Synthesis and Characterization of Tetracycline Loaded Methionine-Coated NiFe2O4 Nanoparticles for Anticancer and Antibacterial Applications

In the present study, nickel ferrite (NiFe2O4)-based smart magnetic nanoparticles were fabricated and coated with methionine. Physiochemical characterization of the obtained Met-NiFe2O4 nanoparticles revealed the presence of methionine coating over the nanoparticle surface. Drug release study indicated that Tet-Met-NiFe2O4 nanoparticles possess pH-responsive controlled drug release behavior for tetracycline (Tet). The drug loading content for Tet was found to be 0.27 mg/L of nanoparticles. In vitro cytotoxicity test showed that the Met-NiFe2O4 nanoparticles is biocompatible. Moreover, this magnetic nanostructured material shown strong anticancer property as these nanomaterials significantly reduced the viability of A375 cells when compared to free Tet solution. In addition, Tet-Met-NiFe2O4 nanoparticles also showed strong antibacterial activity against different bacterial pathogens.


Introduction
In today's world, hospital-acquired infections caused by viral, bacterial, and fungal pathogens remain the primary concern and biggest challenge among the healthcare workers [1][2][3][4][5]. Antimicrobial molecules include antibiotics and biocides having a bactericidal/bacteriostatic effect on bacteria. Antibiotic is an active substance of synthetic or natural origin which is used to eradicate bacterial infections in humans or animals. While biocide is an active chemical molecule to control the growth of or kill bacteria. Hence, antimicrobial activity exhibits an inhibitory or lethal effect of a biocidal product or an antibiotic [6][7][8][9]. Biocides and antibiotics may share some common behavior and properties in their respective activity and in the resistance mechanisms developed by bacteria. Today, it is important to weigh the risks of selecting antibiotic resistant bacteria by biocide use correctly and to have a clear view of the corresponding emerging health risk. Moreover, understanding the selection and dissemination of biocide resistant pathogens is very important for combating the dissemination of health care associated diseases and foodborne pathogens.
Antibiotics are broadly classified as antibacterial, antifungal, or antiviral drugs depending on their target group [6][7][8]. For many decades, antibiotics are widely utilized in medical procedures ranging from organ transplants to chemotherapy [10]. From this, the significance of antibiotics is understood as it protects the host cells from microbial or viral infections [11,12]. Towards this, an antibiotic, Tetracycline (Tet), has been extensively used to treat various bacterial infections [13,14]. In addition, Tet is a well-recognized antibiotic used in the treatment of skin cancer and shown to inhibit several other cancer types.
The functional role of antibiotics is mainly based on the drug delivery system and the mechanism of their controlled delivery to targeted affected cells [15][16][17][18]. Recently, several organic-inorganic hybrid nanoparticles have been extensively used in anticancer therapy. Indeed, methionine is one of the essential amino acids i.e., required for the human growth due to its antioxidant activity. The activated functional groups of methionine (-COOH and -NH 2 ) are used to conjugate the metal atoms, and further the loading and release behavior are investigated. The design and synthesis of organic-inorganic hybrid nanoparticles through combining the advantages of both organic and inorganic counterparts may improve the overall properties, such as particle size, surface charge, and many other physicochemical properties [19,20]. Roca et al. (2012) reported that modified magnetic NPs (MNPs) has shown increased efficacy under in vivo conditions [21].
