Studying of the Antibacterial Properties of Ferric Ions Doped Zinc Oxide Nanoparticles

INTRODUCTION
Nanoparticles (NPs) possess distinctive surface characteristics that render them optimal substitutes for existing materials and technologies in various lifestyle and biomedical applications. Recent studies in nanotechnology have recognized it as an effective instrument for numerous prevalent applications [1]. Due to the diverse range of pathogenic bacteria and their ability to acquire resistance, antibacterial medications are presently a big issue in the biomedical and healthcare industries. Here, a handful of NPs-dubbed “Nanoantibiotics”- have shown enhanced antibacterial action [2,3].
Zinc, iron, copper, and manganese are present in soil and cellular constituents in trace amounts. Consequently, the oxides of these metals are anticipated to offer superior biocompatibility at a reduced cost. Zinc oxide nanoparticles (ZnO NPs) are among the most extensively researched antibacterial agents due to their resistance to many antibiotics [4]. The Minimum Inhibitory Concentration (MIC) of ZnO nanoparticles is superior against many pathogenic strains when compared to other nanoparticles, including silver and copper oxide [5]. The primary antibacterial mechanism of these nanoparticles was the generation of Reactive Oxygen Species (ROS) and the induction of geno-toxicity. Reactive oxygen species (ROS) are unsuitable for medical applications as data indicates that even minimal amounts can harm human cells [6,7]. The antibacterial properties of ZnO have been demonstrated through (i) nanoscale reactive oxygen species (ROS) generation [8], (ii) the liberation of antibacterial Zn2+ ions [9], and (iii) direct engagement with the lipopolysaccharide of cell membranes [10], rendering it a more suitable nanoparticle in this context. Despite worries about its potential harm to human cells, zinc oxide has shown effective against specific types of superbugs [11].
Therefore, ZnO nanoparticles’ potential medical uses are still under active research. It is imperative to lower the MIC values of ZnO Nano antibiotic and to establish a ROS-independent antibacterial mechanism for the effective therapeutic utilization of ZnO. Multiple research confirmed that the antibacterial efficacy of ZnO is contingent upon its size and morphology; hence, modifying the dimensions of ZnO crystals is anticipated to reduce MIC values [12,13]. Recent research from our group indicates that doping transition metal ions impacted the lattice properties, hence altering the mechanism and antibacterial activity of metal ion-based Nano antibiotics [14]. Rather than experiencing cell wall collapse, the bacterial cells were rendered inert. The antibacterial processes are influenced by the types and concentrations of dopants [15].
Furthermore, it is anticipated that metal ions with inherent antibacterial characteristics will synergistically augment the antibacterial efficacy of ZnO nanoparticles. An example is the enhanced antibacterial efficacy of iron-based compounds against diverse illnesses [16]. Iron-based nanoparticles exhibit a more rapid disintegration rate attributable to their magnetic characteristics, hence improving target efficiency for localized antibacterial activity [17]. Furthermore, Zn2+ possesses a larger ionic radius (0.074 nm) compared to Fe3+ (0.06 nm), suggesting it is likely to modify the structural defects of the ZnO lattice to improve its antibacterial efficacy without inducing cytotoxicity [18,19].
Consequently, we synthesized Fe(III) doped ZnO nanoparticles (Fe-ZnO NPs) and examined their antibacterial efficacy and mode of action. The production of bimetallic Fe-ZnO nanoparticles was validated by various physical characterization techniques, including EDX and XRD. SEM was utilized to analyze the physical and chemical properties of Fe-ZnO, focusing on its morphology and structure. Two clinically relevant bacterial species, Gram positive (S. aureus) and Gram negative (K. pneumoniae), were tested to determine the antibacterial activity of the suggested nanoparticles. The MIC values were subsequently calculated. This study is beneficial for understanding the antibacterial activity of Fe-ZnO under standard settings.

MATERIALS AND METHODS
Materials and characterization 
The primary materials utilised for the synthesis of Fe-ZnO nanoparticles were potassium hydroxide (KOH), ferric chloride (FeCl3), and zinc acetate dihydrate (C4H10O6Zn). Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) was among the fluorescent dyes employed to elucidate the antibacterial mechanism.
At ambient temperature, with parameters of 40 kV and 40 mA, employing Cu Kα radiation (d = 1.54 Å), the Bruker D8 X-ray diffraction AXS apparatus was used to acquire the XRD patterns of the nanoparticles. The surface morphology of the materials was investigated using an EI Quanta 400F SEM coupled with an Oxford-Instruments INCA 400 X-Max detector for EDX at 300 magnifications (spot size 1 mm x 1 mm) and an accelerating voltage of 20 kV.

