Fabrication of New Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n Nanohybrid Ferrogels for Antibacterial Applications

Revised: October 17, 2020; Accepted: December 27, 2020 New Fe 3 O 4 /polyvinyl alcohol (PVA)/sodium alginate (C 6 H 7 O 6 Na) n nanohybrid ferrogels for antibacterial applications were fabricated. The crystal and molecular structures along with optical and magnetic properties of the prepared samples were characterized. The antibacterial activity of the ferrogels against Bacillus subtilis and Escherichia coli was investigated using the agar dilution method. X-ray diffraction analysis showed that the Fe 3 O 4 /PVA comprised a PVA amorphous phase and a spinel-structured Fe 3 O 4 crystalline phase. The Fe 3 O 4 /PVA crystallite size was 7.5–9.9 nm and the scanning electron micrographs showed that the Fe 3 O 4 /PVA agglomerated. The ferrogels were superparamagnetic with saturation magnetizations from 14.8 × 10 −3 to 82.1 × 10 −3 emu/g. The absorption of the ferrogels showed a bathochromic effect, accompanied by an increase in the bandgap from 2.09 to 2.18 eV with increasing Fe 3 O 4 content in the ferrogels. The ferrogels demonstrated new potency as antibacterial agents against B. subtilis and E. coli , where their antibacterial performance


Introduction
Over the past few decades, researchers have widely developed methods of preparing Fe 3 O 4 nanoparticles (NPs) in various shapes and sizes and in the forms of powders, films, ferrofluids, and ferrogels. In particular, intensive research on Fe 3 O 4 NPs in the form of ferrogels has been motivated by their advantageous properties compared with those of other forms, even ferrofluids. Theoretically, Fe 3 O 4 ferrogels, which are gels with a cross-linked polymer network containing Fe 3 O 4 NPs as a filler, offer specific advantages over conventional gels with respect to their high flexibility and high sensitivity to an external magnetic field, which implies that ferrogels can be easily positioned inside a living organism 1 .
Fe 3 O 4 NPs exhibit superior advantageous properties, including superparamagnetism, biocompatibility, and low toxicity. They are also inexpensive and easily synthesized 2 , making such materials attractive in the biomedical field 3 , for example, as antibacterial agents for skin infections. In general, skin infections occur and often pose therapeutic challenges because of increasing concerns about multidrug-resistant bacteria 4 . Furthermore, gel-based skin antibacterial agents are particularly attractive because gels have advantages over powders and liquids. However, the Fe 3 O 4 -based antibacterial agents developed thus far have been based on powders. Therefore, the development of Fe 3 O 4 ferrogels as antibacterial agents is strongly desired. Fe 3 O 4 ferrogels based on polyvinyl alcohol (PVA) have recently begun to attract researchers' attention for possible use in various multipurpose applications [5][6][7] . Recent research has shown that Fe 3 O 4 ferrogels with a PVA matrix have excellent prospects for use in biomedical applications 8 . However, for skin antibacterial applications, PVA has a drawback because of its relatively high stiffness, which reduces antibacterial efficacy. Consequently, an appropriate biocompatible crosslinking agent that reduces the stiffness of Fe 3 O 4 /PVA ferrogels is urgently needed.
Sodium alginate, with chemical formula (C 6 H 7 O 6 Na) n , exhibits strong potential for use as a crosslinking agent that supports the antimicrobial performance of Fe 3 O 4 /PVA ferrogels. Theoretically, in addition to reducing the stiffness of PVA 9 , (C 6 H 7 O 6 Na) n offers other advantages of nontoxicity, biocompatibility 10 , bioresorption ability, biodegradability, good mechanical properties, and relatively low cost 11,12 . Therefore, in the present work, we developed new Fe 3 O 4 / PVA/(C 6 H 7 O 6 Na) n nanohybrid ferrogels as antibacterial agents and investigated their crystal and molecular structures, optical properties, bandgaps, magnetic behaviors, and antibacterial efficacy. *e-mail: ahmad.taufiq.fmipa@um.ac.id

Materials
The main precursors were FeCl 2 ·6H 2 O, FeCl 3 ·6H 2 O, HCl (12 M), NH 4 OH (6.5 M), PVA (M w ≈ 60,000), and (C 6 H 7 O 6 Na) n . All precursors were analytical grade to ensure high purity and quality of the samples. All precursors were purchased from Merck and were used without further purification.