Several recent findings have emphasized the therapeutic role of MNPs in treating the disease [22][23][24]. Nanoparticles whose particle size ranging between 10-100 nm have shown properties to improve drug bioavailability and facilitate the targeted accumulation of drugs inside the cell through enhanced permeability and retention (EPR) effect [25,26]. Magnetic spinel ferrite nanoparticles are widely used in biotechnology. The main advantage of magnetic nanoparticles is that can be readily isolated from the solution media using an external magnetic field. NiFe 2 O 4 nanoparticles have gained worldwide acceptance due to their low coercivity, high saturation magnetization, excellent chemical stability, high Curie temperature, and electromagnetic performance [27]. However, to the authors' best knowledge, the utilization of NiFe 2 O 4 magnetic nanoparticles in the field of biomedicine is available. Further, NiFe 2 O 4 nanoparticles are widely used as an in vivo magnetic hyperthermia agent in biomedicine [28]. NiFe 2 O 4 nanoparticles exhibited an inverse spinel structure with Ni +2 in octahedral sites (Ni(OH)) and Fe +3 equally distributed between tetrahedral (Fe (Td)) and octahedral sites (Fe(OH)) of the O 2− FCC (Face-Centered Cubic) cell [29]. The complete crystalline structure belongs to O 7 h space group with oxygen atoms occupying the 32e positions, Fe (Td) atoms occupying the 8a ones and the Ni(OH) and Fe(OH) atoms are distributed on 16d positions [30]. Different methods, including solvothermal, sol-gel, co-precipitation, microemulsion, and thermal decomposition, have been utilized to fabricate magnetic nanostructures of varying morphologies and structural variants [31][32][33][34] which has immense scope and applications in biomedical science.
Various amino acids have been successfully applied in the preparation of magnetic nanoparticles. However, the use of methionine in the fabrication of nickel-ferrite based nanocomposite is not reported yet. Recently, Saykova et al. synthesized magnetic NiFe 2 O 4 @Au crystal nanoparticles using the amino acid methionine as a reducing and stabilizing agent [35]. The nanoparticles obtained in this study after three stages of gold deposition had an average particle size of about 120 nm, which is relatively large for cellular adsorption studies and for biomedical applications thereof. In this study, methionine-coated nickel ferrite nanoparticles with an average particle size of about 27 nm are synthesized in a simple and cost-effective step by reflux method, which is the preferred magnetic nanomaterial for various biological applications.
Recently, non-coated nickel ferrite magnetic nanoparticles were synthesized by Majed et al. and tested in a rat model [36]. However, tetracycline loading on this nanostructure (Met-NiFe 2 O 4 ), and its effect on normal and A-375 cancer cells has not been studied. In addition, the loading of tetracycline on Met-NiFe 2 O 4 as a biocompatible and targeted nanocarrier and its effect on bacterial growth remain unexplored. Compared to previous literature, it can be concluded that we have prepared nickel ferrite nanoparticles coated with methionine in a facile synthetic route which is explored for anticancer and antibacterial applications.
In the present study, a simple and effective strategy is developed to synthesize Met-NiFe 2 O 4 nanoparticles employing a one-step reflux procedure. Various characterization studies are performed to examine the shape, structure, and magnetic characteristics of Met-NiFe 2 O 4 nanoparticles. Tet antibiotic is used to explore the drug loading and release profile of Met-NiFe 2 O 4 nanoparticles, for the first time. Moreover, MTT tests are used to assess the in vitro cytotoxicity of Met-NiFe 2 O 4 nanoparticles before and after Tet loading of varying doses and incubation periods. Furthermore, antibacterial activity of Met-NiFe 2 O 4 nanoparticles against pathogenic bacteria is assessed. To date, the cytotoxicity and antibacterial properties of Met-NiFe 2 O 4 nanoparticles have not been reported.

Fabrication of Met-NiFe 2 O 4 Nanoparticles
Met-NiFe 2 O 4 nanoparticles were synthesized by reflux method (as shown in Figure 1). In brief, 2.431 g of FeCl 3 ·6H 2 O and 1.069 g of NiCl 2 ·6H 2 O were dissolved in 120 mL of distilled water (d.H 2 O), followed by the addition of 1.5 M NaOH solution to adjust the pH to 12. Then, a brown colored solution appeared, and was filtered and washed several times with d.H 2 O until the pH of the filtrate becomes neutral (7). Next, 1.5 g of methionine was added. The resulting mixture was heated to 70-80 • C and refluxed for 3 h. The final product was collected by strong magnetic separation and rinsed three times with absolute ethanol and d.H 2 O. In a similar way, NiFe 2 O 4 nanoparticles were synthesized, but without methionine.

