In-Vitro Cytotoxicity and Anticancer Drug Release Behavior of Methionine-Coated Magnetite Nanoparticles As Carriers


 An innovative and customized drug delivery system for in vitro cancer treatment has been developed successfully by a simple one-step method. A CoFe2O4@Methionine core-shell nanoparticle was prepared by the reflux assay, in which amino acid on the surface makes the ferrite biocompatible, enhances the chemical stability of the compound, and increases the drug loading capacity. The synthesized nanoparticles were evaluated using SEM, TEM, FTIR, and VSM, while XRD and TGA analysis verified the presence of a coating amino acid on the surface of CoFe2O4. The appearance of a new peak for C≡N in the FTIR spectrum validates the synthesis of a letrozole-loaded carrier. Both uncoated CoFe2O4 and methionine-coated CoFe2O4 nanoparticles behave super-paramagnetically at room temperature, with saturation values of 46 emu/g and 16.8 emu/g, respectively. SEM and TEM were used to characterize the morphology and size of samples, revealing that the average particle size was around 28–29 nm. The loading of Letrozole and the effect of pH (5, 7.4) on the release behavior of the carrier were studied. The result of the drug release in pH (5) was about 88% higher than pH (7.4). Also, the preparation has been evaluated for determining its cytotoxicity using MCF-7, MDA-MB-231, and MCF10A cell lines as an in vitro model, and the results of in vitro experiments showed that CoFe2O4@Methionine could significantly reduce cancer in the cell model. These results demonstrate that core-shell nanoparticles were prepared that are biocompatible and have potential use as drug delivery.


Introduction
Breast cancer is one of the most often diagnosed cancers around the world, particularly in women, and its cancer patient population is growing each year [1]. Breast malignancies are widely investigated and acknowledged to be hormone-dependent, with estrogen serving as a critical mediator in the progression and spread of breast cancer [2]. Letrozole is one of the most e cient following non-steroidal aromatase inhibitors (AIs) for inhibiting the body's excessive estrogen production [3]. Letrozole uses as positive drug to treat breast cancers and highly potent drugs due to its estrogen receptor [4].
To develop new technology for cancer treatment, some signi cant steps must be taken. One method of execution cancer cells is through targeted drug delivery, in which the term "targeted" refers to the process of eliminating speci c malignant cells without harming healthy ones [5]. Recently, spinel ferrites have gained considerable interest for their prospective uses in biomedical elds such as magnetic resonance imaging (MRI), targeted hyperthermia, and controlled drug delivery [6-8]. Among them, Cobalt ferrite (CoFe 2 O 4 ) has particular signi cance because of its remarkable magnetization property [9] such as a high coercivity [10], a reasonable saturation magnetization [11], a high Thermal stability [12], a big magnetocrystalline anisotropy [13], a superior mechanical hardness [14], and exceptional chemical stability [15], also appropriate biocompatibility and low toxicity [16]. Furthermore, they can be directly injected into cancer cells and delivered by magnetic eld gradient or delivered by other e cient drug delivery systems to release their drugs [17]. So far, various magnetic nanoparticles with different formulations have been synthesized for cancer therapy which to improve biomedical applications, surface modi cation is necessary to coat them with stimuli-responsive [18]. Methionine is a key and major biocompatible amino acid found in the human body, where it performs a variety of physiological roles [19]. Three active functional groups of Methionine (-COOH, -NH2, and -SH) may easily be conjugated to metal atoms (CoFe2O4) and employed as the surface of a carrier to examine previously unknown loading and release behaviors [20]. Wang et al. discovered a simple way to synthesize nanoparticles of Cobalt Ferrite (CoFe2O4) in the presence of Lcysteine (Lys) that could be used as chemotherapeutic agents [21]. It has been shown that FeMn2O4 nanoparticles coated in TEOS and modi ed with 3-mercaptopropionic acid (MPA) can serve as a suitable delivery vehicle for targeted, site-speci c and controlled anti-cancer therapy [22].
In this study, CoFe2O4 nanoparticles coated with methionine using the re ux assay were synthesized and used as a carrier for an anti-cancer drug in one-step. It was determined that the nanoparticles had been characterized using XRD techniques, as well as other methods such as SEM, TEM, VSM, TGA, and FT-IR.

Preparation of methionine Coated CoFe 2 O 4 Nanoparticles
Using the coprecipitation process, cobalt ferrite nanoparticles were made. In this experiment, 1.42 g of CoCl2.6H2O and 3.24 g of FeCl36H2O were dissolved in 180 ml deionized water and agitated for 30 minutes under N2 pressure before raising the pH to 12 using NaOH (1.5 M). The mixture was then supplemented with 1 gram of Methionine dissolved in deionized water. The mixture was heated to between 70 and 80 ° C and re uxed for three hours; the resulting brown precipitate was collected using magnetic separation and washed with deionized water and ethanol. The process is shown in Fig. 1.

