Preparation of NiFe₂O₄@MIL-101(Fe)/GO as a novel nanocarrier and investigation of its antimicrobial properties

Abstract

In this research, we have investigated a novel magnetic nanocomposite including NiFe₂O₄@MIL-101(Fe)/GO for the delivery of the antibiotic tetracycline (TC). Moreover, the antibacterial activity of NiFe₂O₄@MIL-101(Fe)/GO, NiFe₂O₄@MIL-101(Fe)/GO/TC and pure TC was evaluated by agar well diffusion and minimum inhibitory concentration (MIC) methods on both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. In addition, the cytotoxicity of NiFe₂O₄@MIL-101(Fe)/GO/TC on HeLa cells was determined by an MTT assay which showed good results. The structure of the prepared nanocarrier was investigated by various spectroscopic techniques such as Fourier-transform infrared spectroscopy (FT-IR), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), Brunauer–Emmett–Teller (BET), and thermal gravimetric analysis (TGA). The results of this study showed that 98% of the TC was loaded on the synthesized nanocomposite. Drug release occurred at pH: 7.4 (phosphate buffer saline) and pH: 5.0 (acetate buffer) within 3 days, resulting in 77% and 85% release of the drug, respectively.

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1. Introduction

The design of drug delivery systems is very useful in the diagnosis and treatment of diseases and the controlled and targeted release of drugs. Today, great effort has been made to develop new methods for targeted drug delivery and controlled drug release.¹ The aim of the new drug delivery is to optimize the drug's effects while reducing side effects in the body. Therefore, the use of drug nanocarriers for targeted drug delivery can provide these optimal conditions.²

Recently, numerous compounds have been investigated, such as solid lipid nanoparticles,³ polymer nanoparticles,⁴ micelles,⁵ hydrogel nanoparticles,⁶ liposomes,⁷ and metal–organic frameworks (MOFs)⁸ as drug carriers.

MOFs are a new class of porous materials made from metal ions and organic ligands. They have significant properties such as: low density, high porosity, adjustable pore size, easy synthesis, high stability and high loading capacity, which have made them a promising drug carrier in recent years.^9–11 MIL-100 and MIL-101 are the first group of metal–organic frameworks to be considered as a nanocarrier in the release of ibuprofen.¹ In recent years, different MOFs including ZIF-8, MIL-101(Fe)–NH₂, and UiO-66 have been used for drug delivery of some drugs such as doxorubicin, naproxen, and ciprofloxacin.^12–14

Magnetic nanoparticles (MNPs) with highlighted properties including high stability, easy separation, low toxicity, ease of surface modification and magnetic attributes¹⁵ have been used as highly potential materials in diverse fields like biomedical,¹⁶ biochemical,¹⁷ magnetic imaging,¹⁸ catalysis¹⁹ and industrial applications.²⁰ However, their role in biomedicine, particularly in the field of drug delivery, is important because their inherent magnetism aids many tasks, including targeting, which is very important and necessary in drug delivery.²¹

Among the various ferrites, NiFe₂O₄ with the general formula (AB₂O₄) has attracted significant attention due to its excellent magnetic and catalytic properties, high saturation magnetization, strong chemical stability, and low toxicity.²² The ferromagnetic properties of nickel ferrite (NiFe₂O₄) resulting from the magnetic moments of anti-parallel spins between Fe³⁺ ions at tetrahedral sites and Ni²⁺ ions at octahedral sites make them one of the most widely used spinel ferrites in various biomedical applications.²³

The combination of MNPs and MOFs results in the formation of magnetic frameworks composites (MFCs). Combinations of MOFs with NPs have been made due to their high adsorption capacity, easy functionality, and easy isolation with an external magnetic field.²⁴ There are many ways to improve and modify nanocarrier structures by adding a functional group and surface modification using different materials like PEG,²⁵ chitosan,²⁶ alginate,²⁷ carboxymethyl cellulose²⁸ and graphene oxide.²⁹

