption or the cellular absorption of the drug was not the same as that of a drug loaded onto a nanoparticle. Their cellular absorption was one of the reasons they had varying toxicities. The drug had a shorter cross-sectional effect when taken orally due to the faster release but when administered via nanoparticles, the drug had a longer effect due to the slower release and the overall effect of the drug was improved [36,37]. In high amounts, the microelement cobalt can have toxic implications despite being a cofactor of vitamin B 12 [48]. That signifies that cobalt compounds are not particularly toxic. However, cobalt compounds can cause abnormalities, mutagenesis effects, and anemia in people [49]. Reactive oxygen species can be generated due to cobalt-induced oxidative stress [50]. In this work, the toxicity of cobalt compounds was lowered by PEGylation in various fields.

Conclusions
Recent studies have focused on magnetic formulations with amino acids or polymer shells as a drug delivery mechanism. For Ni 1−x Co x Fe 2 O 4 NPs, the methionine and PEG capping agent improved the particle stabilization, drug delivery potential in vitro characteristics, and uniform distribution of the particle size and biocompatibility. Using naproxen as a model drug at human body temperature in normal pH settings and acidic pH conditions, the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs were shown to have drug delivery capabilities. The naproxen-loaded carrier slowly released the drug over the course of 120 h. Therefore, the Ni 1−x Co x Fe 2 O 4 @Methionine@PEG nanocarrier was pH responsive, which could be a promising drug-releasing agent and facilitate the delivery of drugs to the desired tissue types in patients. The fraction cell viability was characterized by an MTT assay in which the cell death of naproxen was well-controlled through the pH-responsive magnetic nanocarriers. We demonstrated cell viability for two cancer cell lines (MCF-7 and MDA-MB-231) and a normal cell line (MCF10A), which showed that the drug-containing Ni 1−x Co x Fe 2 O 4 @Methionine@PEG NPs were more potent than free naproxen and even more potent than the drug-containing Ni 1−x Co x Fe 2 O 4 @Methionine NPs. There was a greater MCF-7 cell inhibition effect than in the MDA-MB-231 cell. None of the drug-loaded carriers or the free drug inhibited the growth of the normal cell line, MCF10A. The results showed significant differences in the drug-loaded Ni1-xCoxFe 2 O 4 @Methionine@PEG NP and the free drug at the same concentrations because when the drug was loaded into the carrier, which was coated by amino acids and PEG, it provided an NP with a larger surface area available for interactions, which enhanced the cell death effect and thus they imparted to the microorganisms.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.