Characterization
X-ray diffraction (XRD) analysis of the samples is recorded by the STOE STADI-P, θ-2θ angle scan is obtained at a rate of 1°/min over 2θ range from 10° to 80°. The amorphous and crystalline phases were estimated by Transmission Electron Microscopy (TEM). Morphology and size of the nanoparticles were estimated using FESEM (model Zeiss-EHT-10.00 kV Germany) and TEM (model Zeiss-EM10C-100 kV Germany). FTIR data was taken in the spectral range from 400-4000 cm-1 by using a model nexus 870 spectroscopy.
The amount of adsorbed and released drug is monitored as functions of soaking time by Ultravioletvisible (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 eld ranging from -15 kOe to +15 kOe.

Loading Capacity of Letrozole
0.0016 g Letrozole was diluted in 20 ml of methanol, followed by the addition of 0.04 mg Methionine-CoFe2O4 nanoparticles. This mixture was swirled at room temperature for 24 hours to load the drug molecules. After centrifuging the sample dispersion at 6,000 rpm for 12 minutes to collect the Letrozoleloaded nanoparticles, the supernatant was retained for determining the drug loading content. The Letrozole-loaded nanoparticles were collected at room temperature and the supernatants were analyzed using UV-Vis spectroscopy at a wavelength of 239 nm, and the loading capacity was determined using a standard curve with known drug concentrations. The dose of loaded letrozole was determined using Eq (1).
Where C0 represents the initial concentration of Letrozole, Ct represents the drug concentration determined using the Letrozole standard curve, V0 and Vt represent the volume of the liquid phase (ml), is the dilution ratio, and w (mg) is the weight of the nano carrier.

In vitro release study and kinetic modeling
The in-vitro release kinetics of Letrozole were examined by dissolving 15 mg Letrozole-loaded Methionine-CoFe2O4 in 15 ml PBS (phosphate-buffered saline) with varying pH values (5 and 7.4) in the dark and shaking (100 r/min) at a constant temperature (37 °C). The supernatant (2ml) is removed and replaced with the same fresh medium PBS with the same pH value at different time intervals. The percentage of Letrozole released was determined using the UV-Vis method at a wavelength of 239 nm in accordance with Eq (2).
Where Ce (mg/ml) represents the concentration of Letrozole in the supernatant, V (ml) describes the amount of buffer solution, and w (mg) denotes the amount of drug loaded. The drug release data was mathematically examined using models tted to kinetic model equations for the purpose of determining the release kinetics and elucidating the release mechanism. Zero-order kinetics (cumulative percent drug Where D signi es the crystallinity size, β is the complete width at half maximum, K is the shape factor, θ shows the Bragg angle corresponding to the peak and λ is X-ray wavelength. As observed in Fig. 3 (a) and (b), the spherical shapes with nearly uniform sizes of the

TGA Analysis
As illustrated in Fig. 5, the presence of Methionine on the CoFe2O4 nanoparticles was further investigated using thermal analysis, which revealed TGA curves for bare CoFe2O4 and Methionine-coated CoFe2O4 nanoparticles. The initial weight loss of bare CoFe2O4 from room temperature to 150 °C is probably owing to the elimination of surface hydroxyls or physically adsorbed water, but the curve becomes practically constant at 800 °C due to the structure's strong stability. This phenomenon was also seen during the fabrication of L-cysteine-coated cobalt ferrite nanoparticles. 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 with additions of Methionine molecules. So weight loss of Methionine@CoFe 2 O 4 has occurred in a range of 400°C was related to degradation of Methionine molecules.