Preparation of Fe-ZnO Nanoparticle
Iron(III)-doped zinc oxide nanoparticles (Fe-ZnO NPs) were produced via a co-precipitation method. 95 mmol (20.8 g in 300 mL) Zn(OOCCH3)2.2H2O and 5 mmol (0.81 g in 30 mL) FeCl3 were combined with ethanol and agitated thoroughly until complete dissolution of the solutes occurred. After that, 330mL of the mixture solution was subjected to sonication for 3 min. Following sonication, the solution exhibited little turbidity. The sonication process continued for 4 hours, with a gradual dropwise addition of (7.85g in 80mL) potassium hydroxide solution at 60°C, as shown in Fig. 1. The resultant dry powder was repeatedly washed with a 1:1 mixture of distilled water and ethanol, then with acetone, centrifuged at 12,000 rpm, dried, and stored. 

Activity against bacteria
Minimum inhibitory concentration (MIC) determination
The Resazurin Microtitre-plate Assay (REMA) was modified to utilize a 96-well microtiter plate for assessing the MIC of Fe-ZnO nanoparticles against bacteria [20]. The adult male (35 years) wound infection was cultured for S. aureus. Urologists diagnosed an adult female (35 years) patient with a UTI after removing Klebsiella pneumoniae from her urine samples. Dispensed 100 µL of Mueller-Hinton broth (MHB) into each well of the microtiter plates under aseptic conditions. Subsequently, 100 µL of the test material was introduced into the first row of the 96-well plates. The test ingredient, Fe-ZnO NP, was serially diluted by transferring 100 µL from one well to another. The initial concentration of 50 mg/mL was reduced in successive halving increments (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and 1/128). Ten microlitres of bacterial solutions, each containing 1.5×108 CFU/ml of S. aureus and K. pneumoniae, were introduced into each well. The bacterial cultures were cultivated until they attained a 0.5 McFarland standard.
Subsequently, they were incubated at a temperature of 35±2°C for 18-24 hours after being loosely enveloped in Parafilm. The plate was re-incubated for a further 18-24 hours after incubation to observe any color change, after which 10 µl of resazurin solution (Alamar blue) was added to every well [21].

Agar diffusion assay
The antibacterial efficacy of the examined Fe-ZnO composites was evaluated utilizing the agar well diffusion technique. The first step was to mix 100 µl of freshly prepared Muller Hinton Agar (MHA) with 100 µl of 24-hour cultures of S. aureus and K. pneumoniae in BHI broth. The cultures were adjusted to a 0.5 McFarland standard during the cooling phase (42 °C) after autoclaving. The amalgamation was subsequently placed onto Petri dishes. After the liquid solidified, a sterile cork borer was employed to form a single well (7 mm in diameter) in each plate. All tested Fe-ZnO compounds were subjected to the well, at MIC concentration. Following a 24-hour incubation of the plates at 37°C, the diameter of the inhibitory zone was determined in millimeters [22,23].

RESULTS AND DISCUSSION
Nanoparticle characterization
These ZnO nanoparticles with a 0.5% Fe(III) doping are shown in Fig. 2 in the XRD patterns. The Fe(III)-ZnO nanoparticle sample exhibits indexed diffraction peaks that indicate a hexagonal wurtzite structure belonging to the space group (P63mc). The diffraction data showed a high degree of agreement with the ZnO JCPDS card (JCPDS 36-1451). Doped Fe-ZnO samples did not show any evidence of pure Fe -or- Fe2O3 in their diffraction spectra. The efficient distribution of Fe(III) inside the ZnO lattice structure or the small amount of Fe(III) dopant used could be responsible for this [24].
Although undoped ZnO had narrower XRD peaks [25], Fe-ZnO exhibited wider peaks, indicating that crystallisation was hindered by Fe doping inside the ZnO lattice. Lattice instability and stress were both brought about by the addition of Fe(III) dopants to ZnO during crystallisation. Other research [26], has been shown that when smaller particles are combined with ZnO, they develop faster. This effect is caused by the fact that the ionic radii of Fe(III) ions are 0.067 nm and those of Zn(II) ions are 0.083 nm [27]. In XRD analysis, the lattice parameters of Fe-ZnO and ZnO were found to be a = b = 3.248 Å, c = 5.2054 Å and a = b = 3.249 Å, c = 5.206 Å, respectively, as previously reported [28]. The slightly lower lattice parameters of Fe-ZnO compared to ZnO demonstrated that the wurtzite structure could be preserved with the incorporation of Fe ions into the ZnO crystal lattice [29].
Scanning electron microscopy (SEM) produces high-resolution, pseudo-three-dimensional topography images of nanoparticles. Dimensions, form, surface morphology, and aggregation are critical structural characteristics that can be elucidated by scanning electron microscopy (SEM). Fig. 3 presents scanning electron micrographs of ZnO nanoparticles, either in their pure form or doped with iron. The micrographs indicate that the synthesized nanoparticles are aggregated and exhibit a spherical morphology. The doped ZnO displayed minor aggregation, while the photos indicate that the Fe-ZnO particles predominantly assumed a spherical shape. Smaller Fe-ZnO particles are detected in samples with an increased number of Fe3+ ions [30]. The Fe-ZnO sample, exhibiting a particle size range of 16 to 20 nm, serves as a clear illustration of this phenomenon.
EDX was utilized to get a qualitative analyze of Fe-ZnO nanoparticles. This study elucidates the constituents present in the nanoparticle sample. Fig. 4 presents the results of the EDX study for the Fe-ZnO nanoparticle sample. Based on the EDX spectra, the synthetic Fe-ZnO sample contains primarily Zn and O, with very little Fe. The EDX spectral peak for O is observed at 0.5 keV, while Zn is detected at 1 keV, 8.6 keV, and 9.5 keV in the Fe-ZnO sample. The Fe signal manifests as diminutive peaks at 0.7 keV and 6.4 keV [31].

