In vitro cytotoxicity and anti-cancer drug release behavior of methionine-coated magnetite nanoparticles as carriers

A novel and specific drug delivery for in vitro cancer targeted are developed successfully by a simple one-step method. A CoFe2O4@Methionine core–shell nanoparticle was prepared by the reflux assay which amino acid in the surface makes ferrite biocompatible, enhances its chemical stability, and improves the drug-loading capacity. The synthesized nanoparticles were characterized using FTIR, TGA, XRD, SEM, TEM, and VSM which coating amino acid on the surface of CoFe2O4 was confirmed by XRD and TGA. The appearance of a new peak for C≡N confirms the formation of Letrozole-loaded carrier in the FTIR. The vibrating sample magnetometer of both bare CoFe2O4 and Methionine-coated CoFe2O4 nanoparticles exhibited room-temperature superparamagnetic behavior with a saturation value of 46 emu/g and 16.8 emu/g, respectively. The morphology and size of samples were characterized by SEM and TEM that the average size of the particle was around 28–29 nm. The loading of Letrozole and the effect of pH (5, 7.4) on the release behavior of the carrier was studied. The result of the drug release in pH is equal to 5 was about 88% which higher than pH is equal to 7.4. Also, the preparation had been evaluated for determining its cytotoxicity using MCF-7, MDA-MB-231, and MCF10A cells as an in vitro model, and the result vitro experiments showed that CoFe2O4@Methionine could significantly reduce cancer in cells model. These results demonstrate that core–shell nanoparticle was prepared is biocompatible and have potential use as drug delivery.


Introduction
The number of cancers overtaken patients is increasing day by day [1][2][3][4][5][6]. One of the most common types of cancer, especially in women is breast cancer, and, unfortunately, its cancer-suffering patients are increasing each year [7][8][9][10][11][12][13][14]. It is widely studied and accepted that breast cancers are hormone dependent and that is estrogen which is a key mediator in the progression and metastasis of breast cancer [15]. Letrozole is one of the most effective third-generation nonsteroidal aromatase inhibitors (AIs) that can inhibit excess estrogen biosynthesis in the body [4,6,8,14]. Letrozole uses as positive drug to treat breast cancers and highly potent drugs due to its estrogen receptor [16][17][18][19].
Some strong steps for the treatment of cancer are required to develop new technology. One of the ways to kill the cancer cells by using targeted drug delivery in which the word "targeted" is referred to kill only cancerous cells without any harm of healthy cells. Recently, spinel ferrites have attracted much attention for their potential use in biomedical applications like controlled drug delivery, cell separation, magnetic resonance imaging (MRI), and localized hyperthermia. [9][10][11][12][13][20][21][22][23]. Among them, Cobalt ferrite (CoFe 2 O 4 ) has particular significance because its remarkable magnetization property such as high coercivity, moderate saturation magnetization, high Curie temperature, large magneto crystalline anisotropy, high mechanical hardness, and remarkable chemical stability, also appropriates biocompatibility and low toxicity [24][25][26]. Furthermore, they can be directly injected into cancer cells and delivered by magnetic field gradient or delivered by other efficient drug delivery systems to release their drugs [9][10][11][12][13]27].
Acknowledging several robust steps in cancer therapy needs advance guard. Commonly used method in cancer treatment is targeted drug delivery where they are targeted to destroy only cancerous cell by doing no harm to healthy cells. Recent studies on spinel ferrites draw attention to its potency of drug delivery, cell separation, magnetic resonance imaging (MRI), localized hyperthermia, and several other benefits towards biomedical [9][10][11][12][13][20][21][22][23]. Out of all, Cobalt ferrite (CoFe 2 O 4 ) shows notable results for the high coercivity, moderate saturation magnetization, high Curie temperature, large magneto crystalline anisotropy, high mechanical hardness, and outstanding chemical stability, also suitable biocompatibility, and relatively low toxicity [24][25][26]. Additionally due to the importance in cancer treatment, they can be administered straight into tumor cells either by magnetic field gradient or delivered by other efficient drug delivery systems to release their drugs [9][10][11][12][13]27].
So far, various magnetic nanoparticles with different formulations have been synthesized for cancer therapy which to improve biomedical applications, surface modification is necessary to coat them with stimuli responsive [28][29][30]. Methionine is one of the most crucial and primary biocompatible amino acids in the human body, which has specialized in vivo physiological purposes. Three activated functional groups of Methionine (-COOH, -NH 2 , and -SH) could be simply applied for the conjugation of metal atoms(CoFe 2 O 4 ) which can use as the surface of a carrier to examine the loading and release behaviors that have not yet been reported [31,32]. In 2018 Guangzhou Wang, Fei Zhou et al. revealed a facile synthesis of Cobalt Ferrite (CoFe 2 O 4 nanoparticles) in the presence of L-cysteine (Lys) which can serve as a great carrier to cancer therapy [9][10][11][12][13]33]. Also Amoli-Diva et al. reported that FeMn 2 O 4 nanoparticles coated with (TEOS) and modified with 3-mercaptopropionic acid (MPA) can be a suitable candidate for site-specific and controlled anti-cancer delivery.
In this research, synthesis and application of smart core/ shell CoFe 2 O 4 nanoparticles were reported which coated with methionine through the reflux assay in one step and used as a carrier of an anti-cancer drug. Nanoparticles were characterized by XRD, SEM, TEM, VSM, TGA, and FTIR techniques. Letrozole was used as an anti-cancer model drug, and the drug-loading and drug release behavior of methionine-coated CoFe 2 O 4 nanoparticles is reported. Furthermore, it was reported that CoFe 2 O 4 @Methionine in vitro cytotoxicity was evaluated by MTT assays with varying concentrations on two cancer cell lines and normal cell line at 24, 48, and 72 h.