In vitro Loading capacity and release of Letrozole
To calculate the Letrozole loading capacity of the sample at 239 nm wavelength was used UV-Vis spectroscopy. To determine the loading capacity of Letrozole on the Methionine@CoFe 2 O 4 with different initial Letrozole concentrations, the amount of Methionine@CoFe2O4 was transferred to 20 ml of different initial Letrozole concentrations. When the initial drug concentration is 0.08 mg/ml, the highest loading capacity of the Methionine@CoFe2O4 nanoparticle is 0.025 mg/mg, indicating that 0.62 mg of drug is loaded into 1 mg of nanoparticle. The loading capacity determined in this work is highly dependent on the initial drug concentrations, as illustrated in Fig. 7.
Following that, Letrozole-loaded Methionine@CoFe2O4 nanoparticles are suspended in a PBS buffer media with various pH values corresponding to the simulated environment of tumors, namely pH (5) and pH (7.4), which corresponds to the physiological pH of the body at 37 °C, for 72 hours. At both pHs, Fig. 8 depicts the cumulative drug release of Letrozole from Methionine@CoFe2O4. As can be seen, drug release under acid solution circumstances pH (5) is signi cantly more than under neutral solution conditions pH. (7.4). Additionally, this study discovered that the release of Letrozole from the carrier happens rapidly in the rst 8 hours and then gradually slows down to 72 hours. The cause for the quick dissolution of Letrozole on the surface of Methionine @CoFe2O4 nanoparticles is unknown. Following that, the delayed release of Letrozole appears to be caused by physical and chemical interactions between Letrozole and Methionine@CoFe2O4. As stated in the literature, the model delivery system is pH sensitive, which is critical for drug delivery because at neutral pH (7.4), the modest release rate of the medication alleviates anti-cancer drug side effects on normal cells and drug loss through blood transportation. While acidic conditions pH (5) are associated with intracellular lysosomes, endosomes, or malignant tissues, which may facilitate the active release of anticancer drugs [26]. The release behavior of letrozole-loaded Methionine@CoFe2O4 nanoparticles was determined in this study using mathematical models. Each model with a higher linear regression coe cient (closer to 1) represents the optimum sample release's kinetic model. Table 1 shows the coe cient of determination (R 2 ) for each model at various pH values (5 and 7.4). As can be seen, the pH values released correspond to the Korsmeyer-Peppas model. The obtained n values (n=0.45) in the Korsmeyer-Peppas model for these two settings show that letrozole molecules are released from Methionine@CoFe2O4 nanoparticles via the Fickian diffusion mechanism.

In vitro Cytotoxicity Test
As is well known, the cytotoxicity of magnetic nanoparticles is dependent on several parameters, including degree of aggregation, surface area, hydrophobicity, surface coating, and particle size [23]. As demonstrated in cytotoxicity experiments using the MTT test on human breast cancer cells (MCF-7, MDA-MB-231) and normal cells (MCF10A). For 24, 48, and 72 hours, cells were treated with free letrozole, Methionine@CoFe2O4 nanoparticles, and letrozole loaded on Methionine@CoFe2O4 nanoparticles at various doses (0-40 g/ml). The results indicated that Methionine@CoFe2O4 is almost as toxic to cancer cells as free letrozole, indicating that the letrozole-Methionine@CoFe2O4 nanoparticles are more readily internalized via the receptor-mediated endocytosis mechanism, whereas free letrozole is transported into cells via a passive diffusion mechanism [24]. Additionally, it was revealed that letrozole-methionine @CoFe2O4 was more cytotoxic to MCF-7 cells than to MDA-MB-231 cells. Additionally, normal MCF10A cells were treated with Methionine @CoFe2O4 and letrozole-Methionine@CoFe2O4 at the same doses. The results demonstrated that Methionine@CoFe2O4 and letrozole-Methionine@CoFe2O4 exhibited no detectable toxicity on MCF10A cells after 72 hours of treatment, indicating that they are biocompatible enough to be used as a drug delivery system. This nding indicated that loading the drug on a carrier boosted the growth inhibitory effect on cancer cells synergistically, indicating the therapeutic potential of Methionine@CoFe2O4. The viability of MCF-7 cells is shown in Fig. 9, while that of MDA-MB-231 cells is presented in Fig. 10. Table 2 summarizes the IC50 values for the free drug and Letrozole loaded on Methionine@CoFe2O4 formulations against MCF7 and MDA-MB-231 cells.

Conclusion
Methionine@CoFe2O4 nanoparticles were produced in this study and the drug delivery and in vitro cytotoxicity were studied. Magnetic nanoparticles with a methionine coating demonstrated increased colloid stability and biocompatibility. The potential of Methionine@CoFe2O4 nanoparticles to transport drugs in vitro is demonstrated using letrozole as a model drug at body temperature (37 °C), which exhibited pH-dependent release behavior. It was discovered that the e cacy and selectivity of the drug carrier system can bene t the suppression of rapid drug release in neutral blood systems but accelerate drug release in acidic tumor cells. MTT experiments revealed that Methionine@CoFe2O4 as a model carrier had a low cytotoxicity even at high concentrations after 72 hours of treatment, but Letrozole-Methionine@CoFe2O4 exhibited a high cytotoxicity in both types of cancer cells. As a result, the Methionine@CoFe2O4 Nanocarrier is projected to be a viable drug delivery system that might be used in therapy.

Declarations
Availability of Data and Materials: N/A Con ict of Interest: Authors declare no competing interest, intellectual or nancial.  The XRD patterns of CoFe2O4 nanoparticles (a) and Methionine@CoFe2O4 nanoparticles (b).     Curve capacity of Letrozole on carrier at different initial Letrozole concentrations.