Fabrication of Fe 3 O 4 /PVA Nanocomposites
The Fe 3 O 4 NPs were prepared using a coprecipitation method following the procedure described in our previous work 13 . One g of PVA was dissolved in 20 mL of distilled water at 120 °C for 3 h using a magnetic stirrer with a speed of 720 rpm. Various masses of Fe 3 O 4 (0.13, 0.26, 0.39, 0.52, and 0.65 g) were mixed with 2 mL of PVA solution at 120 °C for 30 min using a magnetic stirrer with a speed of 720 rpm to produce Fe 3 O 4 /PVA. The Fe 3 O 4 /PVA was collected after the mixture cooled to room temperature. The samples were labeled S1-S5 according to the respective masses of Fe 3 O 4 (0.13, 0.26, 0.39, 0.52, and 0.65 g).

Fabrication of Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n Ferrogels
The Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n nanohybrid ferrogels were prepared through a sonochemical method. Three hundred mg of (C 6 H 7 O 6 Na) n was dissolved in 20 mL of distilled water at 120 °C under stirring using a magnetic stirrer with a speed of 720 rpm for 4 h, followed by a cooling process to room temperature. Next, 2 mL of (C 6 H 7 O 6 Na) n solution was reacted with the prepared Fe 3 O 4 /PVA (S1-S5 samples) at 120 °C for 30 min under stirring using a magnetic stirrer with a speed of 720 rpm. After the mixtures cooled to room temperature, the final products were obtained as solid-black gels in the form of Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n nanohybrid ferrogels. The prepared Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n nanohybrid ferrogels were labeled SF1-SF5 according to the respective S1-S5 samples. The schematic showing the construction of the Fe 3 O 4 /PVA/ (C 6 H 7 O 6 Na) n nanohybrid ferrogels is shown in Figure 1.

Characterization
The structural, optical, and magnetic properties were characterized by X-ray diffraction (XRD) for S1-S5 samples, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX) for S1-S5 samples, UV-Vis spectrophotometry for SF1-SF5 samples, Fourier transform infrared (FTIR) spectroscopy for SF1-SF5 samples, and vibrating sample magnetometry for SF1-SF5 samples. The antimicrobial performance of the SF1-SF5 samples against Bacillus subtilis (Gram-positive) and Escherichia coli (Gramnegative) was evaluated through the agar diffusion method. All characterizations were conducted at ambient temperature.