Physicochemical Characterization Studies
X-ray diffraction (XRD) analysis NiFe 2 O 4 and Met-NiFe 2 O 4 nanoparticles was carried out using the STOE STADI-P instrument (STOE, & Cie GmbH, Darmstadt, Germany). Field Emission-Scanning Electron Microscopy (FESEM) (Zeiss-EHT-10.00 kV, Carl Zeiss SMT AG Company, Oberkochen, Germany) and High Resolution-Transmission Electron Microscopy (HR-TEM) (Zeiss-EM10C-100 kV, Carl Zeiss SMT AG Company, Oberkochen, Germany) instruments were used to determine morphology and particle size of nanoparticles respectively. The particle size was measured in a dry state through the particle size distribution curve. Next, Fourier Transform Infrared Spectroscopy (FTIR) (Nexus 870, Nicolet, Madison, WI, USA) was also performed. Absorbance measurements at 275 nm through means of UV/visible spectroscopy (Shimadzu UVS-1700, Shimadzu Corporation, Kyoto, Japan) was employed to study the amount of drug adsorbed on nanoparticle surface and the controlled drug release behavior. Thermal properties were analyzed by Thermogravimetric Analyzer (TGA) (Shimadzu TA Q600, Shimadzu TA Instrument SDT Q600, New Castle, DE, USA) in a temperature range of 25 to 800 • C under Nitrogen atmosphere at a constant heating rate. Finally, the magnetic properties were studied by Quantum Design MPMS-XL-7 instrument (MPMS-XL-7), San Diego, CA, USA.

Average Hydrodynamic Size and Zeta Potential Measurement
The average hydrodynamic size (Z-average) (based on light diffraction), particle size distribution (PDI), and average zeta potential (based on electrophoretic movement of particles) of NiFe 2 O 4 , Met-NiFe 2 O 4 and Tet-Met-NiFe 2 O 4 nanoparticles were determined by Malvern Zetasizer 2000 HS instrument (Malvern, UK) where nanoparticles were diluted 100 times in distilled water at 25 ± 0.1 • C prior to experiment.

Tetracycline Loading and Release Behavior
Various concentrations of Tet were added to 0.2 mg of Met-NiFe 2 O 4 nanoparticles and incubated in the dark conditions for 24 h at room temperature. The resulting Tet-Met-NiFe 2 O 4 nanoparticles were pelleted using centrifuge, and the collected supernatant was subjected to absorbance measurements at 275 nm using UV/Visible spectroscopy to determine the drug loading efficiency of Tet ( Figure 1).
Next, 10 mg of Tet-Met-NiFe 2 O 4 nanoparticles was suspended into 10 mL of PBS buffer of pH 5 and 7.4 at 37 • C under dark conditions. Then, 1 mL supernatant was collected at different time intervals and replaced with fresh PBS. Finally, the concentration of released Tet was calculated using UV/Visible spectroscopy (Shimadzu UVS-1700, Shimadzu Corporation, Kyoto, Japan). The drug release data were fitted with different kinetic models' equations for determining the mechanism of drug release from Tet-Met-NiFe 2 O 4 nanoformulations. The most appropriate drug release kinetic model was determined by analyzing the regression coefficients of graphs. According to this, the drug release mechanism was determined from the drug delivery system.

