Synthesis of Ternary Fe3O4/ZnO/ChitosanMagnetic Nanoparticles via an Ultrasound-Assisted Coprecipitation Process for Antibacterial Applications

Institute of Chemistry and Materials, 17 Hoang Sam, Cau Giay, Hanoi, Vietnam Institute of Fundamental and Applied Sciences, Duy Tan University, Ho Chi Minh City 700000, Vietnam Faculty of Natural Science, Duy Tan University, Danang City 550000, Vietnam Ministry of Public Security, 80 Tran Quoc Hoan, Cau Giay, Hanoi City 100000, Vietnam Laboratory of Advanced Materials Chemistry, Advanced Institute of Materials Science, Ton Duc Thang University, Ho Chi Minh City 758307, Vietnam Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City 758307, Vietnam


Introduction
Recently, iron oxide (Fe 3 O 4 ) nanoparticles (ION) have attracted the attention of scientists all over the world due to its various applications, particularly in biomaterial engineering, such as MRI contrast agent, hyperthermia treatment, and drug delivery [1][2][3][4][5]. ION have been synthesized from many routes, including coprecipitation [6,7], solvothermal method [8,9], sol-gel method [10], microwave [11], …. Particularly, ION coated with an organic, inorganic, or polymer layer have been proved not only to prevent the aggregation of particles but also to improve the drug loading capability, solubility, biocompatibility, and selectivity of ION [12,13]. Vu et al. successfully synthesized spherical PEG and citric acid-coated ION with a diameter of 10-15 nm and high saturation magnetization value of 70.52 emu/g [14].
Chitosan, a nontoxic biopolymer structured from glucosamines, has been used as a potential surface-protecting layer for ION in recent years. In addition to its biocompatibility, biodegradability, and adsorption ability, chitosan with amine groups has also shown the ability to control drug release, improve drug diffusion, and enhance drug permeation [15]. Moreover, chitosan has emerged as an antibacterial and antiviral agent due to its positive charge which helps it adhere to negatively charged surfaces and interact with polyanions to form a gel structure [16]. Like chitosan, utilization of ZnO nanoparticles in the controlled drug release and tumour cell destruction has been also reported [17]. ZnO is an n-type semiconductor with a wide band gap (3.1-3.3 eV) and high activated energy (60 meV) at room temperature, which has led to a variety of unique optical and thermal properties of ZnO nanoparticles [18,19]. The combination of chitosan and inorganic nanoparticles like ZnO would form antibacterial materials with multifunctional properties [20]. Yuan et al. reported the synthesis of chitosan-ZnO by a chemical degradation method for targeted drug delivery [21]. The chitosan-ZnO complex was proved to be a more effective antibacterial agent than chitosan [22,23]. Moreover, the combination of ZnO and other polymers like chitosan can prevent the intrinsic aggregation of ZnO nanoparticles due to their small size [24].
A nonconventional ultrasonic method has been known as an effective and environmentally friendly method for chemical modifications of polymers which have a variety of applications. For instance, Barreto et al. reported the fabrication of a chitosan/ZnO complex under high-intensity ultrasonic radiation. The hybrid showed the highest antibacterial activity against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria [25]. The ultrasonicassisted method also exhibited its effectiveness in the preparation of ternary chitosan/ZnO/TiO 2 nanocomposite for crystal violet (CV) dye adsorption reported by Bhanvasea et al. [26]. Similarly, the work performed by Vardikar et al. revealed that the kaolin-chitosan-TiO 2 ternary nanocomposites synthesized by a sonochemical route had many advantages for dye adsorption over those prepared by a conventional method [27]. To the best of our knowledge, there is a lack of reports on the synthesis of ternary Fe 3 O 4 /Z-nO/chitosan nanoparticles via a coprecipitation ultrasonication method for antibacterial and antifungal application.
Therefore, in this research, we synthesized Fe 3 O 4 /ZnO/chitosan nanoparticles using a coprecipitation-ultrasonication method. ZnO nanoparticles were synthesized using rambutan peel extraction and zinc nitrate hexahydrate. The effects of chitosan concentration, magnetic properties, and antibacterial activity were also investigated. ZnO nanoparticles were prepared according to a previous rambutan (Nephelium lappaceum L.) peel extraction method [24,28]. Rambutan peels were washed with water, cut into small pieces, and subsequently placed in a circulating oven at 60°C until completely dried. 3 g of the dried rambutan peels was boiled with a mixture of ethanol and distilled water (1 : 2, v/v) for 30 min during the extraction. The extract was passed through a Whatman No.1 filter and stored in a refrigerator for further use. 0.1 M zinc nitrate hexahydrate was prepared in 50 ml deionized water, and then, 50 ml of rambutan peel extract (Vietnam) was slowly added dropwise into the solution under magnetic stirring at room temperature, and the solution was sonicated for 1 h in an ultrasonic bath (500 W, 20 kHz) to obtain a zinc-ellagate complex. The zinc-ellagate complex was collected by centrifugation at 7000 rpm for 20 min. Then, the solid was washed with distilled water, dried in an oven at 40°C for 8 h, and calcinated in a muffle furnace at 450°C to obtain ZnO nanoparticles.

