Investigating the Impact of Mycosynthesised Iron Nanoparticles Against Candida Albicans Cells and Studying Their Antioxidants Activity

INTRODUCTION
Nowadays, nano and microstructures (that is less than 100 nm), have acquired applications in several fields of science and technology, including physics, chemistry, biology, and biotechnology [1]. Nanoparticles are characterized by distinct chemical compositions, physical attributes, high ratios of surface area to volume, low melting points, strong photoconductivity, catalytic capabilities, and biological features [2]. To date, Iron nanoparticles (FeNPs) are innovative materials with distinctive physicochemical features, including high catalytic activity, strong magnetic, low toxicity, and the ability to absorb microwaves [3]. These particles have diverse applications, such as drug delivery, magnetic targeting, thermal ablation, hyperthermia, stem cell sorting and manipulation, gene therapy, food preservation, environmental remediation, antimicrobial agents, bioseparation, and treatment for cancer [4]. In biochemical applications, it is crucial to synthesize eco-friendly and non-toxic nanoparticles. Iron nanoparticles have been especially desired due to their low toxicity, as iron naturally exists in the body and can be tolerated in higher doses compared to other metals. Chemical and physical techniques in synthesizing nanoparticles have been deemed unsuitable for biochemical applications due to the production of hazardous compounds [5]. The biogenic path to NP synthesis provides a novel avenue to explore and identify new biological sources that can reduce metals to the nanoscale. Certain microorganisms, including bacteria, fungi, and plants, have been discovered to possess the ability to generate NPs. There is a limited number of publications on synthesizing NPs by endophytic microorganisms. This method is considered to be safe, nonhazardous, and ecologically friendly [6,7].
 Among the biological agents, fungi have consistently played a prominent role in providing a more sustainable alternative to chemically synthesized nanoparticles, referred to as mycosynthesis [8]. The preference for the fungal synthesis of iron magnetic nanoparticles over other microorganisms is due to its easy handling, low cost, high tolerance towards heavy metals, and simplified downstream processing. Fungi have been reported to display increased variability in size, improved structural integrity, and a wider variety of dimensions in producing various nanoparticles [9,10]. Various fungi have been successfully employed for the biological production of nanoparticles. However, according to the available literature, it is evident that several species of Fusarium fungi are the prime to scientists [11].
 Apart from that, reactive species are involved a participate in a plethora of other biological activities within every organism, notably cell maturation, immunological defence, and cytotoxicity against pathogens. Nevertheless, they have the potential to cause damage to proteins, lipids, DNA [12], and cell walls [13].
This study aimed to use the endophytic fungus Fusarium graminearum to synthesize FeNPs. FeNPs action against candida albicans cells and their antioxidant activity were analyzed.

MATERIALS AND METHODS 
This study’s chemicals and reagents were procured from the American company Sigma, Ltd. All the experiments were performed at the cell biology laboratory of the Applied Science Department at the University of Technology in Iraq.

Isolation and identification of Fusarium graminearum
Immediately after rinsing with running water, the Salvia rosmarinus leaves were submerged in double-distilled water for 10 minutes. To sterilise the leaves, we submerged them in 70% ethanol for three minutes, then 0.5% sodium hypochlorite (NaOCl) for one minute, and then another thirty seconds. Immersion in 70% ethanol. Finally, they rinsed three times with sterilized distilled water. Petri dishes with Potato Dextrose Agar (PDA) treated with chloramphenicol at a concentration of 250 mgL‒1 were used for the cultivation of sterile leaf fragments measuring 0.5 cm. The plates were thereafter wrapped with parafilm and incubated at a temperature of 250 °C until the endophytic fungus became visible. In order, the developing hyphal tips from the plant segments were isolated and subjected to subculture to get a pure culture [15,16]. The identification of fungal endophytes was achieved using traditional identification protocols. Using macroscopic characteristics such as colony growth, form, and color, alongside microscopic characteristics like colony surface, texture, conidia, conidiophores, and hypha pigmentation [17]. The potential of Fusarium graminearum has been evaluated for its use in the synthesis of FeNPs. 

Preparation of fungal extract 
The inoculum was generated using a modified Wunder media [18]. The components of the medium included glucose (10 gL‒1), polypeptone (1 gL‒1), K2HPO4 (0.125 gL‒1), (NH4)2SO4 (1 gL‒1), MgSO4·7H2O (0.5 gL‒1), KH2PO4 (0.875 gL‒1), CaCl2·2H2O (0.1 gL‒1), NaCl (0.1 gL‒1), MnSO4·H2O (0.02 gL‒1), and FeSO4·7H2O (0.001 gL‒1). Each Erlenmeyer flask was enriched with 170 ml of medium, and six mm diameter discs of the fungus were added, which were previously cultured on PDA medium. The inoculated flasks underwent continuous agitation at a speed of 125 rpm at 28 °C for 7 days. After incubation, the fungal biomass was filtered and used in future experiments.