Graphene oxide (GO) with properties including special surfaces, pore size, non-toxicity and excellent biological compatibility which has oxygen-containing agents, epoxy and hydroxyl, carboxyl, carbonyl, and phenol functional groups, is a suitable substrate for drug loading through hydrogen bonding and physical absorption.³⁰

One of the advantages of this nanomaterial is that graphene oxide can be well-dispersed in water and physiological environments due to its abundant hydrophilic groups, such as hydroxyl, epoxide and carboxylic groups on its large surfaces. In addition, its good biocompatibility and lack of obvious toxicity make it a promising material for drug carrier substances.^31,32

The hydroxyl and epoxy functional groups of GO permit the metal ions in MOFs to be formed as a composite hence, the idea of MOF-GO nanocomposites is developed. So, by merging the complementary features of the two materials, GO becomes dense arrays of layers and nonporous.

Recently, some MOF-GO structures like Fe₃O₄@MOFs/GO and CMC/MOF-5/GO have been used as an efficient drug carrier for the drug release of ibuprofen and doxorubicin, respectively.^33,34

Tetracyclines (TC) are a family of bacteriostatic antibiotics that are active against various Gram-negative and Gram-positive bacteria and are used to treat acne and skin infections.³⁵

Tetracyclines inhibit protein production, they are able to inhibit bacterial protein synthesis by binding reversibly to ribosomal units (Fig. 1).

Fig. 1 The chemical structure of tetracycline.

Considering the significance of the previous research related to the need of new drug delivery systems to find a suitable and practical nanocarrier,^36–38 herein, we were able to report a novel magnetic nanocomposite including NiFe₂O₄@MIL-101(Fe)/GO, as a carrier for the drug delivery of TC (Scheme 1). Moreover, the antibacterial properties of NiFe₂O₄@MIL-101(Fe)/GO, pure TC and the drug-loaded nanocarrier were compared against Escherichia coli and Staphylococcus aureus bacteria by agar well diffusion and MIC methods which showed significant results. An MTT assay was performed to evaluate the toxicity of NiFe₂O₄@MIL-101(Fe)/GO/TC.

Scheme 1 A schematic exhibition of the synthetic procedure of NiFe₂O₄@MIL-101(Fe)/GO as a TC carrier with antibacterial activities.

2. Results and discussion

2.1. Characterization of NiFe₂O₄@MIL-101(Fe)/GO and NiFe₂O₄@MIL-101(Fe)/GO@TC

In this research, the structures of NiFe₂O₄@MIL-101(Fe)/GO and NiFe₂O₄@MIL-101(Fe)/GO@TC were fully characterized by different spectroscopy techniques.

2.1.1 Infrared spectroscopy. The FT-IR spectra of NiFe₂O₄, MIL-101(Fe), NiFe₂O₄@MIL-101(Fe)/GO, NiFe₂O₄@MIL-101(Fe)/GO/TC and the pure TC are shown in Fig. 2. From the FT-IR spectrum of NiFe₂O₄ shown in Fig. 2a, the presence of Ni–O and Fe–O bonds in the magnetic particles was confirmed by the peak appearing at 556 cm⁻¹ and 610 cm⁻¹, respectively. Also, the FT-IR spectrum of MIL-101(Fe) is shown in Fig. 2b, the peak at 748 cm⁻¹ is related to the C–H bond in benzene, and the band at 1392 cm⁻¹ corresponds to aromatic ring. The peaks observed at 1654 cm⁻¹ and 1600 cm⁻¹ are related to the stretching vibration of C=O and C=C of BDC, respectively.

Fig. 2 FT-IR spectra of (a) NiFe₂O₄, (b) MIL-101(Fe), (c) NiFe₂O₄@MIL-101(Fe)/GO, (d) NiFe₂O₄@MIL-101(Fe)/GO/TC, and (e) TC.