Preparation of Fe 3 O 4 /ZnO/Chitosan.
For the synthesis of Fe 3 O 4 /ZnO/chitosan, PEG-Fe 3 O 4 and ZnO (1 : 1, w/w) were firstly dissolved in 100 ml of deionized water. The solution was mixed homogeneously using a magnetic stirrer at temperature for 10 min. Then, the reaction mixture was placed in a sonicator (VCX500; 500 W, 20 kHz) for 30 min. Meanwhile, chitosan was added to 30 ml of 1% aqueous acetic acid and agitated for 30 min to obtain a chitosan solution. The two obtained solutions were mixed together to form a white emulsion. In the preparation of Fe 3 O 4 /ZnO/chitosan magnetic nanoparticles, the emulsion was dropped into the mixture homogenous (PEG-Fe 3 O 4 /ZnO) using a sonicator (VCX500; 500 W, 20 kHz) for 30 min. The Fe 3 O 4 /ZnO/chitosan was separated under an external magnetic field and washed two times with deionized water, then finally dried in a vacuum oven at 60°C. The effect of different mass ratios of CS and PEG-Fe 3 O 4 /ZnO on the magnetic properties of Fe 3 O 4 was investigated by varying different mass percentages of chitosan (5%, 10%, 20%, and 30%). Fe 3 O 4 /ZnO/chitosan samples with different amounts of chitosan were denoted as Fe 3 O 4 /ZnO/chitosan 5%, Fe 3 O 4 /ZnO/chitosan 10%, Fe 3 O 4 /Z-nO/chitosan 20%, and Fe 3 O 4 /ZnO/chitosan 30%. Figure 1 shows the schematic of the preparation of Fe 3 O 4 /ZnO/chitosan magnetic nanoparticles.

2.4.
Characterizations. X-ray diffraction patterns were recorded on a P'Pret Pro-PANalytical X-ray diffractometer operated at 1.8 kW (40 mA/45 kV) using CuKα (λ = 1:5406 Å) radiation. FT-IR spectra were recorded on a Bruker FT-IR spectrometer using the KBr pellet method. FESEM was carried out using a Hitachi S-4800 microscope. To prepare FESEM samples, a small amount of the solid sample was dispersed in ethanol and small drops of the solution were placed on an aluminum grid. The grid was dried for 1-2 h in a vacuum oven at 40°C prior to the FESEM measurement.