Results and Discussion
The XRD patterns of the Fe 3 O 4 /PVA used as the filler of the ferrogels ( Figure 2) show that two phases were formed, i.e., amorphous and crystalline phases. The amorphous phase, with a broad diffraction peak detected at 2θ of approximately 19.5°, was attributed to PVA. This is consistent with the results of the previous work 14  In this crystal structure, the Fe 2+ and Fe 3+ ions are distributed in octahedral and tetrahedral sites. The distribution of these metallic ions determines the magnetic moments of Fe 3 O 4 , where the magnetic moments between ions in octahedral and tetrahedral sites are anti-parallel. Furthermore, the crystallite size (D) of the Fe 3 O 4 of all samples was calculated using the Debye-Scherrer equation: D = 0.9λ/βcosθ, where λ, β, and θ are the wavelength of the X-rays, the full-width at half-maximum of the peak, and the diffraction angle, respectively 16 . The data analysis results in Table 1 show that D increased with increasing Fe 3 O 4 content. The increasing Fe 3 O 4 content tended to increase the extent of aggregation into clusters because of the concomitant increase in the   Figure 4. The Fe-O stretching band originating from Fe 3 O 4 is observed at 420-500 cm −1 for the tetragonal sites and at 700 cm −1 for the octahedral sites. Furthermore, stretching bands characteristic of PVA were detected at 857 cm −1 (CC stretching) 19 , 1047-1147 cm −1 (C-O) 20 , 1350 cm −1 (=C-O-C), and 2966 cm −1 (CH 2 asymmetric stretching) 21 . The stretching bands characteristic of (C 6 H 7 O 6 Na) n were detected in the range 1473-1560 cm −1 (COO symmetric and asymmetric) 22 and at 1708 cm −1 (C=O) 23 . Interestingly, the Fe 3 O 4 , PVA, and (C 6 H 7 O 6 Na) n , as the main components of the ferrogels, all contributed to the broad O-H band between 3305 and 3670 cm −1 .
The contribution of Fe 3 O 4 , PVA, and (C 6 H 7 O 6 Na) n to the functional bonding observed by the FTIR spectra initiates further discussion. The changes in intensities of chemical bands provide a clue on the mechanical reorientation of the molecules 24 . The decreasing intensities of O-H band, CH 2 asymmetric stretching, =C-O-C, and C-O bonding is believed due to the decrease of PVA content. This trend is related to the increase of Fe 3 O 4 and the presence of alginate. From the theoretical viewpoint, the presence of alginate reduces the stiffness of PVA-based system by virtue of its low elongation break 25 . In other words, the long-chain structure of PVA in Fe 3 O 4 /PVA nanocomposites was destroyed by the addition of alginate. Zhang et al. claimed that one of the best ways to reduce the stiffness of fabric-reinforced PVA was by introducing Na-alginate 26 . They discovered that mechanical strength and stiffness could be decreased due to the swelling behavior of composite hydrogels that diminish friction amongst interfibers 26 . Furthermore, alginate is not only effective in reducing the stiffness of PVA-fibers, but also other polymers, such as poly-lactic-co-glycolic acid 27 .
The absorbance spectra, as shown in Figure 5, were recorded to investigate the optical properties of the Fe 3 O 4 / PVA/(C 6 H 7 O 6 Na) n ferrogels. The peaks at 474 and 511 nm are consistent with the previous work 28 . The absorbance intensity is associated with the number of particles present. Meanwhile, the wavelength of maximum absorption is associated with the particle size. Physically, these peaks represent the difference in energy levels between the σ and π* states 29 . In addition, incorporating the Fe 3 O 4 into ferrogels would increase the distribution of particles embedded in the (C 6 H 7 O 6 Na) n crosslinking agent. As a consequence, the conduction electrons on the surface of the Fe 3 O 4 migrate from one atom to neighboring atoms so that the surface plasmon resonance shifts toward lower energies, which is associated with smaller wavelengths 30 . In physics, this phenomenon is described as the bathochromic effect. The bandgap (E g ) of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n was calculated using the equation αhυ = A(hυ − E g ) 1/2 , where α, h, υ, and A are the absorption coefficient, Planck's constant, the frequency, and a constant, respectively 31 . The linear part of the curve was extrapolated to the intersection with the energy axis to determine the bandgap 32 . The data analysis is shown in Figure 6. The E g of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels  . This difference is attributed to the effect of the surface modification of Fe 3 O 4 using PVA, which led to dispersion of the Fe 3 O 4 in (C 6 H 7 O 6 Na) n to form nanohybrid ferrogels.