In the FT-IR spectrum of NiFe₂O₄@MIL-101(Fe)/GO (Fig. 2c), the presence of Ni–O and Fe–O bonds in the magnetic particles was confirmed by the peaks appearing at 556 cm⁻¹ and 610 cm⁻¹, respectively. The peaks observed at 1654 cm⁻¹ and 1600 cm⁻¹ are related to the stretching vibration of C=O and C=C of BDC, respectively, and the band at 1392 cm⁻¹ was from the aromatic carbon.³⁹ The peak at 1019 cm⁻¹ was from C–O, the absorbed water in GO is shown by a broad peak at 3427 cm⁻¹, contributed to by the O–H stretching of the H₂O molecules. This supports the fact that GO is a highly absorptive material.

The FT-IR spectra of NiFe₂O₄@MIL-101(Fe)/GO/TC confirms the presence of TC in the MOF (Fig. 2d). The peak at 3427 cm⁻¹ is associated with the OH group. The absorption band at 3427 cm⁻¹ belongs to the NH₂ group of TC, representing the loading of TC on the nanocomposite. The bands at 1454 cm⁻¹ are related to the stretching vibrations of C–N. Moreover, the peak at 1685 cm⁻¹ refers to the carbonyl group of the drug.

2.1.2 EDX analysis. The chemical purity of the samples and their stoichiometry were determined by EDX. The EDX spectrum of NiFe₂O₄@MIL-101(Fe)/GO includes C, O, Ni, and Fe elements (Fig. 3a). Furthermore, the EDX spectrum of NiFe₂O₄@MIL-101(Fe)/GO/TC contains C, O, Ni, Fe, and N elements, which means that tetracycline is loaded on NiFe₂O₄@MIL-101(Fe)/GO (Fig. 3b).

Fig. 3 EDX analysis of (a) NiFe₂O₄@MIL-101(Fe)/GO and (b) NiFe₂O₄@MIL-101(Fe)/GO/TC.

2.1.3 SEM analysis. The microscopic morphology of the products was visualized by scanning electron microscopy (SEM). This analysis showed that NiFe₂O₄ was composed of relatively uniform quasispherical particles (Fig. 4a).⁴⁰ The SEM image of MIL-101(Fe) showed that this structure has a typical octahedral shape, which was in accordance with previous reports (Fig. 4b).⁴¹ The SEM image of the GO in Fig. 4c showed that the particles of GO look very dense with the layers stacked together as a result of dispersive forces and strong specific interactions between the surface groups on the graphene-like layers.⁴²

Fig. 4 SEM analysis of (a) NiFe₂O₄, (b) MIL-101(Fe), (c) GO, (d) NiFe₂O₄@MIL101(Fe)/GO, and (e) NiFe₂O₄@MIL-101(Fe)/GO/TC.

SEM images for the nanocarrier before and after drug loading are shown in Fig. 4d and e. The image of the NiFe₂O₄@MIL-101(Fe)/GO shows that the particles have a regular and spherical shape (Fig. 4d). After loading, it is shown that the particle size has become larger and the shape has become irregular (Fig. 4e).

2.1.4 Powder X-ray diffraction. The XRD pattern of the NiFe₂O₄ nanoparticles is shown in Fig. 5a. The peaks at 30.29°, 35.70°, 43.36°, 57.36° and 62.92° (2θ°) indicate the formation of the NiFe₂O₄ nanoparticles.³⁹ Also, the peaks at 8.9°, 9.7°, 19.6° and 21.7° (2θ°) show the presence of the MIL-101(Fe) frameworks (Fig. 5b).⁴³ Moreover, it is considerable that the peak at 2θ = 11.1° indicates the construction of the GO (Fig. 5c).⁴³

Fig. 5 XRD patterns of (a) pure NiFe₂O₄, (b) MIL-101(Fe), (c) GO, (d) NiFe₂O₄@MIL-101(Fe)/GO, and (e) NiFe₂O₄@MIL-101(Fe)/GO/TC.

