Toxicity Analysis of Hybrid Nanodiamond/Fe3O4 Nanoparticles on Allium cepa L

Materials and Methods The chemicals of hydrochloric acid, nitric acid, FeCl3.6H2O, FeCl2.4H2O, NaCl, and NaOH (Sigma-Aldrich chemicals, USA) were utilized in this study. A statistical analysis was performed on the results with a prevalence of p < 0.05. Results A novel ND/Fe3O4 nanocomposite material was successfully synthesized by the in-situ method and characterized by various characterization techniques. The analysis of X-ray diffraction indicated the formation of an ND/Fe3O4 nanocomposite with both participating phases. The saturation magnification of the ND/Fe3O4 nanocomposite is 13.2 emu/g, whereas for a pure Fe3O4 nanomaterial, it is 47 emu/g. The weight rates of ND and Fe3O4 existent in the nanocomposite are 28% and 72%, respectively. From the electrical conductivity analysis, ND/Fe3O4 exhibits conductivity in the order of 27 times more compared to ND. Conclusion The result implies that the product ND/Fe3O4 has both magnetic and electrical properties. The biocompatibility of the synthesized ND/Fe3O4 material was studied based on the in-vitro method.


Introduction
Nanocomposites containing two phases of materials in the nanometer range of( < 100 nm) are a critical part of nanotechnology and the active growing areas in materials science and engineering. Solid coupling between various combining parts results in novel physical wonders and improved prospects, which makes these frameworks better than their single-segment partners for application in biomedicine, nanoelectronics, optoelectronics, and spintronics applications [1]. Nanodiamond particles (ND) with a size of 4-5 nm demonstrate an extraordinary powerful application in fluorescence markers, restorative items, polymers, biosensors, and medication transmission [2][3][4]. e ND particles are normally combined with carbon-polluting influences (size of 100-200 nm), which is not possible in particular applications. Deaggregation of ND nanoparticles from micron-level to nano-level is essential. A nanostructured ND composite with various materials was synthesized by different techniques. Shi et al. [5] combined ND/ Cu nanocomposites for synergist applications. Nunes et al. [6] investigated Ni/ND and Ni/graphite composites delivered by mechanical combination and warmth treatment strategy. Bahadar et al. [7] studied cell toxicity, genotoxicity, and immunotoxicity. Sundar et al. [8] orchestrated ND-Ni nanocomposites by using the in-situ strategy and arranged nanocomposite nanofluids for warm applications. Kumar et al. [9] analyzed the drilling execution of nanodiamond bonded SiC composites for CFRRP laminates by a dual-axis grinding wheel system. Abakumov et al. [10] performed high biocompatibility of obtained nanoparticles and the number of in-vitro toxicological tests on human fibroblasts and U251 glioblastoma cells. It was shown that albumin nanoparticles' coating provides a stable and biocompatible shell and prevents cytotoxicity of the magnetite core. On long exposure times (48 hours), cytotoxicity of iron oxide nanoparticles takes place due to free radical production, but this toxic effect may be neutralized by using polyethylene glycol modification. Bao et al. [11] used CuO nanoparticles to study the toxicity using various Saccharomyces cerevisiae (S. cerevisiae) strains, and wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants, were determined and compared. Assadian et al. stated that [12] CuO-NPs have been employed in the pharmaceutical industry, especially in the production of antimicrobial fabric treatments or the prevention of infections caused by Escherichia coli and methicillin-resistant Staphylococcus aureus. Two key potential routes of exposure to CuO-NPs exist through inhalation and skin exposure. Ozgur et al. [13] studied the toxicity analysis of Fe 3 O 4 nanoparticles on the superoxide dismutase (SOD) and catalase (CAT) activities which showed a significant (p < 0.05) decrease after 100 mg/L after exposure to Fe 3 O 4 NPs for 24 h. As the doses of Fe 3 O 4 NPs increased, the levels of malondialdehyde (MDA) and total glutathione (tGSH) significantly (p < 0.05) increased at doses of 400 and 800 mg/L, respectively. e above-mentioned works are related to ND nanocomposites with magnetic and nonmagnetic materials. ere are several applications with magnetic materials such as Fe 2 O 3 , Fe 3 O 4, and Ni especially in drug delivery, MRI contrast agent, and magnetic hyperthermia. [14]. Under atmospheric conditions, the magnetization of Fe 3 O 4 is high compared to the magnetization of Fe 2 O 3 [15,16]. Huang et al. [17] explained the emphasis of nanoparticles that influence cytotoxicity. Identification of those properties may lead to the design of more efficient and safer nanosized products for various industrial purposes and guide the assessment of human and environmental health risks. Rotini et al. [18] studied the toxicity of CuO nanoparticles and observed the physical interactions between Vibrio and CuO nanoparticles. Cando et al. [19] studied the neurotoxicity, and they observed mitochondrial activity starting at 10 μg·mL −1 with a decrease in cellular vitality of 35% and a maximum decrease of 45% at the highest dose (100 μg·mL −1 ).
However, to the best of my knowledge, nanocomposites made of ND with magnetic Fe 3 O 4 have not been intensively investigated so far. is work aims to prepare a high purity ND/Fe 3 O 4 nanocomposite and estimate its magnetic and biocompatibility properties.