In Vitro Cytotoxicity
A standard MTT test was performed to analyze the in vitro cytotoxicity of Tet-Met-NiFe 2 O 4 nanoparticles on A375 and HFF cell lines. These cell lines were cultured using RPMI-1640 fresh medium supplemented with 10% FBS and 1% penicillin/streptomycin in a humid CO 2 incubator with 5% carbon dioxide at 37 • C. Once the cells had achieved 85-95% confluence, the media was aspirated. Detachment of the cell monolayer was performed using trypsin-EDTA (0.25% (w/v)). Then, the detached cells were resuspended in a complete growth medium, labeled with trypan blue, and counted using hemocytometer. Different concentrations of Tet-Met-NiFe 2 O 4 (0-70 µg/mL) were added to the cultured A375 and HFF cells and incubated for 72 h. For cell proliferation studies, the cells were incubated with 0.5 mg/mL of MTT reagent for 4 h. Then, the purple crystal formazan formed was dissolved in 100 µL of DMSO for the colorimetric determination of the cells' oxidoreductase enzymatic activity. The absorbance values of both control and test concentrations were measured at 570 nm while reference blank measurements taken at 630 nm, and then the cell viability and IC 50 values were calculated [7,15,37]. The percentage of cell viability was calculated using Equation (1).

Antibacterial Activity
The antibacterial activities of free Tetracycline and Tet-Met-NiFe 2 O 4 nanoparticles were evaluated against both Gram-positive and Gram-negative bacteria, such as E. coli, P. aeruginosa, S. aureus, and E. faecalis, using microdilution method. Next, the obtained data was used to calculate the minimum bactericidal concentrations (MBC) and minimum inhibitory concentrations (MIC). Different samples were initially made using Mueller-Hinton Broth (MHB), then 100 µL of each sample was added into 96-well microplates. Approximately 0.5 McFarland standard bacterial suspension was prepared and diluted in Mueller-Hinton Broth to achieve a final concentration of 1 × 10 6 colony forming units (CFU)/mL. Then, 100 µL of diluted bacterial suspension was mixed with 100 µL of samples (with different concentrations as mentioned above) in 96-well microplates and incubated at 37 • C for 18 h. Bacterial suspension without the drug acts as positive control, while the negative control was the highest drug concentration without bacteria. MBC test was performed to confirm the results of MIC test. Consequently, 100 µL of clear wells with no visible bacterial growth was transferred to petri plates containing MHA (Mueller-Hinton Agar) medium and incubated for overnight at 37 • C [7,8].

Statistical Analysis
All tests were performed in triplicates (n = 3) and each test repeated at least three independent times. The equality of variance and normality were checked by Brown-Forsythe and Shapiro-Wilk tests, respectively. Then, the data was compared using one way or two-way analysis of variance (ANOVA) with repeated measures, followed by Tukey's or Sidak's post hoc comparison test. The results were given as mean ± SD. The differences were considered significant when a p-value of less than 0.05 is considered statistically significant. * p < 0.05, ** p <0.01, *** p < 0.001.

Results and Discussion
where D denotes the crystalline size, β is the full width at half maximum (FWHM), θ represents the Bragg angle corresponding to the peak, and λ is the wavelength of the X-rays.
where D denotes the crystalline size, β is the full width at half maximum (FWHM), θ represents the Bragg angle corresponding to the peak, and λ is the wavelength of the Xrays.