Fig. 5d shows the XRD pattern of NiFe₂O₄@MIL-101(Fe)/GO. The peaks observed at 2θ = 30.29°, 35.70°, 43.36°, 57.36° and 62.92° are related to the NiFe₂O₄ NPs. Also the peaks at 8.9°, 9.7°, 19.6° and 21.7° (2θ°) show the presence of the MIL-101(Fe) frameworks as well as the peak at 2θ = 11.1° indicated the construction of the GO, which confirms the formation of the nanocomposite.

From this information, it can be concluded that after loading TC, the crystallite structure of NiFe₂O₄@MIL-101(Fe)/GO is preserved although the peak intensities declined (Fig. 5e).

2.1.5 BET analysis. The BET absorption method is an appropriate technique to measure the porosity of the materials. As illustrated in BET-plots in Fig. 6, the available surface area before and after loading TC is about 790 m² g⁻¹ and 618 m² g⁻¹, respectively. The volume of the cavities in the NiFe₂O₄@MIL-101(Fe)/GO is 0.1362 cm³ g⁻¹, which reached 0.0924 cm³ g⁻¹ after drug loading. This data confirms the loading of the drug in the cages of the metal–organic framework composite.

Fig. 6 BET plot of (a) NiFe₂O₄@MIL-101(Fe)/GO and (b) NiFe₂O₄@MIL-101(Fe)/GO/TC.

In addition, the BET method was used to evaluate the porous structure of NiFe₂O₄@MIL-101(Fe)/GO before and after encapsulating TC based on nitrogen adsorption. As shown in Fig. 7, the adsorption–desorption isotherm for NiFe₂O₄@MIL-101(Fe)/GO is a type I sorption isotherm. Furthermore, the result of the BJH (Barrett–Joyner–Halenda) analysis indicates that the average pore diameter of the nanocarrier is 1.21 nm, which indicates the porosity of the composite (Fig. 8).

Fig. 7 Adsorption/desorption of (a) NiFe₂O₄@MIL-101(Fe)/GO and (b) NiFe₂O₄@MIL-101(Fe)/GO/TC.

Fig. 8 BJH-plot of (a) NiFe₂O₄@MIL-101(Fe)/GO and (b) NiFe₂O₄@MIL-101(Fe)/GO/TC.

2.1.6 Thermogravimetric analysis (TGA). The TGA curve of NiFe₂O₄@MIL-101(Fe)/GO/TC shows that around 5% weight loss is due to the evaporation of all the solvents in the range of 0–200 °C (Fig. 9). Also, a weight loss of about 20% starting from 200 °C to 340 °C is attributed to the destruction of tetracycline. The destruction of MIL-101(Fe) MOFs was observed at the range of 340 °C to 450 °C. Eventually, destruction up to 800 °C is attributed to the GO.⁴⁴

Fig. 9 TGA curve of the NiFe₂O₄@MIL-101(Fe)/GO/TC.

2.2. Study on drug release

Before the release, different concentrations of the drug were prepared in two buffer (phosphate buffer and acetate buffer) solutions. Their absorptions were then obtained by a UV-Vis device at 360 nm. Then, their calibration curves were plotted. The drug release diagram at pH: 7.4 and pH: 5 is shown in Fig. 10. As indicated, the structure of NiFe₂O₄@MIL-101(Fe)/GO is unstable in acidic buffer (pH: 5) and degraded very rapidly. For this reason, drug release at pH: 5 is much faster than pH: 7.4 which means it is unsuitable for a desirable drug delivery system. The obtained results show that the rate of drug release at pH: 5 increased to 70% during the first 4 h. Whereas, the release of the drug at the physiological pH of the body (pH: 7.4) was about 77% over 72 h and then fixed. Based on these observations, the TC release of NiFe₂O₄@MIL-101(Fe)/GO is controlled at pH 7.4.

Fig. 10 The release of TC from NiFe₂O₄@MIL-101(Fe)/GO in two buffers at pH: 5 and pH: 7.4.

