2.2. Preparation of Chemical and Natural Magnetic Iron Oxide Nanoparticles

The Institutional Animal Care and Use Committee Research Ethics of Benha University, Faculty of Veterinary Medicine, Egypt, reviewed and approved all experimental protocols (BUFVTM, 01012021; 1 January 2021) conducted in this study. IONPs were prepared in the department of Corn Diseases and Sugar Crops, Agricultural Research Center, Egypt. Chemical iron oxide nanoparticles’ synthesis was conducted as described earlier [34,35]. Chemical-based synthesis methods are mostly adopted due to the low production cost and high yield. Briefly, magnetite was synthesized by adding a base to an aqueous mixture of Fe²⁺ and Fe³⁺ chloride in a 1:2 molar ratio, resulting in a black color [35]. The chemical reaction of Fe₃O₄ precipitation is given in Equations (1) and (2). The overall reaction [36] is written as [Fe²⁺ + 2Fe³⁺ + 8OH → Fe₃O₄ + 4H₂O] (1). Under an oxygen-free environment, complete precipitation of Fe₃O₄ is likely between pH 9 and 14, maintaining a molar ratio of Fe³⁺: Fe²⁺ (2:1). Fe₃O₄ might also be oxidized as [Fe₃O₄ + 0.25O₂ + 4.5H₂O → 3Fe (OH)₃] (2). Magnetic iron oxide nanoparticles (Fe₃O₄NPs) were prepared by using aqueous leaf extracts of Petroselinum crispum as described earlier [22,37]. Briefly, 0.01 M FeCl₃·6H₂O solution was added to the Petroselinum crispum extract in a 1:1 volume ratio. Fe₃O₄NPs were immediately obtained by the reduction process. To obtain a colloidal suspension, the liquid was agitated for 30 min and then allowed to stand at room temperature for another 60 min. The mixture was centrifuged and washed several times with ethanol before being dried at 40 °C under vacuum to yield the Fe₃O₄NPs. Compared to other sections of the plant, Petroselinum crispum leaves have the best reduction potential against ferric chloride, as seen by the external color change. Leaves were chosen for additional procedures based on this observation. The prepared iron oxide nanoparticles (Fe₃O₄NPs) were characterized for their shape, size and dispersity using a transmission electron microscope operated at 100 KV and connected with a CD camera (TEM; Joel 1230, Japan). A Zetasizer system (Nano ZS, Malvern, UK) was used for the measurement of average hydrodynamic diameters and polydispersity indexes. The zeta potential range (mV) was 200:200 mV. Each sample was analyzed in triplicate at 25 °C at a scattering angle of 173°. The size distribution of the particles was estimated from laser particle analyzer (LPA) images by measurement of the diameters.