2
Journal of Nanomaterials Magnetic measurements of the solid samples were performed at room temperature (25°C) using a Magnet B-10 Vibrating Sample Magnetometer (VSM). The surface morphology of the sample was imaged by transmission electron microscopy (TEM-JEM1010). TEM images were acquired at an operating voltage of 80 kV. The size and ζ-potential of the sample were recorded on a Malvern Zetasizer Nano Z instrument (Malvern Instruments, Ltd., UK). Nanoparticle dispersions were prepared in D.I. water at 37°C with a concentration of 1 mg/ml.

Antibacterial Activity
Testing. The antibacterial activity of Fe 3 O 4 /ZnO/chitosan was tested against both grampositive Bacillus subtilis and Saccharomyces cerevisiae and gram-negative (E. coli ATCC 11632) bacteria using a disk diffusion method. The enrichment medium containing meat extract, yeast extract, peptone, glucose, and some mineral salts was used for microbiological cultivation. For comparison, chitosan and positive control disks were also involved in the test. The antibacterial and antifungal activities were evaluated by measuring the zone of inhibition against the tested organisms. The antibacterial activity experiments were conducted as follows: First, the petri dishes containing appropriate media for the growth of bacteria were prepared. Then, two aforementioned testing materials were sprinkled on these dishes to be evaluated by their mortality in the cultured medium, followed by the inoculation of the suspended solutions obtained from each of the three bacterial strains on the petri dishes with appropriate enrichment media.
The control sample was prepared as follows: the testing materials were sprinkled on petri dishes with the inoculated      Journal of Nanomaterials suspensions of microorganisms. Certain amounts of the testing materials were placed in the Eppendorf tubes prior to the aspiration of the bacterial suspensions into the respective tubes. All samples were then well shaken for 20 min. Afterwards, a small quantity of each sample was aspirated and placed on petri dishes containing appropriate media for evaluating the antibacterial activity of the testing materials. All samples were incubated in a container at 37°C for 24 to 72 h. ZnO nanoparticles were synthesized using rambutan peel extract which contains phenolic antioxidants. A zinc-ellagate complex was formed after 1 h sonication of the solution containing rambutan peel extract and zinc nitrate hexahydrate. It was ascribed to the formation of bonding between hydroxyl groups of phenolic compounds and the zinc metal as a metal phenolate complex by the chelating effect in which the ester oxygen atoms and phenolic hydroxyl groups of phenolic compounds form p-track conjugation effect [28]. The decomposition of the zinc-ellagate complex at 450°C led to formation of ZnO nanoparticles [28]. Then, ZnO nanoparticles were attached to the PEG-Fe 3 O 4 via hydrogen bondings between PEG-Fe 3 O 4 and ZnO and externally coated by the CS as shown in Figure 1. The incorporation of ZnO into the material system was aimed at creating a composite of the chitosan-based system to increase the antibacterial ability against gram-negative bacteria. The products were further characterized by XRD, FT-IR, and VSM.  (1)) was used to calculate crystallite size:

