# Mycosynthesis and Physicochemical Characterization of Vanadium Oxide Nanoparticles Using the Cell-Free Filtrate ofFusarium oxysporum and Evaluation of Their Cytotoxic and Antifungal Activities

Authors: Mohammadhassan Gholami-Shabani; Fattah Sotoodehnejadnematalahi; Masoomeh Shams-Ghahfarokhi; Ali Eslamifar; Mehdi Razzaghi-Abyaneh
Journal: Journal of Nanomaterials
 (2021)

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## Abstract

Green nanotechnology is an expanding branch of knowledge in relation to producing efficient antifungal compounds with potential applications as nanomedicines. The aim of the current investigation was to mycosynthesize functional vanadium oxide nanoparticles (V2O5NPs) by Fusarium oxysporum cell-free filtrate using ammonium metavanadate (NH4VO3) as the substrate. Various spectrometric methods and electron microscopy were used to confirm the production of mycosynthesized V2O5NPs. FESEM and TEM images showed that V2O5NPs were in the size ranging from 10 to 20 nm in a spherical shape. The XRD pattern revealed the presence of crystalline, dominantly spherical V2O5NPs in the sample with a size ranging from 10 to 20 nm. The XRD peaks 15.2, 20.1, 21.6, 26.1, 30.9, 32.2, 33.1, 34.2, 41.0, 41.8, 45.3, 47.2, 48.6, 51.1, 51.9, 55.4, and 58.8 can be assigned to the plane of vanadium crystals and indicate that the V2O5NPs were face-centered, cubic, and crystalline in nature. The FTIR results showed the presence of some biomolecules in fungal cell-free filtrate that act as a bioreducing and capping agent for V2O5NP mycosynthesis. DLS showed that the size of V2O5NPs was 10-20 nm. Zeta potential showed −35.09 mV for V2O5NPs with a single peak. Study of antifungal activity of V2O5NPs against various pathogenic fungi in concentrations of 5, 25, 50, and 100 μg/mL showed that V2O5NPs strongly inhibited both mycelium growth (20.3 to 67.3%) and spore germination (64.8 to 89.9%) dose-dependently. V2O5NPs showed strong cytotoxicity against breast cancer cell-line MCF-7 with an IC50 value of 55.89 μg/mL. Microscopy images showed morphological changes and reduction of cancer cell populations in V2O5NP-treated MCF-7 cell-line. Taken together, our results demonstrated that bioactive V2O5NPs successfully synthesized by F. oxysporum could be considered a potential candidate in drug development against life-threatening fungal pathogens and as a feasible anticancer agent.