FT-IR Spectral Analysis
FTIR analysis was used to study the nature of functional groups present on the surface of MNPs. The FTIR spectra of methionine, free Tet, bare NiFe 2 O 4 , Met-NiFe 2 O 4 nanoparticles, and Tet-Met-NiFe 2 O 4 nanoparticles are shown in Figure 2B. The Methionine amino acid spectrum is a combination of carboxylate salts and the first type amine. The symmetric and asymmetric N-H bending assigned in the region at 1517 cm −1 and 1630 cm −1 respectively. Furthermore, the symmetric and asymmetrical stretching of the COO − bond was exhibited at 1419 cm −1 and 1600 cm −1 , respectively [38]. C-O bond represented at 1232-1330 cm −1 . The inherent vibration of tetrahedral and octahedral metal-oxygen complexes of NiFe 2 O 4 is ascribed to regions observed at 400 and 600 cm −1 , respectively, which are mostly dependent on Fe-O distance [38].
FTIR spectrum of Met-NiFe 2 O 4 nanoparticles revealed that methionine was present on the surface of NiFe 2 O 4 nanoparticles. Tet features two small vibration at 500-569 cm −1 in its FT-IR spectra, which is linked to out-of-plane ring deformation. The regions at 1000-1257 cm −1 are associated with C-H in-plane deformation vibrations, while those at 998 cm −1 are associated with C-N stretching. Two bands appeared at 1462 and 1366 cm −1 displaying the bending vibrations of C-H and CH 3 groups, respectively. Band of C=C stretching is observed at 1571-1632 cm −1 . C-H and CH 3 stretching are attributed to the vibrational at 3013-3078 cm −1 and 2819-2949 cm −1 . Bands of N-H and O-H stretching is ascribed to the regions which seen at 3319-3340 cm −1 [39]. After Tet loading, the regions at 998-1257 cm −1 were originated in the FTIR spectrum, which is most likely due to C-N stretching and C-H vibration bands in-plane deformation of Tet. These findings supported that methionine's gets effectively coated onto MNPs, and, thus, possess good potential for drug loading and acts as smart nanocarrier for drug delivery.

Thermogravimetric Analysis
Thermogravimetric analysis (TGA) of methionine, NiFe2O4 nanoparticles, and Met-NiFe2O4 nanoparticles (Figure 4) were recorded. TGA profile showed that the initial weight loss observed at 150 to 260 ℃ could be due to thermal decomposition of carbon dioxide, water and fine molecules trapped between the layers. On the other hand, heatinduced thermal degradation of methionine could be noticed at 260-300 ℃, which is also consistent with TGA profile of amino acid, methionine. Further increase in temperature above 350 ℃ showed no change in weight which could be due to the high stability of the dioxide, water and fine molecules trapped between the layers. On the other hand, heatinduced thermal degradation of methionine could be noticed at 260-300°C, which is also consistent with TGA profile of amino acid, methionine. Further increase in temperature above 350°C showed no change in weight which could be due to the high stability of the nanostructure [33,40]. The weight loss observed near 300 • C for NiFe 2 O 4 nanoparticles and Met-NiFe 2 O 4 nanoparticles were found to be 8.3% and 13.8%, respectively. Difference in the weight loss between NiFe 2 O 4 nanoparticles and Met-NiFe 2 O 4 nanoparticles near 300 • C indicated that methionine was successfully coated onto the surface of the NiFe 2 O 4 nanoparticles.

Size Distribution and Zeta Potential Measurement
The average hydrodynamic size, particle size distribution, and average zeta potential of all nanoparticles were measured at the same concentration and pH values. The particle hydrodynamic size distribution graph was plotted using Gaussian theorem for NiFe2O4, Met-NiFe2O4, and Tet-Met-NiFe2O4 nanoparticles ( Figure 5). All the calculated values are given in a tabulated form (Table 1).

Size Distribution and Zeta Potential Measurement
The average hydrodynamic size, particle size distribution, and average zeta potential of all nanoparticles were measured at the same concentration and pH values. The particle hydrodynamic size distribution graph was plotted using Gaussian theorem for NiFe 2 O 4 , Met-NiFe 2 O 4 , and Tet-Met-NiFe 2 O 4 nanoparticles ( Figure 5). All the calculated values are given in a tabulated form (Table 1).