2.3. Results of the antimicrobial tests

2.3.1 Agar well diffusion method. As shown in Fig. 11, the antibacterial activity of NiFe₂O₄@MIL-101(Fe)/GO/TC against S. aureus and E. coli bacteria was examined. The results were amazing and NiFe₂O₄@MIL-101(Fe)/GO/TC showed excellent antibacterial activity. The MOF structure is gradually degraded and with the release of metal ions and drug TC, the cell wall of the bacteria is destroyed. Measurement results of the diameter of the inhibition zones showed that the halo created on E. coli was 15 mm for the TC drug and 21 mm for the drug loaded on the MOF. Also, for the S. aureus bacterium, it was 11 mm and 23 mm, respectively (Table 1).

Fig. 11 Results of the antibacterial test: (a) the disk containing E. coli bacteria and (b) the disk containing S. aureus bacteria.

Table 1 The antibacterial activity of TC, NiFe₂O₄@MIL-101(Fe)/GO, and NiFe₂O₄@MIL-101(Fe)/GO/TC

Bacteria	TC	NiFe2O4@MIL-101(Fe)/GO	NiFe2O4@MIL-101(Fe)/GO/TC
E. coli	15	0	21
S. aureus	11	0	23

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We provided Table 2 to show a comparison between our research with similar studies.^37,45–48 As can be seen, the newly prepared nanocomposite including NiFe₂O₄@MIL-101(Fe)/GO had a satisfactory drug loading percentage, drug release, and also antibacterial activities in comparison with previous structures.

Table 2 A comparison between the present research and similar previous works

Entry	Nanocomposite	Drug	Drug release (pH: 7.4)	Drug loading	Bacterial strain	Inhibited zone	Ref.
1	Fe3O4@PAA@ZIF-8	Ciprofloxacin	74%	93%	E. coli	32 mm	37
					S. aureus	15 mm
2	ZIF-8/GO/MgFe2O4	Tetracycline	92%	90%	E. coli	22 mm	45
					S. aureus	25 mm
3	ZnO@ZIF-8	Ampicillin	60%		E. coli	12 mm	46
					S. aureus	48 mm
4	GO-PEG	Nigella sativa		88%	E. coli	30 mm	47
					S. aureus	36 mm
5	Zn2(bdc)2(dabco)	Gentamicin	65%		E. coli	16 mm	48
					S. aureus	9 mm
6	NiFe2O4@MIL-101(Fe)/GO	Tetracycline	77%	98%	E. coli	21 mm	This research
					S. aureus	23 mm

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2.3.2 MIC method. Table 3 shows the MIC results of pure tetracycline, NiFe₂O₄@MIL-101(Fe)/GO, and NiFe₂O₄@MIL-101(Fe)/GO/TC. Based on the minimum inhibitory concentration method, the antibacterial activity of tetracycline incorporated into the prepared NiFe₂O₄@MIL-101(Fe)/GO was higher than NiFe₂O₄@MIL-101(Fe)/GO and pure tetracycline.

Table 3 MIC (μg mL⁻¹) of TC, NiFe₂O₄@MIL-101(Fe)/GO, and NiFe₂O₄@MIL-101(Fe)/GO/TC

Bacteria	TC	NiFe2O4@MIL-101(Fe)/GO	NiFe2O4@MIL-101(Fe)/GO/TC
E. coli	3.125	>100	0.19
S. aureus	25	>100	6.25

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2.4. Cytotoxicity of NiFe₂O₄@MIL-101(Fe)/GO/TC

In this research, an MTT assay was used to determine the cytotoxicity of NiFe₂O₄@MIL-101(Fe)/GO/TC. In the MTT assay, only cells which are viable after 24 hours exposure to the sample were capable of metabolizing a dye (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide) efficiently and the purple-colored precipitate which is dissolved in a detergent was analyzed spectrophotometrically. After 24 hours post treatment, HeLa cells showed excellent viability even up to the concentration of 100 μg mL⁻¹ of NiFe₂O₄@MIL-101(Fe)/GO/TC. As shown in Table 4, The IC₅₀ value of the NiFe₂O₄@MIL-101(Fe)/GO/TC was 33.26 (Fig. 12).