The magnetization curves of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels are depicted in Figure 7. The figure shows that all of the samples are likely to exhibit superparamagnetic character, as indicated by the characteristic S-like shape with the coercivity field close to zero 34 . A quantitative analysis was carried out to fit the magnetization curves using the Langevin equation: where M, M s , μ, H, k B , and T are the magnetization, saturation magnetization, magnetic moment, Boltzmann constant, and temperature, respectively. The magnetic parameters obtained from the data analysis are presented in Table 3. The magnetic moment of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels increased by the addition of Fe 3 O 4 content. The magnetic moment plays an essential role in a material's response to an external magnetic field. Interestingly, not only the increase in Fe 3 O 4 content but also the increase in particle size contributed to Figure 6. Bandgaps of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels. . Furthermore, the coercivity field also increased with increasing Fe 3 O 4 content, which is attributable to the accompanying increase in particle size. Previous work has shown the same trend for Fe 3 O 4 /PVA with a ribbon-like structure 36 .
The antibacterial performance of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels against B. subtilis and E. coli is presented in Figure 8. This figure shows that the inhibition-zone diameter increases with increasing Fe 3 O 4 content. The inhibition-zone is associated with the antibacterial performance of the Fe 3 O 4 / PVA/(C 6 H 7 O 6 Na) n ferrogels. Theoretically, the ability to destroy bacteria is strongly correlated with the ability of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n to generate reactive oxygen species (ROS) 37 . ROS generate superoxide as a free radical that inhibits bacterial growth 38 . Furthermore, in the case of Fe 3 O 4 NPs incorporated into ferrogels, the inhibition-zone diameter was larger than that of pure Fe 3 O 4 . The enhanced bactericidal activity of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels is attributed to the better dispersion of the Fe 3 O 4 particles in the ferrogels than in the pure Fe 3 O 4 , which tends to aggregate. In addition to ROS, particle size also plays an important role in bactericidal activity. Other authors have shown that Fe 3 O 4 with small particle size and monodisperse shape exhibits better inhibitory activity against bacteria than Fe 3 O 4 with large particle size and polydisperse shape 39 . Theoretically, Fe 3 O 4 NPs with an excellent particle size distribution are likely to penetrate more easily into the walls or pores of bacteria. As a comparison, Jalali et al. prepared Fe 3 O 4 NPs by doping Co and Zn to strengthen their antibacterial potential; however, they observed an inhibition-zone diameter of only 6.4 mm for E. coli 40 . In line with that, Salem and co-workers fabricated Fe 3 O 4 NPs from marine algae; however, the inhibition zone diameter for E. coli was still approximately 6 mm and that for B. subtilis was approximately 5 mm. These values are still inferior compared with that of the ferrogels investigated in the present study.   The bactericidal mechanism of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels is presented in Figure 9. From a physics perspective, the negative surface charges on the Fe 3 O 4 /PVA nanocomposite particles repel the bacteria under electrostatic interaction mechanism. This repulsion makes the metal oxide NPs less effective in destroying bacteria. However, when Fe 3 O 4 /PVA are dispersed in (C 6 H 7 O 6 Na) n , the net charge at the surface of the particles becomes positive. This change in surface charge is beneficial in terms of catching the bacteria by Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels. Thus, the chance of ROS penetrating into the bacteria increases. This leads to increase in the diameter of the inhibition zone. Thus, the Fe 3 O 4 / PVA/(C 6 H 7 O 6 Na) n nanohybrid ferrogels exhibit superior antibacterial activity. Furthermore, because of the structural complexity of the Fe 3 O 4 ferrogels, a theoretical explanation of the performance of the ferrogels related to their magneticsensitive behavior has not yet been established. Therefore, for the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n ferrogels to be used on a large scale, further investigation is necessary, especially with respect to the optimum filler, matrix, cross-linker contents in the ferrogels.

Conclusion
We synthesized new Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n nanohybrid ferrogels using a combination of coprecipitation and sonochemical methods. The XRD data indicated the presence of two phases: amorphous PVA and crystalline Fe 3 O 4 . The crystallite size and crystallinity increased with the addition of Fe 3 O 4 . The surface of the Fe 3 O 4 /PVA/(C 6 H 7 O 6 Na) n agglomerated because of the increase in Fe 3 O 4 content, preventing PVA from covering it optimally. The nanohybrid ferrogels exhibited superparamagnetism, where the coercivity field and magnetization saturation increased with increasing Fe 3 O 4 content. Remarkably, the antibacterial activity of the ferrogels was observed to increase with increasing Fe 3 O 4 content, which was attributed to an increase in ROS penetrating the bacteria.

Acknowledgments
This work was financially supported by KEMENRISTEKDIKTI 2019 for AT.