Drug Loading and Release Study
Tet was further loaded onto Met-NiFe2O4 nanoparticles, and the drug loading and release profile was assessed. As shown in Figure 6A, the maximum loading capacity of Met-NiFe2O4 nanoparticles reached 0.055 mg/mg, when the initial Tet concentration was 0.09 mg/mL. In other words, 0.27 mg of Tet was loaded onto 1 mg of nanocarrier. The amount of drug loading is enhanced through increasing the initial drug concentration taken for the study. This is possible due to the larger surface area and hydrogen bonding between Tet and Met-NiFe2O4 nanoparticles. To evaluate the drug release profile, the Tet-Met-NiFe2O4 nanoparticles were suspended in the buffer media of pH 5 and 7.4. Figure  6B shows the pH-dependent release of Tet from Met-NiFe2O4 nanoparticles. Drug release was observed at pH values near the tumor's environment (pH 5), while the lower drug release was detected at pH 7.4. The pH-sensitive drug release of the nanocarrier under neutral conditions (pH 7.4) could reduce the drug loss during blood transportation and lessen the side effects of anti-cancer drugs observed in normal cells. In the simulated environment of tumors (pH 5), this above-mentioned property of nanomaterial facilitates efficient anti-cancer drug release [41]. The pH-responsive nano-based delivery tools could lead to site-specific release of therapeutic cargos through cleaving pH-sensitive bonds across the pH gradient and augment an increase in toxicity under acidic conditions prevalent in the tumor regions [42,43].

Drug Loading and Release Study
Tet was further loaded onto Met-NiFe 2 O 4 nanoparticles, and the drug loading and release profile was assessed. As shown in Figure 6A, the maximum loading capacity of Met-NiFe 2 O 4 nanoparticles reached 0.055 mg/mg, when the initial Tet concentration was 0.09 mg/mL. In other words, 0.27 mg of Tet was loaded onto 1 mg of nanocarrier. The amount of drug loading is enhanced through increasing the initial drug concentration taken for the study. This is possible due to the larger surface area and hydrogen bonding between Tet and Met-NiFe 2 O 4 nanoparticles. To evaluate the drug release profile, the Tet-Met-NiFe 2 O 4 nanoparticles were suspended in the buffer media of pH 5 and 7.4. Figure 6B shows the pH-dependent release of Tet from Met-NiFe 2 O 4 nanoparticles. Drug release was observed at pH values near the tumor's environment (pH 5), while the lower drug release was detected at pH 7.4. The pH-sensitive drug release of the nanocarrier under neutral conditions (pH 7.4) could reduce the drug loss during blood transportation and lessen the side effects of anti-cancer drugs observed in normal cells. In the simulated environment of tumors (pH 5), this above-mentioned property of nanomaterial facilitates efficient anti-cancer drug release [41]. The pH-responsive nano-based delivery tools could lead to site-specific release of therapeutic cargos through cleaving pH-sensitive bonds across the pH gradient and augment an increase in toxicity under acidic conditions prevalent in the tumor regions [42,43]. At neutral and acidic pH, the release kinetics of drug-loaded Met-NiFe2O4 nanoparticles were assessed using zero-order, first-order, Higuchi, and Korsmeyer-Peppas models, as shown in Table 2. An ideal kinetic model with a higher linear regression coefficient (close to 1) and determination (R 2 ) describe the drug release mechanism in the best way. The Higuchi model and Korsmeyer-Peppas model described the release kinetics at pH 5 and pH 7.4 (n < 0.45), whereas the Korsmeyer-Peppas model indicated the Fickian diffusion mechanism. It is worth mentioning that the drug gets distributed through diffusion in these cases (Higuchi model and Fickian diffusion mechanism). The pH-dependent drug release is well suited for cancer therapy, where the cancerous cells have an acidic intercellular environment while the healthy cells do not. The mathematical analysis is illustrated at pH 7.4 and pH 5 in Figures 7 and 8. Table 2. The drug release kinetic models and the parameters obtained for optimum nanoformulation.  At neutral and acidic pH, the release kinetics of drug-loaded Met-NiFe 2 O 4 nanoparticles were assessed using zero-order, first-order, Higuchi, and Korsmeyer-Peppas models, as shown in Table 2. An ideal kinetic model with a higher linear regression coefficient (close to 1) and determination (R 2 ) describe the drug release mechanism in the best way. The Higuchi model and Korsmeyer-Peppas model described the release kinetics at pH 5 and pH 7.4 (n < 0.45), whereas the Korsmeyer-Peppas model indicated the Fickian diffusion mechanism. It is worth mentioning that the drug gets distributed through diffusion in these cases (Higuchi model and Fickian diffusion mechanism). The pH-dependent drug release is well suited for cancer therapy, where the cancerous cells have an acidic intercellular environment while the healthy cells do not. The mathematical analysis is illustrated at pH 7.4 and pH 5 in Figures 7 and 8. Table 2. The drug release kinetic models and the parameters obtained for optimum nanoformulation.