Iron Mycosynthesis
The harvested mycelia were separated from the culture broth by subjecting them to be centrifugated at 4500 rpm for 16 minutes. This operation was repeated three to four times, distilled water was used for each wash. Following a three-day incubation period at a temperature of 28.2 °C,100 ml of fungal mycelia is mixed with 100 ml of deionized water containing 0.4mg of Ferrous sulphate (FeSO4.7H2O) [19]. 

Iron Nanoparticles Characterization 
Prior to characterization, ethanol was used for FeNPs extraction from fungal mycelia. Subsequently, ultrasonic dispersion was employed. The study focuses on the bioreduction process of Iron ions in mycelium solution, which is transformed into Iron nanoparticles. The solution changed color within a particular period, suggesting the conversion of Iron ions into FeNPs. A UV-visible spectrophotometer (LAMBDA 365 spectrophotometer-PerkinElmer, Waltham, MA, USA) was used to conduct absorption measurements in the 200 to 800 nm wavelength range. X-ray diffractograms (XRD) were applied using an X-ray diffractometer (HAOYUAN, Zhejiang, China) at 40 kilovolts and 40 milliamperes with Cu(Kilo α) radiation (wavelenght = 1.5406 Å). Diffraction angle (2θ) scanned from 10° to 80° to estimate the diameter and size of the nanoparticles by measuring their peak width and length. The surface charge and persistence were conducted using a Zeta potential analyzer (Zeta, Brookhaven, Deklab County, GA, USA). Fourier Transform Infrared Spectroscopy analysis used an FTIR spectrometer (PerkinElmer, Waltham, MA, USA) to determine the functional groups generated in the FeNPs. The study was conducted at ambient temperature and recorded within the spectral region of 400-4000 cm−1. FeNPs were mixed with potassium bromide at a ratio of 1:100. Then, the functional groups were based on vibrational mode. The shape of the biosynthesized FeNPs was examined using a Field Emission Scanning Electron Microscope (Inspect F50-FE-SEM, FEI, Eindhoven, The Netherlands) with energy-dispersive X-ray (EDX) attachment to analyze the composition and confirm the presence of elemental iron in its natural form. Furthermore, the surface morphology, aggregation, shape, size, and distance of biosynthesized FeNPs were examined using Atomic Force Microscopy (NaioAFM, Nanosurf AG, Liestal, Switzerland) with AFM image analysis software [20, 21].

Treating Candida albicans cells with FeNPs and detecting their effects on the surface morphology
After treating Candida albicans cells with FeNPs, the changes in surface and cellular appearance were examined using FE-SEM. Harvested cells were rinsed by PBS buffer to eradicate the medium, and then fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 3 hours at 25°C. The cells were cultured either without or with the nanoparticles at a concentration of 40 μg/mL until they reached the mid-exponential phase. The cells were washed with phosphate buffer (pH 7.2) two to three times before being postfixed with 1% OsO4 in 0.1 M phosphate buffer for 1 hour at 4°C to prepare them for FSEM, a portion of the cells that had been treated as described above was dehydrated in acetone, then placed onto a round glass coverslip that had been coated with hexamethyldisilazane, allowed to dry, and finally sputtered with gold, The cells were then observed under FE-SEM with a resolution of 3.0 nm at 30 kV and a magnification of 7–1,000,07 [13,22].

Antioxidant activity
The scavenging activities of FeNPs were assessed using stable DPPH radicals [23]. Adding 0.5 ml of FeNPs at various concentrations (0.0, 0.01, 0.1, 1, 10, and 100 µgml‒1) onto DPPH solution (0.5 millilitres of diphenylpicrylhydrazyl and 3.3 millilitres of 100% ethanol were combined to create the DPPH solution.). Spectroscopy was employed to determine the alteration in color at a specific wavelength of 515 nm, while the sample was subjected to a temperature of 25°C for 90 min. The blank solution was made by combining 0.5 ml of the sample with 3.3 ml of 100% ethanol. A control was employed, consisting of a tube containing a mixture of 100% ethanol (3.3 ml) and DPPH (0.5 ml). The percentage of elimination was determined using an equation for antioxidant activity.