Particle Synthesis
Fe(III)-ZnO nanoparticles are synthesized by introducing a precipitating agent into a solution that contains dissolved zinc and iron precursors. The processes of metal hydroxide nucleation, growth, and calcination result in the development of a stable hexagonal wurtzite crystal structure with iron substitution. To prepare a homogeneous solution, dissolve the Zn2+ and Fe3+ ions in equal volumes of ethanol. The decomposition of the precursors into free metal ions in the solution yields solvated cations of Zn2+ and Fe3+. A concentrated KOH solution is introduced dropwise while maintaining vigorous magnetic stirring. The introduction of OH- ions to a solution results in a quick increase in pH. As seen in reactions 1 and 2, this results in the simultaneous precipitation of insoluble hydroxides, specifically 5% Fe(OH)3 and 95% Zn(OH)2.

Zn2+  + 2OH- → Zn(OH)2                (1)

Fe3+ + 3OH- → Fe(OH)3                 (2)

Hydroxide undergoes fast nucleation when its concentration exceeds its solubility threshold. Due to the similarity between the ionic radius of Fe(III) (0.64 Å) and Zn(II) (0.83 Å) [27], the growing crystal lattice can accommodate Fe(III) ions instead of Zn(II) ions, leading to the creation of a framework doped with Fe(III).

Assessment of MIC Values
The effective was the synthesized nanoparticles against S. aureus and K. pneumoniae. We found out using the micro dilution method. The experiment started with a concentration of 50 mg/mL and was diluted twice. By observing the wells’ consistent purple/blue coloration in Fig. 5, we were able to determine the MIC, which was found to prevent the development of identifiable bacteria. The third dilution showed the highest inhibitory action against S. aureus against Fe(III)-doped ZnO nanoparticles, with an estimated MIC value of 12.5 mg/mL. According to the MIC values for K. pneumoniae, Fe-ZnO nanoparticles demonstrated antibacterial efficacy at the initial dilution. This indicates a moderate inhibitory effect, with a MIC of around 50 mg / mL. According to the minimum inhibitory concentration (MIC) results, S. aureus exhibits greater susceptibility to Fe-ZnO nanoparticles than K. pneumoniae.

Agar diffusion assay
The antibacterial efficacy against S. aureus and K. pneumoniae was assessed utilizing the agar well diffusion technique with the synthesized Fe-ZnO nanoparticle, as shown in Fig. 6. The antibacterial efficacy of Fe(II)-doped ZnO nanoparticles against K. pneumoniae was demonstrated by the presence of distinct inhibition zones surrounding the wells. The lack of inhibitory zones for S. aureus indicates that Fe(II)-doped ZnO nanoparticles exhibited no measurable antibacterial activity under identical testing conditions. To determine how effective, the chemicals were against bacteria, we used the standard agar diffusion approach, which involves evaluating the presence and relative size of inhibition zones.

CONCLUSION
The co-precipitation method was employed to synthesize doped nanoparticles with mixed properties using zinc and ferric ions as dopants. Miniature Fe-ZnO nanoparticles, approximately 16 nm in size, were synthesized by gradually introducing a basic hydroxide solution into a solution containing selected transition metal ions. Characterization tests, including XRD, of Fe-ZnO samples have revealed a more prominent hexagonal wurtzite structure. Conversely, EDX analysis offered a comprehensive evaluation of the presence and quantities of each element in every sample. The detection of Fe(III) ions in Fe-ZnO particles signifies the successful execution of the process involving these nanoparticles, as recently disclosed. The results indicated that K. pneumoniae exhibited greater resistance to the majority of nanoparticle formulations than S. aureus, with MIC values of 50 mg/mL and 12.5 mg/mL, respectively. 

ACKNOWLEDGMENT
The authors would like to express their gratitude to the Iraqi Ministry of Higher Education and Scientific Research, specifically the Faculty of Pharmacy at Al Muthanna University, for providing financial assistance for this paper.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.