Preparation of methionine-coated CoFe 2 O 4 nanoparticles
Cobalt ferrite nanoparticles were synthesized by the coprecipitation method. In this experiment, 1.42 g of CoCl 2 .6H 2 O and 3.24 g of FeCl 3 .6H 2 O with molar ratio 1:2 were dissolved in 180 ml deionized water and stirred for 30 min under N 2 atmosphere and then pH raised to 12 with adding NaOH (1.5 M). Then 1 g of Methionine is dissolved in deionized water, added to the mixture. The mixture was heated to 70-80 °C and refluxed for 3 h; the final formed brown precipitate was collected by magnetic separation and washed with deionized water and ethanol. The procedure is shown in Fig. 1.

Characterization
X-ray diffraction (XRD) analysis of the samples is recorded by an STOE STADI-P with Cu Kα radiation (λ = 1.54060 Å) and with the 2θ range of 10-80° at room temperature. Field emission Scanning Electron Microscopy (FESEM) (model Zeiss-EHT-10.00 kV Germany) and transmission electron microscope (TEM) (model Zeiss-EM10C-100 kV Germany) were used for size and morphology measurement of the methionine-CoFe 2 O 4 nanoparticles. Fourier transform infrared spectroscopy (FTIR) data were taken in the spectral range from 400 to 4000 cm −1 by using a model nexus 870 spectroscopy. The amount of adsorbed and released drug is monitored as functions of soaking time by Ultraviolet-visible (UV-Vis) spectra were obtained with a Shimadzu UVS-1700 at 239 nm. The thermal properties (TGA) were performed by a Shimadzu TA Q600 (USA) system from 25 to 800 °C in the nitrogen atmosphere at a constant heating rate. The magnetic properties of the synthesized Methionine-coated CoFe 2 O 4 nanoparticles and magnetic nanoparticles CoFe 2 O 4 were measured at room temperature by a Quantum Design MPMS-XL-7 superconducting quantum interference device (SQUID) with an external magnetic field ranging from −15 to + 15 kOe.
Loading capacity of letrozole 0.0016 g Letrozole was dissolved in 20 ml methanol, and then 0.04 mg of Methionine-CoFe 2 O 4 nanoparticles was added to this solution. This mixture was stirred for 24 h at room temperature to load drug molecules. Then the dispersion of the sample was centrifuged at 6,000 rpm for 12 min to collect the Letrozole-loaded nanoparticles and kept the supernatant for calculating the drug-loading content. The Letrozole-loaded nanoparticles which collect dried at room temperature and supernatants were collected measured by UV-Vis spectroscopy at a wavelength of 239 nm, and the loading capacity was calculated according to the standard curve with different known drug concentrations. The amount of loaded Letrozole was calculated according to Eq. (1).
where C 0 is the initial concentration of Letrozole, C t is the concentration of the drug which calculated by standard curve of Letrozole, V 0 and V t are volume of the liquid phase (ml), α is dilution ratio, and w (mg) is the weight of the nano-carrier. where C e (mg/ml) is the concentration of Letrozole in the supernatant, V (ml) is the volume of buffer solution, and w (mg) is the amount of drug loading. The drug release data were analyzed mathematically according to the models fitted in kinetic models' equations for the release kinetic studies and to investigate the release mechanism. The linear form diagrams which used for models are zero-order kinetics (cumulative % drug released vs. time), first-order kinetics (log % drug retained vs. time), Higuchi model (cumulative % drug released vs. square root of time), and Korsmeyer-Peppas equation (log amount

