Evaluation of antibacterial property of hydroxyapatite and zirconium oxide‐modificated magnetic nanoparticles against Staphylococcus aureus and Escherichia coli

Abstract

In the first section of this research, superparamagnetic nanoparticles (NPs) (Fe₃ O₄) modified with hydroxyapatite (HAP) and zirconium oxide (ZrO₂) and thereby Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs were synthesised through co‐precipitation method. Then Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs characterised with various techniques such as X‐ray photoelectron spectroscopy, X‐ray diffraction, scanning electron microscopy, energy dispersive X‐ray analysis, Brunauer–Emmett–Teller, Fourier transform infrared, and vibrating sample magnetometer. Observed results confirmed the successful synthesis of desired NPs. In the second section, the antibacterial activity of synthesised magnetic NPs (MNPs) was investigated. This investigation performed with multiple microbial cultivations on the two bacteria; Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Obtained results proved that although both MNPs have good antibacterial properties, however, Fe₃ O₄ /HAP NP has greater antibacterial performance than the other. Based on minimum inhibitory concentration and minimum bactericidal concentration evaluations, S. aureus bacteria are more sensitive to both NPs. These nanocomposites combine the advantages of MNP and antibacterial effects, with distinctive merits including easy preparation, high inactivation capacity, and easy isolation from sample solutions by the application of an external magnetic field.

1 Introduction

Nowadays, antibiotic resistance, due to human misuse and disregard, becomes a serious public health concern throughout the world [1, 2]. Between them, nanomaterials have an important role in this research area. However, for consideration of nanoparticles (NPs) in biological applications, such as antibacterial therapeutics, several key requirements have to be fulfilled. The first is to deal with the engineered NPs of biocompatibility. The second implies manipulation of the simple carriage to the desired location. Finally, the most crucial requirement is their easy separation from the biological environment. Despite the very fast expansion of the bio‐nanotechnology in recent years, there are many challenges facing these three requirements. Relevant works that aimed at correlating synthesis, stabilisation and surface modification of NPs with their biological effects and decreased toxicity have shown that there is no general role.

Application of NPs offers many advantages due to their unique size and physical properties [3]. For most types of uses, to impede biocompatible particles and their stability in the carrier liquids, complex structure type magnetic core/non‐magnetic shell is required. Magnetic NPs (MNPs) have a wide range of uses in various disciplines, such as biomedical applications catalysis, biotechnology, biomedicine, magnetic resonance imaging, and environmental remediation [4]. In this regard, using MNPs in controlling bacterial infection is carried out by many researchers [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. Iron oxides are somewhat stable against oxidation and have a significant magnetisation, and also due to their lack of toxicity being preferred in various uses [11, 20, 21]. Superparamagnetic NPs have been extremely studied due to their varied applications, in both physico‐chemical and biomedical fields. Candidate MNPs for biomedical uses are mainly biocompatible Fe₃ O₄ NPs which can be coated with organic and inorganic agents able to ensure active biochemical agents grafting on the functionalised surface of MNPs.

The antibacterial effects of ZnO, Al₂ O₃, CuO, TiO₂, NiO and iron oxide NPs have been previously reported [22, 23, 24, 25, 26, 27, 28, 29]. Metallic NPs, as a result of their antibacterial properties, act as an effective agent against bacterial resistance [30]. MNPs because of their special physical properties such as ease of preparing nanosize material and good magnetic effect and the possibility to attach with microbial cells, have a lot of applications in biomedicine and biologic systems. The synergistic effect of MNPs as antimicrobial agents is previously studied [5, 11]. Recently, antibacterial agent‐modificated MNPs, were used to improve their activity and easy separation of them from environment by applying an external magnetic field, such as Ag NPs [7, 9, 14, 16, 18], Au NPs [12, 31], N‐halamine [8], polyrhodanine [13], poly(vinyl pyrrolidone) [15], alumina [19], cetyltrimethylammonium bromide [32], and TiO₂ [6].

Other interesting agents that have been used for antibacterial application are hydroxyapatite (HAP) [33, 34] and zirconium oxide (ZrO₂) [35, 36, 37]. It seems highly promising to modification of MNPs with them and enhanced the antibacterial property and dedicate to them magnetic property to easy separation. In spite of this interesting combination, there is no report on the preparation of ZrO₂ and HAP‐modified MNPs as antibacterial agents against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli).