Results and Discussion
where d is the mean crystallite size (nm), k is the crystallite shape constant (approximately taken as 0.89), λ is the X-   Journal of Nanomaterials ray wavelength (nm), β is the full width at half maximum height of the X-ray diffraction peak, and θ is the Bragg angle (degree). * The distance between atomic layers of a crystal was calculated by Bragg's law: where n is any integer; d hkl is the distance between atomic layers of a crystal (Å); θ hkl is the incident angle, the angle between the incident rays, and the scatter plane (degree); and λ is the wavelength of the incident X-ray beam (nm). Based on the function of Miller Indices and lattice parameters, we can determine the formula for interplanar spacing of Fe 3 O 4 cubic crystals with a = b = c; α = β = γ = 90°as follows: Meanwhile, the formula for interplanar spacing of ZnO hexagonal crystals with a = b, c; α = β = 90°, γ = 120°can be determined as follows:  The FT-IR spectra of all samples showed absorption peaks at around 3440 cm −1 and 1634 cm −1 which are related to the OH groups or NH 2 groups. Additionally, the CS (1) and PEG-Fe 3 O 4 /ZnO/CS (3) spectra exhibited the characteristic IR peaks of β (1)(2)(3)(4) glycosidic bands in the polysaccharide unit at 1110 cm -1 and 1065 cm -1 indicated the stretching vibration of C-O-C in glucose circle [28]. However, these peaks were absent from PEG-Fe 3 O 4 /ZnO (2) spectra. Instead, it showed strong absorption in the fingerprint range at 564 cm -1 , indicating the presence of Fe-O bond in the Fe 3 O 4 -PEG sample [13]. The Fe-O-C bond interaction identified by the absorption around 1100 cm -1 indicated that Fe 3 O 4 was successfully coated with PEG [30]. The FT-IR spectra of PEG-Fe 3 O 4 /ZnO/CS (3) showed absorption peaks at around 431 cm −1 which are the stretching vibrations of N-Zn and Zn-O [32]. As compared to the spectra of (2), the bands at 1100 cm -1 and 564 cm -1 in the (3) sample have a reduced intensity as a result of the external coating of the CS gel on the PEG-Fe 3 O 4 /ZnO core, which is shown in Figure 1.
Magnetic measurements were conducted using a vibrating sample magnetometer (Figure 4). Magnetization curves were recorded at room temperature, and parameters such as coercive field (Hc) and initial susceptibility (χi) were obtained. The saturation magnetization (Ms) was obtained by extrapolation to the infinite field of the experimental data obtained in the high-field range where the magnetization varied linearly with the inverse of the applied field. Saturation magnetization values were normalized by taking into account the percentage of CS contained in the samples.
The saturation magnetization of bare Fe 3 O 4 nanoparticles was reported as 68.9 emu/g [4]. After coating Fe 3 O 4 with PEG, the saturation magnetization of PEG-Fe 3 O 4 was determined as 65.71 emu/g [2]. The saturation magnetization of  3.2. Antibacterial Activity. The survivability of microorganisms (fungi and bacteria) after contacting with testing materials is shown in Figure 6. It could be clearly observed that all two petri dishes showed no signs of bacterial or fungal growth on the nutrient medium surfaces when certain amounts of testing samples were sprinkled on the nutrient media and incubated in suitable conditions for 72 h (Figures 6(c)-6(h)). The results indicated that Fe 3 O 4 /ZnO/chitosan reached its best capability to inhibit the growth of Bacillus subtilis, Saccharomyces cerevisiae, and E.coli. Also, clear zones (nonbacterial zones) were established at the sprinkled points of Fe 3 O 4 /ZnO/chitosan on the Bacillus   subtilis and Saccharomyces cerevisiae inoculated petri dishes. Moreover, the suspensions were evenly spread on the controlled dishes without testing materials. Growth of microorganisms to a smooth and thick layer on the dish surfaces was observed (Figures 6(i), 6(l), and 6(o)). However, in the presence of Fe 3 O 4 /ZnO/chitosan, the cellular density of Bacillus, Saccharomyces cerevisiae, and E. coli was significantly decreased. Therefore, it can be concluded that

Conclusions
We have successfully prepared Fe 3 O 4 /ZnO/chitosan magnetic nanoparticles using the ultrasound-assisted coprecipitation method. Based on the XRD data of the samples  7 Journal of Nanomaterials obtained with the variation of CS content, it can be concluded that the crystallite size of Fe 3 O 4 nanoparticles and their lattice parameters were almost no different from the PEG-Fe 3 O 4 sample while the crystal size of ZnO increased as compared to the ZnO substance sample. The modified surface of nanoparticles resulted in the softening of magnetization. The results from studying the survivability when in contact with testing materials proved the ability of Fe 3 O 4 /Z-nO/chitosan to inhibit the growth of a large number of microorganisms. Fe 3 O 4 /ZnO/chitosan possessed the ability to eliminate not only bacterial cells but also fungal (mold) cells on the material surfaces and worked well with the bacterial Bacillus subtilis strains.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.