RESULTS AND DISCUSSION 
Endophytic fungi derived from medicinal plants, mostly belonging to the Ascomycota and their sexual forms, have been collected. These fungi, commonly called herbaceous plants, can infect and establish a symbiotic relationship with many herbaceous plants worldwide [24, 25].
 Applying endophytes in a biosynthetic approach is an innovative method for developing safe and economical technologies for NP production [26]. Fungi have the ability to generate enzymes and metabolites that function as reductive and capping agents. enabling them to create metal NPs with stable and controlled shapes. Research has demonstrated that enzymes or metabolites produced by fungi can reduce the metallic content of NPs by interfering with the main reaction that is dependent on the reduction or oxidation of the substrate. So, this makes it easier to make colloidal structures. [27]. 
 The bottom-up approach uses fungi to reduce FeNPs, which are advantageous over other organisms. Their proteins and enzymes, such as reductive, can be used for fast and sustainable nanoparticle synthesis. Nevertheless, the top-down approaches aren’t as suitable as the bottom-up approaches for generating particles of petite sizes. In bottom-up methods, nanostructures are constructed by adding atoms, molecules, or clusters. This enables us to produce and design materials with predefined features [28]. The biosynthesis of FeNPs can be categorized into two methods: (i) intracellular and (ii) extracellular mechanisms. However, extracellular synthesis offers more advantages owing to its simplified downstream processing [29]. During extracellular synthesis, Iron ions underwent reduction to form FeNPs. This process occurs in the presence of extracellular enzymes, secondary metabolites, and biomolecules created by fungal cells [30]. However, the biosynthesis of NPs by fungi requires three steps: gathering metal ions near fungal cells, releasing enzymes that reduce iron ions, and stabilizing NPs using fungal peptides and proteins [31].

Mycosynthesised nanoparticle characterization and analysis
UV-Vis Spectrophotometry
The first sign of FeNPs synthesis was the color shift and UV-Vis spectroscopic detection of maximal SPR. The transition of color from yellowish-orange to dark brown during the production of FeNPs might be ascribed to surface Plasmon resonance (SPR), which is a distinctive characteristic of NPs. The prepared FeNPs were characterized by measuring their absorbance using UV-Vis spectrometry in the 200 to 800nm wavelength range, as shown in Fig. 1. The peak observed at 303nm indicated the formation of FeNPs by the endophytic fungi. Furthermore, the broadening of the peak is related to several distinct factors; the shape, size, and polydispersity of NPs [32], These findings are in line with the previously documented absorption peak of FeNPs at 300 nm synthesized from Penicillium oxalicum [33]. The endophytic fungal isolate of Fusarium graminearum had the maximum color intensity with an absorption peak of 303 nm, which aligns with the surface Plasmon resonance (SPR) for FeNPs. 

FT-IR Analysis
The FTIR spectra of iron NPs produced using Fusarium graminearum are shown in Fig. 2. In order to determine whether iron NPs contained phytocompounds derived from a fungal extract and whether they had a role in the reduction of iron ions, Fourier transform infrared (FTIR) tests were conducted. The transmittance peaks detected at 424.34 cm−1, 609.51 cm−1, 848.68 cm−1,1024.20 cm−1,1060.85 cm−1, 1342.46 cm−1, 1533.41 cm−1, 3385.07 cm−1, respectively. The vibrational frequency at 609.51 cm−1 is linked with O2 (Fe–O) stretching bonds [34]. A weak peak obtained at 1060.85 cm−1 corresponds to the stretching vibrations of C-O-C as reported in reference [35]. 
 The bands observed at 3385.07 cm−1 and 1533.41 cm−1 correspond to O–H and C=C stretching vibrations in water, respectively. The band at 1342.46 cm−1 is equivalent to the stretching vibration of C–N bonds in aliphatic and aromatic amines, this band indicates the existence of phenolic compounds that might be involved in the NP formation. Thus, fungal extracts include diverse organic compounds and act as a great supply of capping and reducing agents in producing FeNPs [36]. The –OH groups are implicated in the reduction process by oxidizing them to carbonyl groups. Additionally, particle stability is achieved by the involvement of carbonyl and carboxylate groups [37]. 

XRD Analysis
The XRD is the most efficient technique for analyzing materials. Findings and patterns in the XRD analysis’s output tell us anything about the particles’ nature. The X-ray diffraction (XRD) pattern of the FeNPs obtained by mycosynthesis is presented in Fig. 3. The pattern exhibited absorption peaks corresponding to the crystallographic planes (012), (104), (006), and (113). These peaks aligned with the Bragg diffraction at 2θ values of 24.84°, 33.52°, 39.12°, and 42.31° indicating the presence of the crystalline phase of FeNPs. This observation was compared to the JCPDS standard card No. 06-0362. These findings are consistent with those observed in previous studies on FeNPs [38].