Met-NiFe 2 O 4 Nanoparticles (pH-5)
Zero-Order C t = C 0 + K 0 t 0. At neutral and acidic pH, the release kinetics of drug-loaded Met-NiFe2O4 nanoparticles were assessed using zero-order, first-order, Higuchi, and Korsmeyer-Peppas models, as shown in Table 2. An ideal kinetic model with a higher linear regression coefficient (close to 1) and determination (R 2 ) describe the drug release mechanism in the best way. The Higuchi model and Korsmeyer-Peppas model described the release kinetics at pH 5 and pH 7.4 (n < 0.45), whereas the Korsmeyer-Peppas model indicated the Fickian diffusion mechanism. It is worth mentioning that the drug gets distributed through diffusion in these cases (Higuchi model and Fickian diffusion mechanism). The pH-dependent drug release is well suited for cancer therapy, where the cancerous cells have an acidic intercellular environment while the healthy cells do not. The mathematical analysis is illustrated at pH 7.4 and pH 5 in Figures 7 and 8.  At neutral and acidic pH, the release kinetics of drug-loaded Met-NiFe2O4 nanoparticles were assessed using zero-order, first-order, Higuchi, and Korsmeyer-Peppas models, as shown in Table 2. An ideal kinetic model with a higher linear regression coefficient (close to 1) and determination (R 2 ) describe the drug release mechanism in the best way. The Higuchi model and Korsmeyer-Peppas model described the release kinetics at pH 5 and pH 7.4 (n < 0.45), whereas the Korsmeyer-Peppas model indicated the Fickian diffusion mechanism. It is worth mentioning that the drug gets distributed through diffusion in these cases (Higuchi model and Fickian diffusion mechanism). The pH-dependent drug release is well suited for cancer therapy, where the cancerous cells have an acidic intercellular environment while the healthy cells do not. The mathematical analysis is illustrated at pH 7.4 and pH 5 in Figures 7 and 8.

Cytotoxicity Studies
The cytotoxicity of Tet-Met-NiFe 2 O 4 nanoparticles and free Tet were investigated using MTT test thrice for the concentration range of 0-70 µg/mL. Melanoma (A375 cells) and Human Foreskin Fibroblasts (HFF cells) were selected for the MTT assay. Results indicated that both free Tet and Tet-Met-NiFe 2 O 4 nanoparticles showed cytotoxicity on cancer cells while toxicity was not observed with normal cells. As shown in Figure 9B, the cytotoxicity of Tet-Met-NiFe 2 O 4 nanoparticles (IC 50 = 32.55 ± 7.31 µg/mL) was higher than free Tet (IC 50 = 76.58 ± 5.78 µg/mL) at 72 h. One possible reason for the differences in toxicity is due to difference in intracellular uptake of drug between the free drug and drug-loaded nanoparticles. More importantly, when the free drug is taken by cells, its release is faster, which means it is available to cell in less time owing to cross-sectional effect. When the drug is loaded onto nanoparticles, it exhibits controlled release, sustained effect, and as a result, the overall therapeutic efficacy is significantly improved [22,44]. Furthermore, Tet-Met-NiFe 2 O 4 nanoparticles displayed no cytotoxicity on HFF cells ( Figure 9A) due to excellent biocompatibility observed through coating the MNPs with amino acid methionine. These results finally showed that the biocompatible Tet-Met-NiFe 2 O 4 nanoparticles synergistically improved the growth inhibitory effect on cancer cells.