This work aims to synthesise of Fe₃ O₄ MNPs by chemical co‐precipitation method and to modify its surface with ZrO₂ and HAP, i.e. Fe₃ O₄ /ZrO₂ and Fe₃ O₄ /HAP nanocomposites. After that, characterisation of nanocomposites by X‐ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), vibrating sample magnetometer (VSM) techniques and subsequently, evaluation of their antibacterial activity by treating with S. aureus and E. coli. To the best of our knowledge, the synergistic influence of modified MNPs (Fe₃ O₄ /ZrO₂ and Fe₃ O₄ /HAP) as an antibacterial agent is not yet reported in the literature.

2 Experimental

2.1 Chemicals and materials

All reagents were of analytical reagent‐grade and used as supplied. Ferric chloride (FeCl₃ ·6H₂ O), ferrous chloride (FeCl₂ ·4H₂ O), calcium nitrate (Ca(NO₃)₂ ·4H₂ O), potassium di‐hydrogen phosphate (KH₂ PO₄), zirconium chloride (ZrCl₄), barium chloride (BaCl₂), sulphuric acid (H₂ SO₄), hydrochloric acid (HCl), perchloric acid (HClO₄), acetic acid (CH₃ COOH), sodium hydroxide (NaOH), Nutrient broth and nutrient agar were purchased from Merck^®. All solutions were prepared with purified water (18 MΩ cm, Millipore‐MilliQ, Millipore Inc.).

Two bacteria chosen for investigations of the antibacterial assay were one Gram‐positive bacteria such as S. aureus ATCC 25923 and one Gram‐negative bacteria E. coli ATCC 25922 supplied from microbiology department, Esfahan University, Esfahan, Iran. Bacterial cultures were prepared in Nutrient Broth and maintained for 24 h at 37°C. The cell suspensions were diluted with sterile nutrient broth to provide an initial cell count of about 10⁶ CFU/ml.

2.2 Preparation of Fe₃ O₄ MNPs

The Fe₃ O₄ NPs were prepared by chemical coprecipitation methods [38, 39]. Briefly, 5.2 g of FeCl₃ ·6H₂ O, 2.0 g of FeCl₂ ·4H₂ O were dissolved in 25 ml 1 M of HCl (degassed with nitrogen gas before use for 20 min). Then, the solution was added dropwise to 250 ml of 1.5 M NaOH solution under vigorous stirring using a magnetic stirrer at 80°C. The obtained Fe₃ O₄ NPs were separated from the reaction medium by magnetic field, and washed with 200 ml deionised water four times, then resuspended in 100 ml deionised water and used for the next section.

2.3 Preparation of Fe₃ O₄ /ZrO₂ NPs

The Fe₃ O₄ /ZrO₂ NPs were synthesised based on our previous reported work [40]. Briefly, fresh solution of Zr(IV), prepared from zirconium tetrachloride in 0.23 M in HClO₄ solution, was added dropwise to the Fe₃ O₄ suspension and the pH value was adjusted to 8.0 by the addition of 2 M NaOH solution within 1 h. Stirring of the mixture carried on for 2 h after the addition. In the whole of process, temperature maintained at 80°C and for removal of oxygen, nitrogen gas bubbled in the suspension. The mass ratio of Fe₃ O₄ to ZrO₂ prepared in this work was 2:5. Finally, the formed NPs thoroughly washed and resuspended in deionised water.

2.4 Preparation of Fe₃ O₄ /HAP MNPs

For preparation of the Fe₃ O₄ /HAP, firstly 16.530 gr Ca(NO₃)₂. 4H₂ O and 5.715 gr KH₂ PO₄ were weighed and dissolved in a 100 ml volumetric flask. Then, 2.1 ml glacial acetic acid added to this solution and after that, diluted carefully to 100 ml with deionised water. 1 gr Fe₃ O₄ NPs prepared in the previous section, added to this freshly prepared solution and the resulted suspension mixed thoroughly for 30 min. Thereafter, NaOH 2 M solution dropwise added to the suspension and the pH value of the mixture was adjusted to 13. Suspension preserved 6 h in 37°C. Finally, the formed Fe₃ O₄ /HAP NPs thoroughly washed with deionised water and resuspended in deionised water and then separated by a permanent magnet. The mass ratio of Fe₃ O₄ to HAP prepared in this work was 1:5.