FE-SEM micrographs of Iron nanoparticles 
Fig. 4 exhibits representative images at various degrees of magnification. Image A provides a general overview (20.00k) of mycosynthesized FeNPs, while image-B offers a higher magnification (50.00k). The SEM technique was used to investigate the size, shape, and aggregation of FeNPs, which influence their metabolic processes. It is evident that most of the FeNPs are spherical with a smooth surface and show some particle aggregation. The dimensions of the structures ranged from 45.89 nm to 89.05 nm, which is related to the findings of the XRD analysis. The activity of the NPs increased as the size diminished. Additionally, the shape of NPs impacts their behavior [39,40]. The observed aggregation of FeNPs may be attributed to the electrostatic contact between layers of the NP surface. Furthermore, NPs tend to form agglomerates suspended due to their elevated surface area-to-volume ratio [41].
The energy-dispersive X-ray (EDX) confirms the existence of iron elements in the compound. The EDX spectra demonstrated the presence of iron peaks in three separate regions (0.7, 6.4, and 7.0). The weight percentage of Fe in the FeNPs was 55.2% of the total sample components, with 30.9% of oxygen, 6.9% of carbon, with a minimal percentage of calcium and chlorine were also found in these FeNPs sample, which may be attributed to the fungal extract (Fig. 5) [42].

Size and Zeta Potential Measurements and Atomic Force Microscope (AFM)
The findings of the values of the polydispersity index (PDI) and hydrodynamic diameter (DLS) for FeNPs were shown in Fig. 6. It shows the mean size was 63.7 nm with PDI of 0.35, confirming the nanoscale size of the FeNPs. The PDI is an essential indicator for evaluating the distribution of particle sizes in FeNPs [46]. 
Shafiq et al. (2016) [7] and Aja et al. (2018) [47] described Fusarium graminaerum biosynthesised FeNPs in various sizes, which were sluggish to consume AFM. 

Cellular and intracellular imaging
To investigate how 40 μg/mL (MIC90) FeNPs affected the surface morphology of Candida albicans cells, Fe-SEM was employed. Untreated cells seemed to have a smooth exterior in the matching SEM micrographs (Fig. 8), while cells treated with FeNPs displayed noticeable surface changes and outer cell walls that were rough and wrinkled (Fig. 9).
Disruptions to membrane potential, cellular ultrastructure, and apoptosis triggering were the only negative effects of Nps on Candida cells that were documented in a small number of investigations [44]. 
The cell wall and membrane of Candida albicans cells were clearly visible and undamaged when no FeNp treatment was applied (control) (Fig. 8). In contrast, our FE-SEM findings were supported by Fig. 9, which showed that Candida cells treated with 40 μg/mL FeNps had reduced cell wall and membrane integrity, which is consistent with prior results [45]. Pathogenic fungi rely on their cell envelope, which consists of the cell wall and membrane, to be stable, rigid, and resistant to physical stressors [46]. The physical condition and, by extension, the fluidity of the cell membrane may be affected by the effects of FeNps on the cell wall and membrane.

Antioxidant action of FeNps
The stable free radical DPPH, at room temperature, exhibits a rich violet colour with a 517 nm, which represents the absorbed wavelength when dissolved in organic solvents. The inclusion of selenium nanoparticles (SeNPs) in the analysis resulted in a decrease in DPPH stability and a change in colour from violet to yellow, which can be attributed to the phenolic OH groups present [47]. 
The scavenging of DPPH was seen to be directly related to the concentrations of the FeNPs. Specifically, at concentrations of 0.01, 0.1, 1, 10, and 100 gm/ml of FeNPs, the DPPH free radicals’ scavenging capabilities were found to be 4.48%, 11.315 24.6%, 46.9%, and 100%, respectively (Fig. 10). FeNPs2 demonstrated the most pronounced scavenging activity when present at a concentration of 100%.

CONCLUSION
It may be inferred that the use of endophytic fungi fusarium for the biosynthesis of FeNPs is a straightforward, expeditious, and efficient method with a diameter of 45.89–80.05 nm nanometers; furthermore, the utilisation of fungal cell filtrate renders it even more environmentally sustainable and less detrimental to human health and the surrounding ecosystem. It has been proven that iron nanoparticles (FeNPs) exhibit scavenging activity increased with concentration.

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