Cytotoxicity Studies
The cytotoxicity of Tet-Met-NiFe2O4 nanoparticles and free Tet were investigated using MTT test thrice for the concentration range of 0-70 μg/mL. Melanoma (A375 cells) and Human Foreskin Fibroblasts (HFF cells) were selected for the MTT assay. Results indicated that both free Tet and Tet-Met-NiFe2O4 nanoparticles showed cytotoxicity on cancer cells while toxicity was not observed with normal cells. As shown in Figure 9B, the cytotoxicity of Tet-Met-NiFe2O4 nanoparticles (IC50 = 32.55 ± 7.31 μg/mL) was higher than free Tet (IC50 = 76.58 ± 5.78 μg/mL) at 72 h. One possible reason for the differences in toxicity is due to difference in intracellular uptake of drug between the free drug and drug-loaded nanoparticles. More importantly, when the free drug is taken by cells, its release is faster, which means it is available to cell in less time owing to cross-sectional effect. When the drug is loaded onto nanoparticles, it exhibits controlled release, sustained effect, and as a result, the overall therapeutic efficacy is significantly improved [22,44]. Furthermore, Tet-Met-NiFe2O4 nanoparticles displayed no cytotoxicity on HFF cells ( Figure 9A) due to excellent biocompatibility observed through coating the MNPs with amino acid methionine. These results finally showed that the biocompatible Tet-Met-NiFe2O4 nanoparticles synergistically improved the growth inhibitory effect on cancer cells.

Antibacterial Activity
Microdilution method was used to evaluate the antibacterial activity of free Tet and Tet-Met-NiFe 2 O 4 nanoparticles. Table 3 showed that MIC of Tet-Met-NiFe 2 O 4 nanoparticles against pathogenic bacteria were between 1 to 8 µg/mL. However, Tet-Met-NiFe 2 O 4 nanoparticles had potent antibacterial activity against Gram-negative bacteria with a significant reduction in MIC and MBC values i.e., 2-3-folds as compared to that of free Tet (Table 3). This was probably due to more penetration of Tet-Met-NiFe 2 O 4 nanoparticles into Gram-negative bacterial cells [45]. In fact, Gram-negative bacteria possess very thin peptidoglycan layer than Gram-positive bacteria, and this structure may facilitate the penetration of nanoparticles into bacterial cells [46][47][48][49]. These data supported the idea that Met-NiFe 2 O 4 nanoparticles can be utilized as carriers of antibiotics for antibacterial applications. Another probable antibacterial mechanism of Met-NiFe 2 O 4 nanoparticles could be due to enhanced bacterial outer membrane permeability, which is due to the interaction of nanoparticles with the bacterial cell walls, which, in turn, alters the intrinsic membrane potential [50].

Conclusions
MNPs have been broadly explored for the development of drug delivery systems in recent years. In this study, Tet-Met-NiFe 2 O 4 nanoparticles was fabricated for cancer therapeutics. Results suggested that methionine coating onto magnetic NiFe 2 O 4 nanoparticles stabilize the nanoparticles and, thus, lead to more effective drug loading. Evidently, Tet was released rapidly into the cancer cells. In vitro cytotoxicity test indicated the excellent biocompatibility of magnetic nanocarriers due to the surface coating with methionine. When Met-NiFe 2 O 4 nanoparticles were tested, no cytotoxicity was observed on both cell lines. Tet-Met-NiFe 2 O 4 nanoparticles showed significant cytotoxicity on A375 cells, even higher than free Tet solution.
Furthermore, strong bactericidal activity was seen, when Tet-Met-NiFe 2 O 4 nanoparticles was tested against different Gram-positive and Gram-negative bacterial pathogens. Collectively, these results presented that Met-NiFe 2 O 4 nanoparticles like biocompatible nanocarriers could be potentially utilized for treating bacterial infections and cancer.

Conflicts of Interest:
The authors declare no conflict of interest.