Study Area.
e study was executed at the Department of Genetics & Biotechnology, Osmania University, from January 2020 to March 2021. e plant material and nanoparticles were obtained from the same institution.

Deaggregation of ND Particles.
Commercially available ND soot aggregates with amorphous carbon impurities of microbe size. It is important to deaggregate and purify these ND particles into almost single ND crystals. For decollection, ND ash was broken up in a fluid NaCl arrangement and pursued by tip sonication for up to 5 hrs, separated and washed a few times with refined water, and after that solidified and dried. To functionalize and remove nondiamond carbon impurities, the deaggregated ND soot was treated with a strongly acidic medium containing 1 : 3 molar ratios of hydrochloric corrosive and nitric corrosive [20] for up to 72 hours under attractive mixing at 60°C. en, the particles were washed a few times with refined water and dried in the stove at 80°C for up to 24 hrs. e benefit of this method is the removal of nondiamond carbon impurities and the formation of carboxyl bunches on the surface of the ND molecule.

Preparation of the ND/Fe 3 O 4 Magnetic Composite.
e ND/Fe 3 O 4 composite was prepared by the in-situ method. is method includes a dispersion of 0.3 g of treated ND in 30 m of refined water under attractive mixing for 60 minutes. After that, FeCl 3 .6H 2 O (0.33 g) and FeCl 2 . 4H 2 O (0.165 g) were added in the molar ratio of 2 : 1. After full dispersion of iron salts, an aqueous NaOH solution was added drop by drop, and the arrangement pH was kept at 12. After vigorous stirring, the formation of a black coloured solution was observed. It indicates the reaction was completed, and the resulting black precipitate was washed a few times with refined water and dried in a broiler at 80°C for 24 h. e same procedure was used to synthesize the Fe 3 O 4 nanoparticles without treating ND in distilled water for comparison purposes.