Table 4 The cytotoxic effect of NiFe₂O₄@MIL-101(Fe)/GO/TC

Concentration (μg mL^−1)	Absorbance at 570 nm			Average	Average-blank	% viability	IC50 (μg mL^−1)
100	0.211	0.241	0.238	0.23	0.222	18.5	33.26
75	0.355	0.312	0.347	0.338	0.33	27.5
50	0.577	0.561	0.558	0.565333	0.557333	46.44
25	0.798	0.778	0.781	0.785667	0.777667	64.801
10	0.877	0.845	0.863	0.861667	0.853667	71.14
5	1.008	1.001	0.998	1.002333	0.994333	82.86
Control (untreated)	1.214	1.197	1.202	1.208	1.2	100
Blank	0.008	0.009	0.007	0.008	0

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Fig. 12 The cytotoxic effect of NiFe₂O₄@MIL-101(Fe)/GO/TC on HeLa cells.

The results of the MTT assay showed that the TC extract in a concentration-dependent manner could inhibit cell proliferation. In this way, by increasing its concentration, the proliferation of HELA cancer cells is inhibited and makes a significant difference compared to the control group. By determining the inhibitory concentration of IC₅₀, it was found that the TC concentration was 33.26 μg mL⁻¹ in 24 hours, which resulted in the death of 50% of HELA cancer cells. Also, the results of the MTT assay to evaluate the effect of TC on the normal HEK-293 cell line showed that the TC extract has a less toxic effect on the HEK-293 cell line than the HELA cancer cell line (Fig. 13).

Fig. 13 The MTT assay on HEK293 cells to evaluate the effect of the TC.

3. Experimental section

3.1. Materials

Chemicals were purchased from Sigma-Aldrich and Merck in high purity. All of the materials were of commercial reagent grade and were used without further purification. FT-IR spectra were recorded on the Magna-IR spectrometer 550. Powder X-ray diffraction (XRD) was carried out on a Philips diffractometer of X'pert company with mono chromatized Cu Kα radiation (λ = 1.5406 Å). The compositional analysis was done by X-ray energy dispersive analysis (EDX, Kevex (Newark, DE) Delta Class I). The microscopic morphology of products was visualized by SEM (LEO 1455VP). Thermogravimetric analysis (TGA) was performed on a Mettler Toledo TGA under argon and heated from room temperature to 825 °C. The approximate sample weight was 10 mg in the TG experiment with a 10 °C min⁻¹ heating rate. Nitrogen adsorption–desorption isotherms were measured at 196 °C using a Belsorp mini automatic adsorption instrument after degassing the samples at 150 °C for 5 h. Absorption spectra were recorded in the range 200–800 nm on a Shimadzu model 1601 PC UV-visible spectrophotometer (Shimadzu, Tokyo, Japan).

3.2. Preparation of NiFe₂O₄ nanoparticles

NiFe₂O₄ was prepared using a method described by Zhang et al.⁴⁰ via a coprecipitation method. Briefly, FeCl₃·6H₂O (1.35 g, 5 mmol) and NiCl₂·6H₂O (0.595 g, 2.5 mmol) were dissolved in distilled water (10 mL) and then sonicated to form a clear solution. Then, 20 mL of 5 mol L⁻¹ NaOH solution was added into the mixture and stirred for 15 min. The resulting mixture was transferred into a 50 mL Teflon-lined autoclave and heated at 180 °C for 12 h. After cooling slowly to room temperature, the black product was washed several times with distilled water and ethanol and then dried at 50 °C for 24 h.