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## Body

## 1. Introduction

Nowadays, nanoparticles (NPs) have been widely studied for their antimicrobial activities and anticancer properties and to combat any life-threatening disease as medicine [1–3]. Most of the current approaches for manufacturing NPs are based on the usage of chemicals, including strong reducing agents, organic solvents, or surfactants [4–6]. Although chemically synthesized NPs possess unique properties with potentially broad-ranging application, there is a clear tendency toward synthesis of NPs by nontoxic safe green approaches using microorganisms and plants [7–9]. Development of clean and ecofriendly procedures is essential in the nanotechnology field, as many organisms like plant, fungi, and bacteria have the capability to produce inorganic nanostructures and organic NPs [10]. A wide array of established and newly developed physical, chemical, physicochemical, and biological methods have been successfully used for producing various NPs [11, 12].Fungi are being employed in nanotechnology for the manufacture of NPs; biosynthesis using various fungi has displayed that this environmentally benign and renewable source can be operated as an effective bioreducing agent for the biosynthesis of NPs [13]. The biosynthesis of NPs by fungi (yeasts, molds, and mushrooms) and their subsequent application, exclusively in medical science, are studied under “myconanotechnology.” The possible use of fungi because they are simple to culture in bulk form [14]. Extracellular secretion of enzymes/proteins to produce NPs has an additional advantage in the handling of biomass and downstream processing [15]. Extracellularly secreted fungal biomolecules are able to trap heavy metals and metal salts on the cell surface and subsequently bioreducing them by enzymes/proteins to easily recover from the process, while in intracellular biosynthesis, heavy metal ions are transported into the microbial cell to produce NPs in the attendance of enzymes, and the NPs are difficult to recover in pure form [16].Vanadium (V) metal was first isolated in 1869 by the English chemist named Henry Enfield Roscoe. V generally has diverse oxidation numbers that give various oxide structures, for instance, VO, V2O3, VO2, and V2O5, of which the latter is possibly the most investigated one, due to its thermodynamic stability. Vanadium oxide (V2O5) appears as a yellow to red crystalline powder or orange solid which plays an important role in the industry, agriculture, and medicine especially in producing biologically active nanomaterials [17–21].Vanadium nanoparticles have received major consideration due to their unique characteristics and various applications in the structure of sensors, electrode materials for electrochemical capacitors, electrochromic and optical switching devices, and windows for solar cells [22]. Vanadium oxide nanoparticles (V2O5NPs) have been successfully synthesized by conventional approaches using chemical compounds as the base source and capping agent [23]. With the growth of knowledge in science and technology, V2O5NPs can be now biosynthesized with safe and effective green chemistry using microorganisms and plants. To our knowledge, green synthesis of V2O5NPs is still limited, for instance, to Saccharomyces cerevisiae, leaf extract of Moringa oleifera, and Foeniculum vulgare stem extract [24, 25].The present study brings the information about mycosynthesis on V2O5NPs using a cell-free filtrate of the filamentous fungus, F. oxysporum PTCC 5115, as a bioreducing and stabilizing agent for the first time. Successfully synthesized V2O5NPs were characterized using different techniques such as UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), field emission scanning electron microscope (FESEM), X-ray diffraction (XRD), and dynamic light scattering (DLS) techniques. Furthermore, the antifungal activity of mycosynthesized V2O5NPs was evaluated against different plant and human fungal pathogens, and their cytotoxic effects against the human breast cancer MCF-7 cell-line was investigated.


## 2. Materials and Methods

### 2.1. Chemicals

Ammonium metavanadate (NH4VO3) was purchased from Sigma-Aldrich, USA. Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), potato dextrose broth (PDB), glucose, peptone, malt, and yeast extract were purchased from Merck, Germany. All other solvents and reagents were of analytical grade prepared from international companies.


### 2.2. Strain, Culture Conditions, and Preparation of Fungal Cell-Free Extracts

Fusarium oxysporum PTCC 5115 (CBS 620.87) was obtained from the Iran Research Organization for Science and Technology, Tehran, Iran (https://irost.org/ptcc/ptccdb/node/649). The fungus was grown on SDA slants for 5 days at 28°C. The mycelia were separated from the culture medium and aseptically transferred to a liquid medium containing glucose 10 g, peptone 5 g, yeast extract 3 g, malt extract 3 g, and 1000 mL distilled water [26]. The submerged cultures were incubated on a shaker incubator (LABTECH, Korea) for 4 days at 26°C with shaking (200 rpm). Fungal biomass was washed three times with sterile distilled water under aseptic conditions to remove any medium residual component from the respective biomass and subcultured to new cultures in sterile distilled water at the above conditions for 2 days. Finally, cultures were centrifuged (5000×g, 20 min, 4°C, AWEL Centrifuge, France) to remove fungal mycelia. The clear cell-free was obtained by filtration (Whatman filter paper) and stored at −70°C for further use in V2O5NP synthesis.


### 2.3. Mycosynthesis of V2O5NPs

The extracellular mycosynthesis of the V2O5NPs was carried out using cell-free filtrate of F. oxysporum with some modifications [27, 28]. 1 mL of fungal cell-free filtrate was mixed with 1 mL of NH4VO3 (1 mM solution). The mixture was shaken for further 96 h at room temperature under dark conditions. The secretion of large amounts of bioactive substances into the supernatant led to the mycosynthesis of V2O5NPs. Absorption spectra of the supernatant contained V2O5NPs were recorded on a PerkinElmer EZ301 UV-visible spectrophotometer.