Characterization Techniques.
e ND/Fe 3 O 4 composite was portrayed using an X-beam diffractometer (Siemens D-500, 45 kV, and 40 mA), SEM (Hitachi; SU-70), and micro-Raman (Jobin-Yvon LabRam; 514 nm argon-ion laser). FTIR spectra were recorded using a Bruker Equinox V70 FTIR spectrometer in dry KBr pullet in the scope of 400-4000 cm −1 and atomic power microscopy (AFM, NT-MDT, NTEGRA Aura, and Nanotec's AFM with Dulcinea Electronic). e immersion polarization of the composite was investigated using a vibrating test magnetometer (VSM), Cryogenic, UK. e round fully grown onions were used for the analysis, and they were washed with water before they were used in the equipment. e temperature of the onions were s maintained at 28 ± 0.5°C. Meristematic root tips of 2-3 cm were obtained with a sharp cutting-edge knife. e square root tips are turned to different sides, those are placed in round-mouthed measuring glasses by using 5 μg·mL −1 of Co 3 O 4 , 10 μg·mL −1 of cND, and 20 μg·mL −1 of cND-Co 3 O 4 , and those are treated with distilled water for 15 min. At around 4 h of time, the root tips were rinsed 3 times with water. e root tips were placed in a 5 m tube along with Cornoy's liquid (acidic corrosive: ethanol in a 1 : 3 ratio) to capture the dynamic mitosis. Root tips set in cylinders with Milli-Q water were treated as the control group, and three duplicates had been made for every target.

Root Tip Squash Preparation and Light
Microscopy. e structure was adopted for the root tip squash planning and light microscopy, as shown by Kumari et al. in their illustration from [21]. In a nutshell, settled root tips were individually corrosive hydrolyzed in 0.1 N hydrochloric corrosive for 2 min at 60°C to degenerate solidifying material between the cells. After recoloring corrosive hydrolyzed pull tips with acetocarmine for 4-6 minutes, root tip squash was made by applying pressure to separate slides with coverslips. Using a light microscope, arranged slides that had accidentally been treated with nail polish were examined for the distinctive cyto-hereditary traits described in the current study. e number of separating cells per 1,000 observed cells was used to calculate the mitotic cells list, while alternative cells were also examined based on how many cells were uploaded and how many were scored in each combination.

Data Analysis.
e mean and standard error (SE) of statistical analyses were verified, and Student's t-test was used to test for drift. At p 0.05, the degree of significance for each result was confirmed.

Characterization.
e produced ND, Fe 3 O 4, and ND-Fe 3 O 4 composites were examined by XRD analysis, and the patterns were presented in Figure 1 Table 1 demonstrates the X-ray diffraction positions (2θ) and the interplanar spacing values (d hkl) of the Fe 3 O 4 sample (blue color line), Table 2 furnishes the X-ray diffraction positions (2θ) and the interplanar spacing values (d hkl) of the ND sample (black color line), and Table 3 (1) D is crystalline size (nm), λ is wavelength (nm), β is full width at half maximum intensity peak in radians, and θ is the angle of diversion reciprocal to peak. e crystallite size of Fe 3 O 4 was calculated from the high intensity peak, and it is noticed at 45.2 nm. e crystallite size of ND was calculated at a high-intensity peak, and it was observed as 6.96 nm. e final ND-Fe 3 O 4 nanoparticles' crystalline size was calculated at a high-intensity peak, and it is found as 53. e FTIR spectra of ND soot and considered ND particles reveals that the peak at ∼1735 cm −1 is ascribed to the C � O stretch of carboxylic (COOH) (Figure 1(c)), which is verification of a covenant connection of carboxylic gathering on the surface of the ND molecule. Notwithstanding, this pinnacle is not seen in the ND/Fe 3 O 4 composite amid the insitu development, which implies that the carboxylic gatherings get lessened amid the arrangement of Fe 3 O 4 nanoparticles at first glance of ND. Figure 1(d) shows the Raman spectra of treated ND and ND/Fe 3 O 4 composites. A diamond peak in ND/Fe 3 O 4 is observed at 1328 cm −1 with a shoulder at 1130 cm −1 that starts from littler ND particles or littler rational dissipating spaces isolated by imperfections in bigger ND particles [8]. Each sample contains an amount of 80 mg powder and is made into a circular (diameter 1 cm; inset in Figure 1(a)) pellet for electrical measurements by using the four-point probe technique [25] which is typically used for highly conductive materials, where the contact resistance between sample and connection wires could play an important role, leading to a wrong estimate of the real material resistance     Journal of Toxicology 5 [26]. To perform I-V measurements, a setup already tested was chosen and used for the estimation of ND soot, ND-treated, ND/Fe 3 O 4, and Fe 3 O 4 samples at room temperature. To make the associations specifically onto our examples surface in a simple, quick, and reproducible way, the silver conductive glue was picked [27], which does not require specific gear, for example, metals sputtering or warm evaporator gadgets.