3.3. Synthesis of NiFe₂O₄@MIL-101(Fe)

Synthesis of NiFe₂O₄@MIl-101(Fe) was performed by a solvothermal method. In this way, terephthalic acid (0.415 g, 2.5 mmol) and FeCl₃·6H₂O (1.35 g, 5 mmol) were added into 30 mL of DMF. Then, 0.12 g of the prepared NiFe₂O₄ was added to it. The mixture was suspended under ultrasonic irradiation for 15 min. Next, the mixture was transferred to a Teflon autoclave at 120 °C for 20 h. When the reaction was completed, the obtained residue was washed with DMF and EtOH to eliminate unreacted materials. The purified product was dried in an oven for 30 min at 70 °C and then activated at 150 °C after10 h. The obtained NiFe₂O₄@MIL-101(Fe) was 0.51 g which proved that the content of the MOF in the composite is about 0.39 g.

3.4. Synthesis of GO

GO was synthesized from graphite powder according to the Hummers' method.⁴⁹ In this regard, graphite (2 g) and NaNO₃ (2 g) were mixed in 50 mL of H₂SO₄ (98%) and kept in an ice bath (0–5 °C) with continuous stirring. The mixture was stirred for 2 hours at this temperature and KMnO₄ (6 g) was added to the suspension very slowly. The rate of addition was carefully controlled to keep the reaction temperature lower than 15 °C. The ice bath was then removed, and the mixture was stirred at 35 °C until it became pasty brownish and kept under stirring for 2 days. It is then diluted with a slow addition of 100 mL of water. The reaction temperature was rapidly increased to 98 °C. Further, this solution was diluted by adding additional 200 mL of water under continuous stirring, followed by a slow addition of 10 mL of H₂O₂ to terminate the reaction by the appearance of a yellow color. Finally, the mixture was washed with HCl (10%) and deionized water several times. The product was dried at 50 °C for 24 h to obtain GO.

3.5. Synthesis of NiFe₂O₄@MIL-101(Fe)/GO

A mixture of GO (0.05 g) and NiFe₂O₄@MIL-101(Fe) (0.1 g) was dispersed in DMF (30 mL) under ultrasonic treatment for 20 min. Then, the mixture was transferred to a Teflon autoclave at 120 °C for 12 h. When the reaction was completed, the product was washed with DMF, deionized water and EtOH. Finally, it was dried at 70 °C for 24 h.

3.6. Loading TC on the NiFe₂O₄@MIL-101(Fe)/GO

Drug loading of NiFe₂O₄@MIL-101(Fe)/GO was as follows: 0.1 g of TC was dissolved in 5 mL of deionized water which continued by the addition of NiFe₂O₄@MIL-101(Fe)/GO (0.05 g). This solution was stirred for the duration of five days. After completion of the loading, the prepared NiFe₂O₄@MIl-101(Fe)/GO/TC was washed three times with deionized water to remove unloaded drug, then dried in an oven at 60 °C for 24 h. The amount of drug loading was 98% based on eqn (1):

Drug loading (%) = (TC weight in sample/total weight of sample) × 100% (1)

3.7. Drug release from NiFe₂O₄@MIL-101(Fe)/GO

The release of the drug was performed in two buffers, phosphate buffer saline (pH: 7.4) and buffer acetate (pH: 5). In this procedure, 0.02 g of NiFe₂O₄@MIL-101(Fe)/GO/TC was added to 50 mL of each buffer separately. Then, the solution was stirred at 37 °C for 3 days. Each time, 5 mL of the solution was removed and quickly replaced with the same amounts of fresh buffer. Further, measurement of the TC released from NiFe₂O₄@MIL-101(Fe)/GO was done by an instrument of spectroscopy UV/Vis at 360 nm. Finally, the release percentage was expressed by eqn (2):

Release percentage (%) = m_r (amount of released TC)/m_l (total amount of loaded TC) (2)

3.8. Assays for antibacterial activity

3.8.1 Agar well diffusion test. S. aureus and E. coli were used as the standard strains in this study. To determine the susceptibility of the microorganisms, antibiotics were used from Mueller Hinton Agar culture media.⁵⁰ Firstly, a concentration of the materials (10 μg mL⁻¹) was prepared in HCl solutions (0.1 mol mL⁻¹). Then, 50 μg mL⁻¹ concentrations of tested agents were injected into the well. Finally, the plates were incubated at 35 °C for 24 hours and the zone of inhibition was measured (mm).