In vitro release study and kinetic modeling
of drug released vs. log time). The correlation coefficient (r) values for the linear curve were calculated obtained by regression of the above plots.

In vitro cytotoxicity
To investigate the cytotoxicity effects of the Methionine-CoFe 2 O 4 nanoparticles on the cancer cell lines (MCF-7, MAD-MB-231) and normal cell line (MCF10A), MTT assay was used. The cells in the 96-well plate seeded at a density of 2 × 10 4 cells per well and cultivated in a medium containing 1% penicillin/streptomycin and fetal bovine serum (FBS, 10%) at 37 °C in a humidified incubator with 5% CO 2 . After 24 h of incubation, the suspensions of Methionine-CoFe 2 O 4 with various concentrations (0-80 μg/ml) were added to the medium and continuously incubated for 24 h, 48 h, and 72 h, respectively. Then, the contents of the 96-well plates were removed, and 0.05 ml of MTT solution was added to each well following another 4 h of incubation in a 5% CO 2 atmosphere and at 37 °C. The medium was then replaced with 0.05 ml of dimethyl sulfoxide (DMSO) that was added to each well to dissolve the purple formazan crystals [34,35]. Finally, the absorbance of each well was measured using a microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, VT) at a wavenumber of 570 nm. Also, half-maximal inhibitory concentration (IC 50 ) was calculated and the rate of cytotoxicity was calculated according to Eq. (3).
(3) Cell survival rate = absorbance of control cells absorbance of treated cells × 100

XRD analysis
The structure of nanoparticles was characterized by X-ray diffraction. Figure 2  where D denotes the crystalline size, β is the full width at half maximum, K is the shape factor, θ represents the Bragg angle corresponding to the peak, and λ is the wavelength of the X rays.

Morphologic studies of methionine@CoFe 2 O 4 nanoparticles
FESEM micrographs of the synthesized Methionine@ CoFe 2 O 4 nanoparticles have been shown in Fig. 3. As observed in Fig. 3a and b, the spherical shapes with nearly uniform sizes of the Methionine@CoFe 2 O 4 nanoparticles are exhibited from the SEM images in which the average size of the spheres is around 28-29 nm. Figure 3c and d shows the TEM micrographs of Methionine@CoFe 2 O 4 nanoparticles with slight agglomeration which may be due to the strong magnetic interactions between nanoparticles.

Magnetic studies
The magnetic hysteresis loops of the prepared core/shell Methionine@CoFe 2 O 4 nanoparticles and bare CoFe 2 O 4 were measured at room temperature by SQUID in an external magnetic field ranging from −15 to + 15 kOe as depicted in Fig. 4