2.5 Apparatuses and methods

The XRD patterns and the crystal structure of prepared NPs were analysed by X‐ray diffractometer (XRD, Bruker Advanced D8 model), using CuKα radiation (λ  = 1.5406 Å). Morphology and elemental analysis of the samples determined by scanning electron microscope (SEM) equipped with EDXA (Philips XL‐30). The size of the as‐prepared particles was characterised using transmission electron microscope, TEM, (Philips‐EM‐208S). Magnetic properties were analysed using a vibrating sample magnetometer (VSM, Daghigh Meghnatis Kashan Co., Kashan, Iran). FTIR spectroscopy analysis was performed with a Nicolet Impact 400D Model spectrophotometer (Nicolet Impact, Madison, USA). pH meter (EDT Instrument, England), incubator (Heraeus, Germany), Oven (Memert, Germany), Autoclave (model A‐1000, Abzar teb, Iran) were also used in antibacterial experiments. Optical density (OD) of the samples was determined by UV–vis Cintra Model 404.

Standard of 0.5 McFarland barium sulphate prepared by adding 0.5 ml 0.048 M of BaCl₂ solution to the 99.5 ml 0.18 M of sulphuric acid solution and after that by consecutive mixing, a suspension prepared. Exact density of standard turbidity determined using a spectrophotometer and recording absorption value in 625 nm, which (must be in the range of 0.08–0.13). 5 ml of BaCl₂ suspension transferred to the tubes with equal size and maintained in the dark and room temperature.

2.6 Preparation of culture medium

According to the procedure of supplier, 8 g of nutrient broth powder and 30 g of nutrient agar weighed and separately dissolved in 1 l deionised water and then transferred to the tubes and subsequently by closing tubes inlet by cotton, sterilised during 15 min by an autoclave in 121°C and 15 Pa. For investigation of bactericidal effects of nanomaterials, from each of two bacteria and after night cultivation, a turbidity equal to 0.5 McFarland (OD₆₀₀  = 0.132) prepared. Then from each one up to 10% Vol secondary culture media impregnated to the tubes contain nutrient broth in three groups. Distribution of NPs in three groups of tubes were, 0.075, 0.15, 0.3, 0.6 and 0.9% of Fe₃ O₄, 0.075, 0.15, 0.3, 0.6 and 0.9% of Fe₃ O₄ /HAP and 0.075, 0.15, 0.3, 0.6 and 0.9% of Fe₃ O₄ /ZrO₂ NP. For blank sample also this proportion of bacteria added to the tubes contain culture media without NPs. If NPs create turbidity in the suspension, to compensate it one group consider as a negative control. An inlet of tubes closed with cotton and after shaking placed in the incubator for night culture at 37°C. A mentioned examination carried out in three series. Samples also examined for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests. Statistical evaluations were performed by using Minitab 16 software and balanced ANOVA method.

2.7 Antibacterial assay of NPs

The bactericidal efficacy was assayed by measuring OD of incubated samples with a UV–vis spectrophotometer [41].

After 24 h incubation, firstly MIC of samples evaluated and after that for determining MBC, all tubes without turbidity separately cultured on the nutrient agar plate and subsequently incubated 24 h in 37°C. In the next step, the OD value of samples undergoes first incubation determined. For this reason, firstly NP separated by a permanent magnet and then OD at 600 nm in comparison with blank and control samples determined. This test performed on the three series of samples and the average value of each sample was determined. After 24 h incubation at 37°C, to determining MBC value, plates were inspected and minimal concentration which growing of bacteria not occurred reported as MBC value in the presence and absence of Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs.

3 Results and discussion

3.1 Characterisation of prepared NPs

XRD : The XRD patterns of the as‐prepared Fe₃ O₄ /HAP (b) and Fe₃ O₄ /ZrO₂ (b) NPs in comparison to pure Fe₃ O₄ (a) are shown in Fig. 1. Comparison of the XRD patterns of the pure sample Fe₃ O₄ and modified Fe₃ O₄ with HAP and ZrO₂ MNPs revealed the existence of HAP and ZrO₂ in addition to Fe₃ O₄ structure in the resulting particles. As shown in the pattern (b) the diffraction peaks at d ‐values of 2θ  = 22°, 25.8°, 28.6°, 32.7°, 34° and 50° were indexed to (100), (200), (210), (300), (202) and (310) planes of HAP, respectively. It is also, the diffraction peaks at d ‐values of 2θ  = 30.4°, 35.2°, 50.6° and 60.3° were indexed to (111), (200), (220), and (311) planes of zirconium oxide, respectively [40]. The diffraction peaks observed at d ‐values of 2θ  = 30.0°, 35.5°, 57.2°, 62.7° and 89.9° were indexed to (220), (311), (511), (440), and (731) planes of magnetite, in which demonstrated no impurity peaks are detected and the high purity Fe₃ O₄ spinel structure is synthesised (pattern (a)). From the XRD patterns, the average size of dispersed Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs were calculated using Scherer equation as 27 ± 1 and 10 ± 2 nm, respectively.