### 2.4. Characterization of Mycosynthesized V2O5NPs

Characterization of mycosynthesized V2O5NPs was performed using different approaches including the UV-visible spectroscopy, FTIR, DLS, FESEM, and TEM.


#### 2.4.1. UV-Visible Spectrophotometry

UV-Vis spectroscopy was used to confirm mycosynthesized V2O5NPs. The wavelength of green-synthesized V2O5NPs based on light absorbance (300 to 700 nm) with resolution of 1 nm was measured by loading 2 mL of sample in a quartz cuvette in a UV-Vis spectroscope (PerkinElmer, United States). The reaction was monitored by measuring the absorbance at 1, 8, 16, 32, 48, 72, and 96 h [24, 25].


#### 2.4.2. DLS Analysis

The dynamic light scattering (DLS) particle size analysis was considered a very fast, reproducible, and reliable technique to measure a wide range of NPs [29]. In the present study, the DLS (Malvern Zetasizer Nano ZEN3600, United Kingdom) was used to measure the size and distribution pattern of mycosynthesized V2O5NPs. A sample of 50 μg of V2O5NPs was dispersed in 20 mL ultrapure water and sonicated for 20 min. 5 mL of the dispersed V2O5NPs was loaded on DLS to determine the size of mycosynthesized V2O5NPs. The stability of the V2O5NPs was assessed in terms of zeta potential using the above DLS allowing to run from -200 mV to +200 mV and plotting the data in a graph.


#### 2.4.3. FESEM Examination

The field emission scanning electron microscope (FESEM-FEI NanoSEM 450) was used to image the V2O5NPs. A sample of 50 μg of V2O5NPs was dispersed in 20 mL ultrapure water and sonicated for 20 min. A small drop of sample was used, and the images were captured at various magnifications [30].


#### 2.4.4. TEM Observations

Transmission electron microscopy (TEM-Philips CM200) was used to image the mycosynthesized V2O5NPs. For the above purpose of the pasteurizing image, V2O5NPs were dispersed in ultrapure water and sonicated for 20 min. The resultant solution (a small drop) was placed on a 300-mesh lacy carbon-coated copper grid and dried, and the images were captured [31].


#### 2.4.5. XRD Analysis

The air-dried V2O5NPs were coated onto an X-ray diffraction (XRD) grid and analyzed for the formation of V2O5NPs by a Philips PW 1390 X-ray diffractometer at a voltage of 20-100 kV. Two grams of fine-powder V2O5NPs with thickness of 0.2 cm in a uniform layer on one side was used for tacking diffractograms. The diffracted intensities were recorded from 2θ angles, and a graph was drawn by the standard method [24, 31].


#### 2.4.6. FTIR Analysis

For the FTIR analysis, a sample of 50μg of V2O5NPs was used, and the spectra were scanned (400–4000 cm-1, resolution of 4 cm-1) for the characterization of chemical functional groups present over the surface of mycosynthesized V2O5NPs. The FTIR data measures the interaction between V2O5 and biomolecules [24, 25].