Biocompatibility.
Once in ecological chambers, either emphatically or unintentionally, the destiny and effect of nanoparticles on real biota must be lit up, as this information can be an unequivocal factor in the success of nanotechnology. As already mentioned, the chromosomes of plants and living things have comparable morphology and comparative reactions to mutagens [28]. In this study, Allium cepa L. was used as a test model to clarify the potential cytogenotoxicity of single nanoparticles of  (Table 1). However, a dose-dependent effect on the mitotic index was noted for Fe 3 O 4 and Fe 3 O 4 -cND. In particular, the mitotic records were observed to be 58.07 ± 1.7, 37.8 ± 1.2, and 28.6 ± 0.8% upon presentation to 5, 10, and 20 μg·mL −1 , respectively. Remarkably, diminishes in MI were irrelevant at these convergences of cND with estimations of 68.3 ± 2.0, 65.7 ± 1.9, and 59.0 ± 1.7, separately, as it may be seen in Table 4 very well. e ameliorative impact of cogathered effects is shown by low (5 μg·mL −1 ) and moderate (10 μg·mL −1 ) concentrations of cND-Fe 3 O 4 . is shows if unintentionally discharged into condition, cND-Fe 3 O 4 would be alright for biotic life to the most extreme grouping of 10 μg·mL −1 .
e observed cogathered cyto-genotoxic outcomes agree with comparable prior investigations, where cooxide nanoparticles were assumed to destroy the entire cell digestion and phases of cell division for the most part by blocking water channels through adsorption as well as by affecting hereditary material by causing different sorts of chromosomal mutilation.
Anjum et al. [29] previously explained the expanded amount of aggregate chromosomal abnormalities with increasing test groupings of different nanoparticles. Sticky chromosomes are primarily thought of as a type of chromatid aberration that results from the corruption or depolymerization of chromosomal DNA [27]. Instead, sticky chromosomes were among them, and they were observed as frequently as possible throughout the anaphase and telophase stages of mitosis in the root tips of the A. cepa plant (onion). In summary, the minor alterations in MI with a moderate concentration (10 g·mL −1 ) of cND-Fe3O4 also support the notion that this compound had a significant interference with the normal progression of mitosis, primarily due to its inability to prevent cells from entering the prophase and obstruction of the mitotic cycle during the entomb stage restraining DNA/protein combination.

Conclusions
e novel ND/Fe 3 O 4 nanocomposite has been effectively arranged by the in-situ technique. e produced ND/ Fe 3 O 4 nanocomposite shows a magnetic hysteresis loop, indicating it might be used as a possibility to obtain a bright magnetically separable photo-catalyst for the expulsion of natural contamination from water. e weight level of Fe 3 O 4 is 28% and that of ND is 72% in the ND/ Fe 3 O 4 composite. e ND/Fe 3 O 4 composite also shows electrical conductivity of the order of 26 times compared to ND soot. We are also in the process of preparing the ND/Fe 3 O 4 composite cross-breed nanofluids for warm applications. Nonetheless, chromosome variations (for example, stickings, prospect, and dispersed metaphase) are inconsistent for the cND-Fe 3 O 4 nanocomposite at a convergence of 10 g·mL −1 , in contrast to 20 g·mL −1 of cND-Fe 3 O 4 and 5, 10, and 20 g·mL −1 of Fe 3 O 4 , clearly indicating that at this concentration, cND-Fe 3 O 4 is environmentally ecofriendly.

Data Availability
All relevant data are within the paper and their supporting information files.