Fig. 1.

XRD patterns of the as prepared Fe₃ O₄ (a), Fe₃ O₄ /ZrO₂ (b) and Fe₃ O₄ /HAP(c)

FTIR: To further prove successful preparation of nanomaterials, the FTIR spectroscopy was employed to examine the surfaces of the as‐made Fe₃ O₄, Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs. The characteristic band of Fe₃ O₄ appears at ~576 cm⁻¹ (Fig. 2 spectra a) [42]. The FTIR spectrum of the Fe₃ O₄ /HAP NPs (Fig. 2 spectra b) shows characteristic bands at 603, 566, 1035 and 1420–1576 cm⁻¹, which is due to the stretching and bending modes of PO₄ ^3– the bond of HAP [43]. Furthermore, observed peak around 872 cm⁻¹ is related to P–O band. The FTIR spectrum of the Fe₃ O₄ /ZrO₂ NPs (Fig. 2 spectrum c) shows bands at 500–850 cm⁻¹, which is the characteristic bond of Zr–O [37].

Fig. 2.

FTIR spectrums of the as prepared Fe₃ O₄ (a), Fe₃ O₄ /ZrO₂ (b) and Fe₃ O₄ /HAP(c)

Hence, these FTIR spectroscopy results provide additional evidence for the successful synthesis of NPs. The results support the presence of HAP and ZrO₂ on the surface of MNPs, and thus, modifications took place on the Fe₃ O₄ NPs, i.e. the resulted Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ MNPs have unique behaviours of HAP and ZrO₂ in addition to magnetic property, respectively.

SEM and EDXA : The morphology and elemental structure of as‐prepared Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ particles were further examined by SEM (Fig. 3) and EDXA (Fig. 4). As displayed in Fig. 3 A, the morphology of bulk Fe₃ O₄ particles is irregular and predicted agglomeration take place between them. Observed SEM images for bulk Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ particles in Figs. 3 B and C, respectively, showed condensed irregular particles. Chemical analysis using EDXA shows the presence of Zr atom in the Fe₃ O₄ /ZrO₂, as shown in Fig. 4 A, and Ca, O and P elements in the Fe₃ O₄ /HAP particles, as shown in Fig. 4 B. Obtained evidence from EDXA along with XRD and FTIR results confirmed successful preparation of Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ particles.

Fig. 3.

SEM images of

(A) As‐prepared Fe₃ O₄, (B) Fe₃ O₄ /ZrO₂ and (C) Fe₃ O₄ /HAP. The scale bars are 3, 3 and 10 µm in SEM images (A), (B) and (C), respectively

Fig. 4.

EDXA of

(A) As‐prepared Fe₃ O₄ /HAP, and (B) Fe₃ O₄ /ZrO₂

TEM : Figs. 5 A and B show the typical TEM images of the as‐prepared Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs, respectively. As displayed, the particles exhibited spherical morphology but with a small tendency to agglomeration and also the as‐prepared particles still in nanometre size during the preparation process. From TEM images, the average size of dispersed Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs was estimated as 35 ± 5 and 12 ± 4 nm, respectively. The TEM images and corresponding XRD patterns and EDXA, strongly suggested that the spherical, HAP and ZrO₂ particles are made up of a large number of small‐sized tetragonal Fe₃ O₄ nanocrystallites.

Fig. 5.