TGA analysis
As shown in Fig. 5, existence of Methionine on the CoFe 2 O 4 nanoparticles was further examined by thermal analysis that demonstrates TGA curves of the bare CoFe 2 O 4 and Methionine-coated CoFe 2 O 4 nanoparticles. The weight of bare CoFe 2 O 4 , initial weight loss from room temperature up to 150 °C, is probably due to the removal of surface hydroxyls or physically adsorbed water but by increasing the temperature to 800 °C due to the high stability of structure, the curve is almost constant. The occurrence of this report was also found in the synthesis of L-cysteine-coated cobalt ferrite nanoparticles [33]. In the second sample, which Methionine coated cobalt ferrite nanoparticles; the TGA curve shows that the weight loss of 13.83% is observed at 400 °C which is related to thermal decomposition of surface-treated CoFe 2 O 4 (4) D = K cos

In vitro Loading capacity and release of Letrozole
To calculate the Letrozole loading capacity of the sample at 239 nm wavelength UV-Vis spectroscopy was used. To determine the loading capacity of Letrozole on the Methio-nine@CoFe 2 O 4 with different initial Letrozole concentrations, the amount of Methionine@CoFe 2 O 4 was transferred to 20 ml of different initial Letrozole concentrations. The maximum loading capacity of the Methionine@CoFe 2 O 4 when the initial drug concentration is 0.08 mg/ml can reach 0.025 mg/mg which is in another word obvious that in 1 mg of nanoparticle, 0.62 mg of the drug is loaded. The result loading capacity in this study strongly depends on the initial drug concentrations which is shown in Fig. 7. Afterward, to evaluate the drug release behaviors, Letrozole-loaded Methionine@CoFe 2 O 4 nanoparticles are suspended in a PBS buffer media with various pH values for the simulated environment of tumors, which are pH5 and pH7.4 which are a physiological pH of the body at temperature (37 °C) over 72 h measured. Figure 8 shows the cumulative drug release of Letrozole from Methionine@ CoFe 2 O 4 at both pHs. As can be seen, drug release at acid solution conditions (pH 5) in 72 h shows higher release than neutral conditions (pH 7.4). Also, this study observed that the release of Letrozole from the carrier in the beginning 8 h occurs in a rapid manner after which the process gradually slows down up to 72 h. The reason for the rapid manner can be related to the immediate dissolution of Letrozole on the surface of Methionine @CoFe 2 O 4 nanoparticles. After that, the slower release of the Letrozole takes place in the structure of these nanocarriers seems due to the physical and chemical interactions between Letrozole and Methionine@ CoFe 2 O 4 .
As reported in the literature, the model delivery system is sensitive to pH, which is very important and useful for drug delivery because in the neutral conditions (pH 7.4), the low release rate of drug reduces relieves the side effects of antitumor medications to healthy cells and the losing of drugs in the blood transportation system, and at the same ,time intracellular lysosomes, endosomes, or cancerous tissues are tuned with acidic (pH 5) setting corresponding to facilitate anti-cancer drug active release [26]. To govern the release behavior of letrozole letrozole-loaded Methionine@ CoFe 2 O 4 nanoparticles, mathematical models were cast-off with higher linear regression coefficient for each (closer to 1), to designate the kinetic model of the ideal sample release. Table1 attends to the coefficient of determination (R 2 ) for each model at different pH values (5 and 7.4). According to which, the release data for pH values follow the Korsmeyer-Peppas model, and the obtained n values (n < 0.45) in Korsmeyer-Peppas model for these two conditions suggest that the Fickian diffusion mechanism determines the release of letrozole molecules from Methionine@CoFe 2 O 4 nanoparticles.

In vitro cytotoxicity test
The cytotoxicity of magnetic nanoparticles as known generally depends on some factors, such as aggregation degree, surface area, hydrophobicity, surface coating, and particle size [36].   formulations to MCF-7 and MDA-MB-231 cells are summarized in Table 2.

Conclusions
In this study, we synthesized Methionine@CoFe 2 O 4 nanoparticles and study drug delivery and in vitro cytotoxicity. The methionine coating improved the colloid stability and biocompatibility of magnetic nanoparticles. The in vitro drug delivery ability of Methionine@CoFe 2 O 4 nanoparticles is confirmed by using letrozole as a model drug at body temperature (37 °C) which showed a pH-sensitive release behavior. It was found that the effectiveness and selectivity of the drug carrier system can be beneficial for the inhibition of quick release for the anti-cancer drugs in neutral blood systems but the acceleration of drug release at acidic tumor cells.