Magnetic core‐shell Fe3O4@TiO2 nanocomposites for broad spectrum antibacterial applications

Abstract The authors have synthesised a core‐shell Fe3O4@TiO2 nanocomposite consisting of Fe3O4 as a magnetic core, and TiO2 as its external shell. The TiO2 shell is primarily intended for use as a biocompatible and antimicrobial carrier for drug delivery and possible other applications such as wastewater remediation purposes because of its known antibacterial and photocatalytic properties. The magnetic core enables quick and easy concentration and separation of nanoparticles. The magnetite nanoparticles were synthesized by a hydrothermal route using ferric chloride as a single‐source precursor. The magnetite nanoparticles were then coated with titanium dioxide using titanium butoxide as a precursor. The core‐shell Fe3O4@TiO2 nanostructure particles were characterized by XRD, UV spectroscopy, and FT‐IR, TEM, and VSM techniques. The saturation magnetization of Fe3O4 nanoparticles was significantly reduced from 74.2 to 13.7 emu/g after the TiO2 coating. The antibacterial studies of magnetic nanoparticles and the titania‐coated magnetic nanocomposite were carried out against gram+ve, and gram–ve bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Shigella flexneri , Escherichia coli, and Salmonella typhi) using well diffusion technique. The inhibition zone for E. coli (17 mm after 24 h) was higher than the other bacterial strains; nevertheless, both the uncoated and TiO2‐coated magnetite nanocomposites showed admirable antibacterial activity against each of the above bacterial strains.


| INTRODUCTION
Metal oxide nanostructured particles are of great significance in the field of antibacterial studies because of large surface areas with extremely active sites. A very discrete type of binary-metal oxide having good magnetization and unique biocompatibility is magnetite (Fe 3 O 4 ) especially with crystal size on the nanoscale. Fe 3 O 4 nanoparticles have been widely used because of some of its excellent optical and magnetic properties [1][2][3][4]. In addition to these, plural cationic oxidation states in Fe 3 O 4 nanoparticles provide an extra edge over other materials for physicochemical applications [5][6][7]. Various methods have been used by researchers to synthesize high-quality monodisperse magnetic nanoparticles such as chemical coprecipitation, solvothermal, hydrothermal, microemulsion, thermolysis, decomposition of precursors, sol-gel, and polyol methods [8][9][10][11][12][13][14][15][16]. The agglomeration of crystals is an undesirable occurrence during the synthesis of magnetic nanoparticles. Functionalization and coating of nucleated crystals with ligands can prevent the agglomeration of nanoparticles. Nanoparticles can also have a stronger interaction with the biological cells' surface because of their high surface area to volume ratio compared with bulkier crystals [17]. Some nanoparticles like ZnO, CdSe, TiO 2 , ZnS, and SiO 2 have shown considerable antibacterial/antimicrobial activity, and selective toxicity in biological systems has previously reported [18]. The antibacterial activity of TiO 2 has been attributed mainly to its capability to activate free hydroxyl radicals (OH À ) under the action of sunlight/ultraviolet radiation [19].
Combining Fe 3 O 4 and TiO 2 nanoparticles to achieve the easy recovery and recycling of TiO 2 nanoparticles permits one solution of remediating the contamination of the environment by wastewater. The synthesized nanocomposites could also be easily removed using a magnet after antibacterial application allowing for repeated use of the nanomaterial. The biochemical interaction that occurs between the nanoparticles and the microbes has been attributed to the positive charge on nanoparticles and the negative charge on the outer cell walls of the microbes, which results in the oxidation of microbes and quick death [20]. Cell lysis mechanism also involves the reaction between the ions generated by nanostructures particles and protein on the bacterial cell [21]. The mechanism of antibacterial action by the nanoparticles thus is also through the oxidative stress due to Reactive Oxygen Species (ROS) that are generated by the nanoparticles [22,23]. The singlet oxygen produced could also damage the proteins or the DNA in the bacteria and thus lead to the bacteria's degradation. Kim et al. studied the generation of H 2 O 2 when Fe 2þ reacted with dissolved oxygen. The reaction between the Fe 2þ and H 2 O 2 also creates the • OH radical, which harms the biological macromolecules' structures [24].
Magnetite (Fe 3 O 4 ) nanoparticles were prepared by using a single precursor. Afterwards, the magnetite nanoparticles were coated with titania using titanium butoxide as a precursor. We have explored here the effect of these Fe 3 O 4 @TiO 2 nanostructured composite particles as antibacterial agents on five different pathogen bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Shigella flexneri, Escherichia coli, and Salmonella typhi. A unique attraction for this study was that these nanocomposites can be quickly concentrated, recovered, and recycled using a simple magnetic separation technique, in-vitro and in-vivo.