TEM images of

(A) As‐prepared Fe₃ O₄ /HAP, and (B) Fe₃ O₄ /ZrO₂. The scale bars are 200 and 100 nm in TEM images (A) and (B), respectively

VSM : MNPs synthesised here possess superparamagnetic behaviour with strong magnetic power. Figs. 6 A and B show the VSM magnetisation curves of Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ MNPs. The saturation magnetisation curves as 5.1 and 10.04 emu g⁻¹ were observed for Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ MNPs, respectively. Those saturation magnetisation curves were high enough for magnetic separation. The results proved the MNPs exhibited typical superparamagnetic behaviour, characterised with strong magnetic susceptibility and no hysteresis, remanence and coercivity (Table 1). The reaction of prepared MNPs in a magnetic field by applied a permanent magnet also presented in Fig. 6 B. These results verified the successful synthesis of Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ MNPs and superparamagnetic behaviour of them.

Fig. 6.

The magnetic responses

(A) Magnetic hysteresis loops of the as prepared Fe₃ O₄ /ZrO₂ (a), and Fe₃ O₄ /HAP (b), (B) Reaction of prepared MNPs in a magnetic field by applied a permanent magnet. The suspension of nanomaterials transparent after 10 min (left picture)

Table 1.

MIC and MBC results for each NP and bacteria

Sample	Microorganism	MIC, %	MBC, %
Fe3 O4	S. aureus	0.9	1.2
	E. coli	0.9	1.5
Fe3 O4 /HAP	S. aureus	0.15	0.3
	E. coli	0.15	0.6
Fe3 O4 /ZrO2	S. aureus	0.3	0.6
	E. coli	0.3	0.9

3.2 Antibacterial studies

Prepared MNPs, Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂, were tested as antibacterial agents. For this purpose, Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ MNPs were added to the S. aureus and E. coli bacteria culture medium in different concentration. Figs. 7, 8, 9 show the growth rate of S. aureus and E. coli bacteria in nutrient broth medium in different percentage of Fe₃ O₄, Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂, respectively. The balanced ANOVA by using Minitab 16 software was used to compare the observed results in Figs. 7, 8, 9. P value ≤0.05 was considered as a signifier.

Fig. 7.

Effect of different concentration of Fe₃ O₄ on S. aureus and E. coli activity

Fig. 8.

Effect of different concentration of Fe₃ O₄ /HAP on S. aureus and E. coli activity

Fig. 9.

Effect of different concentration of Fe₃ O₄ /ZrO₂ on S. aureus and E. coli activity

Statistically, there was no significant difference between the type of bacteria, S. aureus and E. coli, (P >0.05), indicating that prepared nanomaterials have the same effect on the S. aureus and E. coli. However, there was a significant difference between the types of used nanomaterials. Based on the P value, Fe₃ O₄ /HAP NP has greater antibacterial performance than Fe₃ O₄ (P  = 0.05) and there was no significant difference between Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ (P  = 0.18), and Fe₃ O₄ and Fe₃ O₄ /ZrO₂ (P  = 0.57). Furthermore, the MBC/MIC test was performed for approval that Fe₃ O₄ /HAP has more strength bactericidal property than Fe₃ O₄ /ZrO₂ and Fe₃ O₄. After 24 h incubation of plates in 37°C, MIC and MBC of Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs were investigated and thereby lowest concentration in which bacterial growth not occurred reported as MBC value (Table 1). According to the obtained results and refer to Figs. 7, 8, 9, it is obvious that bactericidal effects of Fe₃ O₄ were weaker but when its surface modified with HAP and ZrO₂, this effect significantly increased and the threshold limit of concentration was lower than Fe₃ O₄ NPs. Also, it's obvious that the bactericidal effect of Fe₃ O₄ /HAP is better than Fe₃ O₄ /ZrO₂ and this effect against S. aureus is greater than E. coli.

3.3 Antibacterial mechanism of Fe₃ O₄ /HAP and Fe₃ O₄ /ZrO₂ NPs

Various investigations showed that the cell wall of bacteria has a great role against NPs. Generally, in gram (+) and (−) bacteria its behaviour is different. NPs via the assistance of electrostatic interaction, can connect to the outer membrane of bacteria and cause to disturbing the membrane, suppressing of preplasmic enzymes, rupture of bacteria cell in the culture media, and ultimately restraining of DNA and RNA and protein synthesis. When NPs enter the bacterial cell, it forms a low‐molecular‐weight region in the centre of the bacteria to which the bacteria conglomerates, thus, protecting the DNA from the metal. The NPs preferably attack the respiratory chain, the cell division finally leading to cell death [44, 45, 46]. Furthermore, functionalised NPs release their metallic ions in the vicinity of cell wall which would cause diffuse into the cell and then based upon toxicity mechanism cause injury of bacteria [47, 48].