### 2.5. Effects of V2O5NPs on Fungal Radial Growth and Spore Germination

The effect of the V2O5NPs on the growth of selected fungi Fusarium oxysporum PTCC 5115, Fusarium graminearum PFCC 573, Aspergillus fumigatus PTCC 5009, Aspergillus niger PTCC 5010, Aspergillus flavus PFCC 113, Alternaria alternata PFCC 436, and Penicillium citrinum PFCC 549 obtained from Iran Research Organization for Science and Technology, Tehran, Iran (PTCC strains), and Pathogenic Fungi Culture Collection of the Pasteur Institute of Iran, Tehran, Iran (PFCC strains), was studied using the food-poisoning process [32]. V2O5NPs at variable concentrations were poured into each sterile Petri dish; a PDA medium was added to each sterile Petri dish which gave a PDA-V2O5NP mixture with corresponding 5, 25, 50, and 100 μg/mL V2O5NP concentrations. These Petri dishes were softly rotated to ensure dispersion of the V2O5NPs. The agar-V2O5NP combination was allowed to solidify. All Petri dishes were inoculated at the center with a thickness of 4 mm diameter agar containing a pure culture of each fungus. The control trial was used with distilled water without the addition of V2O5NPs. Then, Petri dishes were incubated at 28°C for 5 days. Finally, the fungal growth inhibition induced by V2O5NPs was calculated according to the technique described by Ebadzadsahrai et al. [32].The effect of various concentrations of the V2O5NPs on spore germination of selected fungi was investigated. Spore suspensions of selected pathogenic fungi were prepared from 7-day-old pure cultures on potato dextrose agar (PDA) slants by gentle rubbing of culture surfaces using a sterile glass rod. Spore suspension of each fungus (103 spores/mL) was exposed to V2O5NPs in various concentrations (5, 25, 50, and 100 μg/mL) in Falcon tubes containing potato dextrose broth (PDB). After 30 min incubation of tubes at 28°C, one drop of lactophenol cotton blue was separately added to the spore suspension of each fungus on glass slides, and the percentage of spore germination was recorded under microscope (×400) according to Begum et al. [33].


### 2.6. Cytotoxicity Effect of Mycosynthesized V2O5NPs

#### 2.6.1. MTT Assay of Mycosynthesized V2O5NPs

The cytotoxicity effect of V2O5NPs was determined by the MTT assay [34]. The MCF-7 cell-lines were obtained from the National Cell Bank (NCBI), Pasteur Institute of Iran, Tehran, Iran. The cancerous MCF-7 cell-lines were cultured into Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (10%, FBS), glutamine (2 mM), and antibiotics (amphotericin B, penicillin G, and streptomycin, 50, 60, and 100 mg/L, respectively) under a relatively humid atmosphere (37°C, 5% CO2). Briefly, 1×105 cells/well were treated with diverse concentrations of V2O5NPs (10-100 μg/mL). After a 24 h incubation, the cells were washed two times by sterile phosphate-buffered saline (PBS), and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-triphenyltetrazolium bromide (MTT or thiazolyl blue; 0.5 mg/mL PBS) was added to the wells and incubated at 37°C for 4 h. The blue formazan crystals that formed were dissolved through adding dimethyl sulfoxide (100 μL/well), and the absorbance was read by a microplate scanning spectrophotometer at 570 nm (ELISA reader, Organon Teknika, Netherlands). The IC50 values of V2O5NPs on cancerous cells lines were then determined.


#### 2.6.2. Cell Cytotoxicity Analysis by Optical Microscopy

Optical microscopic evaluation of the MCF-7 cells was performed to observe the morphological changes after exposure to V2O5NPs [35, 36]. MCF-7 cells were grown in a well plate and treated with V2O5NPs at the IC50 concentration for 24 h. The morphological changes of cells were observed under an invert optical microscope (Olympus, Japan).


#### 2.6.3. Cell Cytotoxicity Analysis Using AO/EB Staining

Study of acridine orange (AO)/ethidium bromide (EB) fluorescent staining was performed according to the previous method with a few modifications [35]. Briefly, the MCF-7 cells (1×105/well) were deposited in a plate and treated by the IC50 concentration of V2O5NPs at 37°C for 24 h. The cells were collected, washed once with sterile PBS, and then suspended with sterile PBS. For AO/EB fluorescent staining, 1 μL of AO/EB (Sigma) dye mixture (10 mg/mL of AO/EB in PBS) was added to 9 μL of cell suspension and then covered with a cover slip on a clean slide. After incubation for 3 min, the cells were observed under a fluorescence microscope (Olympus, Japan).