| Materials
Iron (III) chloride, NH 4 Ac, ethylene glycol (EG), tetracycline, polyvinyl pyrrolidone, and LB agar were all purchased commercially (MERCK). Titanium butoxide was purchased from Sigma Aldrich. Five bacterial species, that is, S. aureus, P. aeruginosa, Shigella flexneri, E. coli, and S. typhi, were acquired locally from the Department of Biotechnology, DCRUST University, Sonepat, India. Following 2 h stirring at room temperature, the ultrasonication was done for 2 h. The suspension was put into an autoclave for hydrothermal action at 200°C for 20 h. The sample was taken apart by a magnet, rinsed with ethanol, and dried at 70°C.

| Antibacterial activities screening using the well diffusion method
250 ml of LB agar was prepared. The media was poured into five Petri plates. Once the medium was solidified, 500 μl of cultures of age 18-24 h were spread on solidified media in all Petri plates. Then uniform holes were made, and 50 mg/ml concentration of nanoparticles was poured into the holes. The enclosed parafilm plates were set aside into the incubator at 37 o C for 24 h. A negative control (ethanol) and positive control (15 μl of 10 mg/ml tetracycline) was used. The zone of inhibition was noted in mm [26]. Figure 1 shows the X-ray diffraction pattern from the magnetite synthesized by the chemical co-precipitation process. These nanoparticles have been investigated using XRD. The XRD chart of magnetite nanostructure particles corresponds to a sequence of peaks at 2θ of 30.  [27].

| Fe 3 O 4 nanoparticles
The highly crystalline structure of the magnetite is shown by the sharp peaks of the XRD graph in Figure 1.

| Fe 3 O 4 @TiO 2 nanoparticles
The XRD graph of these nanocomposites shows peaks at 2θ of 25.3 o , 37.8 o , 48.2 o , that is corresponding to the reflection of (101), (104), and (200) planes of TiO 2 nanostructures particles, respectively [28]. Some peaks of Fe 3 O 4 cores and the intense peaks of anatase form of titania are shown in the XRD graph, confirming the presence of TiO 2 coating on magnetite nanostructured particles (Figure 2), which reduces the relative peak intensity of the magnetite phase in the diffraction pattern.

| Fe 3 O 4 nanoparticles
The FTIR spectrum represents hydroxyl group, which is recognized as absorption at 3429 cm À 1 . The band at 2929 cm À 1 can be attributed to the CH 2 stretching bond. The band at 2365 cm À 1 is due to CO 2 in the atmosphere. In the spectrum, absorption at 590 cm À 1 shows a Fe-O bond. The peak at 1636 cm À 1 is reported as an amino group [29] ( Figure 3).

| Fe 3 O 4 @TiO 2 nanoparticles
The FTIR spectrum showed a band at 3390 cm À 1 , which is recognized as an O-H bond. The band at 2360 cm À 1 is ascribed to the presence of CO 2 in the chamber atmosphere.
The band at 1630 cm À 1 is recognized as NH bending. The band at 660 cm À 1 is assigned to the Ti-O-Ti stretching vibrations [30] (Figure 4). Figure 5 shows the absorption spectrum of magnetite and titania-coated Fe 3 O 4 nanocomposite. When titania nanoparticles were coated on the Fe 3 O 4 nanoparticles, the absorption peak(s) was shifted into the visible region.

| TEM characterization
Both single-phase Fe 3 O 4 nanoparticles and Fe 3 O 4 @TiO 2 coreshell nanostructures, were synthesized using a single precursor by the hydrothermal process. A lot of nanostructured crystalline granules of titania appear to enclose a magnetite core. The titanium dioxide is present as the externally deposited film on the Fe 3 O 4 nanostructure particles, creating the Fe 3 O 4 @TiO 2 nanostructure [31] (Figure 6).

| VSM characterization
The magnetic features of Fe 3 O 4 nanoparticles and Fe 3 O 4 @-TiO 2 nanostructure composites have been studied using the VSM technique (Figure 7).
The saturation magnetization (M s ) of magnetite nanostructure particles and Fe 3 O 4 @TiO 2 nanostructure composites were 74.268 emu/g and 13.755 emu/g, respectively. The saturation magnetization (M s ) reduced because of the non-magnetic TiO 2 outside film on nanoparticles (Table 1).