Because of their complex cell wall structure, gram (−) bacteria are more resistant against NPs and in this regard may be more amount of NPs and more retention time needed for better bactericidal effect. Also, some gram (+) bacteria, such as S. aureus with the formation of biofilm creates more resistance against NPs. It is possible that released ions from nanomaterials react with thiol groups (−SH) of bacteria cells surface proteins. The function of some of these cell membrane proteins is minerals transferring from the cell membrane. The NPs effect on this protein caused inactivation and impenetrable of the cell wall [49]. Inactivation of membrane permeability caused the death of the cell. Metal oxide NPs, based on their surface‐to‐volume ratio showed different bactericidal properties. Attachment of some metallic ions to DNA of bacteria would prevent certain important transferring processes such as carrying of phosphate and succinate agents and can interact with cell oxidation processes and breathing chains [50, 51, 52].

Recent studies on Fe₃ O₄ NPs shows that they can cause to leakage of lactate dehydrogenase from the cell membrane, a disorder in mitochondria function (or operation), the aggregate of chromosomes and creation of oxygen free radicals.

Formation of the oxygen free radicals, cause to disorder in oxidative pressure, cell antioxidant system and also peroxidation of membrane lipids, oxidation of enzymes and structural proteins, breaking of the DNA molecule and finally death of cells [53, 54, 55]. These particles caused erosion of plasmatic membrane, the creation of bubbles towards out of cell and disorder in permeability of plasmatic membrane [56, 57, 58] – microorganisms attachment to the host cells and tissues used by the surface adhesion factors. Attachment of Fe₃ O₄ NPs to the cell wall and surface factors of microorganisms leading to the occupation and inactivation of adhesion factors and as a result prevent its connection to the host cell substrate. This effect proved in S. aureus cells [59].

The existence of magnetic fields in iron oxide NPs helps it to penetrate the biofilm of bacteria. However, Fe₃ O₄ NPs are physicochemically unstable. These substances in the presence of oxygen quickly oxidised and converted to Fe₂ O₃ and because of their magnetic and hydrophobic properties are very unstable in aqueous and biological media. So its surface for better use needed to be coated [60, 61].

Herein, Fe₃ O₄ NPs modified with HAP and ZrO₂. Antimicrobial activity of HAP is related to the release of OH⁻ ions in an aqueous environment. Hydroxyl ions are highly oxidant free radicals that show extreme reactivity, reacting with several biomolecules. This reactivity is high and indiscriminate, so this free radical rarely diffuses away from sites of generation. Their lethal effects on bacterial cells are probably due to the following mechanisms: damage to the bacterial cytoplasmic membrane, protein denaturation, and damage to the DNA. The mechanisms above of HAP in contribution to the Fe₃ O₄ NPs antibacterial mechanism showed outstanding results in this work. On the other hand, zirconium has good cytocompatibility and antibacterial properties, and zirconium oxide having the ability to inhibit bacterial colonisation on the surface. The probable mechanism action of ZrO₂ comes from their interaction with thiol group compounds found in the respiratory enzymes of bacterial cells. Zirconium binds to the bacterial cell wall and cell membrane and inhibits the respiration process.

Due to their extremely large surface area, which provides better contact with microorganisms, it's expected that nanosized Fe₃ O₄ /ZrO₂ show high efficient antimicrobial property. The NPs get attached to the cell membrane and also penetrate inside the bacteria. The bacterial membrane contains sulphur‐containing proteins and the NPs interact with these proteins in the cell as well as with the phosphorus‐containing compounds like DNA.

4 Conclusion

Herein, HAP and ZrO₂ modificated MNPs (Fe₃ O₄) were synthesised and characterised by using physical and chemical techniques such as XPS, XRD, SEM, EDXA, BET, FTIR, and VSM. The antibacterial activity of NPs against S. aureus and E. coli was investigated. Obtained results proved that although both MNPs have good antibacterial properties, however, Fe₃ O₄ /HAP NP has greater antibacterial performance than Fe₃ O₄ /ZrO₂ and Fe₃ O₄. Also, S. aureus bacteria are more sensitive to both NPs based on MIC and MBC studies. These nanocomposites combine the advantages of MNP and antibacterial effects, with distinctive merits including easy preparation, high inactivation capacity, and easy isolation from sample solutions by the application of an external magnetic field.