3.8.2 MIC assay. Bacteria colonies of S. aureus and E. coli were suspended in tryptic soy broth (TSB, 30 g L⁻¹, 5 mL) and solutions were propagated for 16 h in a shaking incubator at 37 °C. The solutions were diluted with TSB to a concentration of 10⁹ bacteria per mL, where the bacterial density was determined by measuring OD values at 600 nm. Further dilution with TSB was made until a final concentration of 10⁶ bacteria per mL was achieved. The resulting bacterial solution was then transferred into a 96-well plate with a volume of 100 μL per well. The bacteria were then treated with pure tetracycline, NiFe₂O₄@MIL-101(Fe)/GO, and NiFe₂O₄@MIL-101(Fe)/GO/TC (100 μg mL⁻¹ stock solution). The final concentrations were 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 and 0.19 μg mL⁻¹. Negative control wells received 10 μL of TSB only. MIC was determined to be the drug concentration that completely inhibited bacterial growth with respect to the positive control. The lowest concentration of growth inhibitor is considered as MIC.⁵¹

3.9. MTT assay

The cytotoxicity of NiFe₂O₄@MIL-101(Fe)/GO/TC on HeLa cells was determined by MTT assay. Approximately 1 × 10⁵ mL⁻¹ HeLa cells in their exponential growth phase were seeded in a flat-bottomed 96-well polystyrene coated plate and were incubated for 24 h at 37 °C in a 5% CO₂ incubator. A series of dilutions (5, 10, 25, 50, 75 and 100 μg mL⁻¹) of NiFe₂O₄@MIL-101(Fe)/GO/TC in the medium was added to the plate in hexaplets. After 24 hours of incubation, 10 μL of the MTT reagent was added to each well and further incubation was carried out for 4 hours. Formazan crystals formed after 4 hours in each well, which were dissolved in 150 μL of detergent and the plates were read immediately in a microplate reader (BIO-RAD microplate reader-550) at 570 nm. Wells with complete medium, NiFe₂O₄@MIL-101(Fe)/GO/TC and MTT reagent, without cells were used as blanks. Untreated HeLa cells as well as the cell treated with (5, 10, 25, 50, 75 and 100 μg mL⁻¹) concentrations of NiFe₂O₄@MIL-101(Fe)/GO/TC for 24 h were subjected to the MTT assay for cell viability determination.⁵²

4. Conclusions

In this study, a novel nanocomposite including NiFe₂O₄@MIL-101(Fe)/GO was successfully produced and its structure was confirmed by FT-IR, EDX, XRD, SEM, BET and TGA techniques. In addition, tetracycline as an antibiotic drug was encapsulated into the NiFe₂O₄@MIL-101(Fe)/GO with a high loading of 98% due to the structure, high surface area and cavities in the structure of the nanocarrier. Furthermore, the drug release from the nanocomposite was tested at pH: 7.4 and pH: 5.0. The results of this release indicated that pH: 7.4 is more appropriate because of the controlled release and withdrawal of the drug from the nanocarrier. Furthermore, E. coli and S. aureus were used for antibacterial testing, and the results showed that the TC drug loaded on the NiFe₂O₄@MIL-101(Fe)/GO better inhibited the growth of the bacterium. As a result, NiFe₂O₄@MIL-101(Fe)/GO/TC has a stronger antibacterial property than pure TC. At the end, MTT studies exhibited that NiFe₂O₄@MIL-101(Fe)/GO/TC was of low toxicity.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The author gratefully acknowledges the financial support of this work by the Research Affairs Office of the Islamic Azad University, Qom Branch, Qom, I. R. Iran [grant number 2019-2898].

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