| Antibacterial activities
TiO 2 -coated magnetite exhibited bactericidal activity in resistance to both gram þve (positive) and gram -ve (negative) bacteria. The antibacterial activity by the core-shell nanocomposite is likely due to the oxidative stress produced by reactive oxygen species (ROS) which include superoxide radical (O À 2 ), hydroxyl radical ( -OH), and also hydrogen peroxide (H 2 O 2 ) (Figure 8). Collin [32] showed how hydrogen peroxide (H 2 O 2 ) and other ROS were generated when Fe 2þ /Fe 3þ reacted with oxygen. The biochemical interaction occurs between H 2 O 2 and membrane proteins or between the substance formed due to magnetite nanoparticles and bacteria's external bilayer. Then H 2 O 2 enters the outer bilayer of bacteria and destroys them [33]. The various oxidation and reduction reactions occur when Fe 3 O 4 @TiO 2 nanocomposites disperse within the media, known as the Fenton reaction in case of the iron ionic species. These reactions generate different reactive oxygen species because of the Fe 3þ and Fe 2þ present in magnetite [34,35]. It has also been reported that Fe 3þ doping of TiO 2 reduces agglomeration resulting in high photocatalytic efficiency and a reduced bandgap of about 2.6 eV [36]. Several oxidation-reduction and photocatalytic reactions can occur simultaneously in this combination: OH • and HO 2 • produced in the reaction are reactive free radicals. Magnetite (Fe 3 O 4 ) NPs can slowly be oxidized to maghemite (γ-Fe 2 O 3 ). This oxidation is a critical part of the origin of oxidative stress to the cell of bacteria, resulting in the bacterial cell's death. Table 2

| Antibacterial index of magnetite nanoparticles
The antibacterial index of magnetite nanostructures particles and Fe 3 O 4 @TiO 2 nanocomposite was shown in Figure 10. The Fe 3 O 4 @TiO 2 nanocomposite exhibited a better bactericidal activity for E. coli as compared with other bacterial strains.
The magnetite nanoparticles, as core materials, showed good bactericidal activity. The maximum antibacterial activity was observed for TiO 2 -coated nanoparticles because of its inherent antibacterial properties [37]. The core-shell Fe 3 O 4 @TiO 2 nanocomposite showed a better antibacterial effect than Fe 3 O 4 nanoparticles. The activity of Fe 3 O 4 nanoparticles to inactivate bacterial strains was improved significantly after coating with TiO 2 .
The core-shell Fe 3 O 4 @TiO 2 nanocomposite could be used for drug delivery because of the inherent antibacterial property of TiO 2 and the magnetic property of magnetite nanoparticles. The ability to kill cancer cells has also been previously studied [38,39]. The TiO 2 nanoparticles have the capability to both oxidize the pollutants and kill the microorganisms [40]. The antibacterial effect of core-shell Fe 3 O 4 @TiO 2 nanocomposite was studied, which combined the treatment of microbial contamination and magnetic separation property of magnetite, synthesized. The antibacterial activity of TiO 2 nanoparticles using as a shell was better than that reported in previous studies [41,42].

| CONCLUSION
Magnetite superparamagnetic nanostructures particles were fabricated using a single precursor by the hydrothermal method and by using a reducing agent. The Fe 3 O 4 nanostructured particles were coated with titania using a separate hydrothermal process. The effect of the TiO 2 shell in the coreshell Fe 3 O 4 @TiO 2 nanocomposite as a broad-spectrum antibacterial agent was investigated, in comparison to the similar effect of the uncoated core magnetite itself. The antibacterial effects of magnetite core and the titania shell were studied on five bacteria strains: S. aureus, P. aeruginosa, Shigella flexneri, E. coli, and S. typhi. The Fe 3 O 4 @TiO 2 nanocomposites showed superior antibacterial action on each bacterium, while the uncoated magnetite's effect was significantly lesser in comparison. The TiO 2 -coated magnetite nanoparticles revealed better and more effective antibacterial activity against the E. coli strain of bacteria than the other strains. It can be concluded that Fe 3 O 4 @TiO 2 is a very active antibacterial agent, as verified by the large diameter of the inhibition zone. The results indicated that nanocomposites synthesized in this work could also be suitable materials for drug delivery applications. These can also be practical and cost-effective agents in cleaning a microbe-polluted water environment where the magnetic core lends itself to easy recycling. The results reveal that bacterial illness could be treated by core-shell Fe 3 O 4 @-TiO 2 nanostructure particles employing TiO 2 as a shell due to its inherent antibacterial properties. Fe 3 O 4 @TiO 2 nanocomposites could be efficient and recyclable antibacterial agents because of their magnetic, anti-bacterial and photocatalytic features.