#### 2.6.4. Cell Cytotoxicity Analysis Using SEM and TEM

MCF-7 cells were prepared for scanning (SEM) and transmission electron microscopy (TEM) analysis according to the previous method with a few modifications [37]. Briefly, the cells were incubated for 24 h with mycosynthesized V2O5NPs at the IC50 concentration. The cells were harvested and fixed in 2.5% glutaraldehyde, washed with sodium cacodylate buffer, postfixed with 1% osmium tetroxide, dehydrated using gradual concentrations of ethanol, and dried using hexamethyldisilazane. Finally, the morphological changes of the cells were observed using SEM and TEM microscopy.


#### 2.6.5. Cell Cytotoxicity Analysis by DNA Fragmentation

DNA fragmentation was performed according to a previous method [35]. An amount of 1×105 MCF-7 cells was exposed to V2O5NPs at IC50 concentrations at 37°C for 24 h. After slight harvesting with centrifugation, the cells were suspended in 10 mL of 10 mM TE buffer solution (pH 8.0, 10 mM EDTA, 10 mM Tris-HCl, 2% SDS, and 20 mg/mL proteinase K). The mixture was incubated for 3 h at 37°C, followed by DNA extraction. The DNA was treated with DNase-free RNase (20 mg/mL concentration, 4°C for 45 min) and precipitated with sodium acetate (100 mL, 2.5 M) and three volumes of absolute ethanol. A DNA fragmentation study was carried out with 10 μL of DNA prepared from treated cells by mycosynthesized V2O5NPs at the IC50 concentration and analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide for a period of 45 min at 100 V.


### 2.7. Statistical Analysis

The data were analyzed using the GraphPad Prism software Version 9.0, andP values under 0.05 were considered significant.


## 2.1. Chemicals

Ammonium metavanadate (NH4VO3) was purchased from Sigma-Aldrich, USA. Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), potato dextrose broth (PDB), glucose, peptone, malt, and yeast extract were purchased from Merck, Germany. All other solvents and reagents were of analytical grade prepared from international companies.


## 2.2. Strain, Culture Conditions, and Preparation of Fungal Cell-Free Extracts

Fusarium oxysporum PTCC 5115 (CBS 620.87) was obtained from the Iran Research Organization for Science and Technology, Tehran, Iran (https://irost.org/ptcc/ptccdb/node/649). The fungus was grown on SDA slants for 5 days at 28°C. The mycelia were separated from the culture medium and aseptically transferred to a liquid medium containing glucose 10 g, peptone 5 g, yeast extract 3 g, malt extract 3 g, and 1000 mL distilled water [26]. The submerged cultures were incubated on a shaker incubator (LABTECH, Korea) for 4 days at 26°C with shaking (200 rpm). Fungal biomass was washed three times with sterile distilled water under aseptic conditions to remove any medium residual component from the respective biomass and subcultured to new cultures in sterile distilled water at the above conditions for 2 days. Finally, cultures were centrifuged (5000×g, 20 min, 4°C, AWEL Centrifuge, France) to remove fungal mycelia. The clear cell-free was obtained by filtration (Whatman filter paper) and stored at −70°C for further use in V2O5NP synthesis.


## 2.3. Mycosynthesis of V2O5NPs

The extracellular mycosynthesis of the V2O5NPs was carried out using cell-free filtrate of F. oxysporum with some modifications [27, 28]. 1 mL of fungal cell-free filtrate was mixed with 1 mL of NH4VO3 (1 mM solution). The mixture was shaken for further 96 h at room temperature under dark conditions. The secretion of large amounts of bioactive substances into the supernatant led to the mycosynthesis of V2O5NPs. Absorption spectra of the supernatant contained V2O5NPs were recorded on a PerkinElmer EZ301 UV-visible spectrophotometer.


## 2.4. Characterization of Mycosynthesized V2O5NPs

Characterization of mycosynthesized V2O5NPs was performed using different approaches including the UV-visible spectroscopy, FTIR, DLS, FESEM, and TEM.


### 2.4.1. UV-Visible Spectrophotometry

UV-Vis spectroscopy was used to confirm mycosynthesized V2O5NPs. The wavelength of green-synthesized V2O5NPs based on light absorbance (300 to 700 nm) with resolution of 1 nm was measured by loading 2 mL of sample in a quartz cuvette in a UV-Vis spectroscope (PerkinElmer, United States). The reaction was monitored by measuring the absorbance at 1, 8, 16, 32, 48, 72, and 96 h [24, 25].


### 2.4.2. DLS Analysis

The dynamic light scattering (DLS) particle size analysis was considered a very fast, reproducible, and reliable technique to measure a wide range of NPs [29]. In the present study, the DLS (Malvern Zetasizer Nano ZEN3600, United Kingdom) was used to measure the size and distribution pattern of mycosynthesized V2O5NPs. A sample of 50 μg of V2O5NPs was dispersed in 20 mL ultrapure water and sonicated for 20 min. 5 mL of the dispersed V2O5NPs was loaded on DLS to determine the size of mycosynthesized V2O5NPs. The stability of the V2O5NPs was assessed in terms of zeta potential using the above DLS allowing to run from -200 mV to +200 mV and plotting the data in a graph.


### 2.4.3. FESEM Examination

The field emission scanning electron microscope (FESEM-FEI NanoSEM 450) was used to image the V2O5NPs. A sample of 50 μg of V2O5NPs was dispersed in 20 mL ultrapure water and sonicated for 20 min. A small drop of sample was used, and the images were captured at various magnifications [30].


### 2.4.4. TEM Observations

Transmission electron microscopy (TEM-Philips CM200) was used to image the mycosynthesized V2O5NPs. For the above purpose of the pasteurizing image, V2O5NPs were dispersed in ultrapure water and sonicated for 20 min. The resultant solution (a small drop) was placed on a 300-mesh lacy carbon-coated copper grid and dried, and the images were captured [31].


### 2.4.5. XRD Analysis

The air-dried V2O5NPs were coated onto an X-ray diffraction (XRD) grid and analyzed for the formation of V2O5NPs by a Philips PW 1390 X-ray diffractometer at a voltage of 20-100 kV. Two grams of fine-powder V2O5NPs with thickness of 0.2 cm in a uniform layer on one side was used for tacking diffractograms. The diffracted intensities were recorded from 2θ angles, and a graph was drawn by the standard method [24, 31].


### 2.4.6. FTIR Analysis

For the FTIR analysis, a sample of 50μg of V2O5NPs was used, and the spectra were scanned (400–4000 cm-1, resolution of 4 cm-1) for the characterization of chemical functional groups present over the surface of mycosynthesized V2O5NPs. The FTIR data measures the interaction between V2O5 and biomolecules [24, 25].


## 2.4.1. UV-Visible Spectrophotometry

UV-Vis spectroscopy was used to confirm mycosynthesized V2O5NPs. The wavelength of green-synthesized V2O5NPs based on light absorbance (300 to 700 nm) with resolution of 1 nm was measured by loading 2 mL of sample in a quartz cuvette in a UV-Vis spectroscope (PerkinElmer, United States). The reaction was monitored by measuring the absorbance at 1, 8, 16, 32, 48, 72, and 96 h [24, 25].


## 2.4.2. DLS Analysis

The dynamic light scattering (DLS) particle size analysis was considered a very fast, reproducible, and reliable technique to measure a wide range of NPs [29]. In the present study, the DLS (Malvern Zetasizer Nano ZEN3600, United Kingdom) was used to measure the size and distribution pattern of mycosynthesized V2O5NPs. A sample of 50 μg of V2O5NPs was dispersed in 20 mL ultrapure water and sonicated for 20 min. 5 mL of the dispersed V2O5NPs was loaded on DLS to determine the size of mycosynthesized V2O5NPs. The stability of the V2O5NPs was assessed in terms of zeta potential using the above DLS allowing to run from -200 mV to +200 mV and plotting the data in a graph.


## 2.4.3. FESEM Examination

The field emission scanning electron microscope (FESEM-FEI NanoSEM 450) was used to image the V2O5NPs. A sample of 50 μg of V2O5NPs was dispersed in 20 mL ultrapure water and sonicated for 20 min. A small drop of sample was used, and the images were captured at various magnifications [30].


## 2.4.4. TEM Observations

Transmission electron microscopy (TEM-Philips CM200) was used to image the mycosynthesized V2O5NPs. For the above purpose of the pasteurizing image, V2O5NPs were dispersed in ultrapure water and sonicated for 20 min. The resultant solution (a small drop) was placed on a 300-mesh lacy carbon-coated copper grid and dried, and the images were captured [31].


## 2.4.5. XRD Analysis

The air-dried V2O5NPs were coated onto an X-ray diffraction (XRD) grid and analyzed for the formation of V2O5NPs by a Philips PW 1390 X-ray diffractometer at a voltage of 20-100 kV. Two grams of fine-powder V2O5NPs with thickness of 0.2 cm in a uniform layer on one side was used for tacking diffractograms. The diffracted intensities were recorded from 2θ angles, and a graph was drawn by the standard method [24, 31].


## 2.4.6. FTIR Analysis

For the FTIR analysis, a sample of 50μg of V2O5NPs was used, and the spectra were scanned (400–4000 cm-1, resolution of 4 cm-1) for the characterization of chemical functional groups present over the surface of mycosynthesized V2O5NPs. The FTIR data measures the interaction between V2O5 and biomolecules [24, 25].


## 2.5. Effects of V2O5NPs on Fungal Radial Growth and Spore Germination

The effect of the V2O5NPs on the growth of selected fungi Fusarium oxysporum PTCC 5115, Fusarium graminearum PFCC 573, Aspergillus fumigatus PTCC 5009, Aspergillus niger PTCC 5010, Aspergillus flavus PFCC 113, Alternaria alternata PFCC 436, and Penicillium citrinum PFCC 549 obtained from Iran Research Organization for Science and Technology, Tehran, Iran (PTCC strains), and Pathogenic Fungi Culture Collection of the Pasteur Institute of Iran, Tehran, Iran (PFCC strains), was studied using the food-poisoning process [32]. V2O5NPs at variable concentrations were poured into each sterile Petri dish; a PDA medium was added to each sterile Petri dish which gave a PDA-V2O5NP mixture with corresponding 5, 25, 50, and 100 μg/mL V2O5NP concentrations. These Petri dishes were softly rotated to ensure dispersion of the V2O5NPs. The agar-V2O5NP combination was allowed to solidify. All Petri dishes were inoculated at the center with a thickness of 4 mm diameter agar containing a pure culture of each fungus. The control trial was used with distilled water without the addition of V2O5NPs. Then, Petri dishes were incubated at 28°C for 5 days. Finally, the fungal growth inhibition induced by V2O5NPs was calculated according to the technique described by Ebadzadsahrai et al. [32].The effect of various concentrations of the V2O5NPs on spore germination of selected fungi was investigated. Spore suspensions of selected pathogenic fungi were prepared from 7-day-old pure cultures on potato dextrose agar (PDA) slants by gentle rubbing of culture surfaces using a sterile glass rod. Spore suspension of each fungus (103 spores/mL) was exposed to V2O5NPs in various concentrations (5, 25, 50, and 100 μg/mL) in Falcon tubes containing potato dextrose broth (PDB). After 30 min incubation of tubes at 28°C, one drop of lactophenol cotton blue was separately added to the spore suspension of each fungus on glass slides, and the percentage of spore germination was recorded under microscope (×400) according to Begum et al. [33].


## 2.6. Cytotoxicity Effect of Mycosynthesized V2O5NPs

### 2.6.1. MTT Assay of Mycosynthesized V2O5NPs

The cytotoxicity effect of V2O5NPs was determined by the MTT assay [34]. The MCF-7 cell-lines were obtained from the National Cell Bank (NCBI), Pasteur Institute of Iran, Tehran, Iran. The cancerous MCF-7 cell-lines were cultured into Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (10%, FBS), glutamine (2 mM), and antibiotics (amphotericin B, penicillin G, and streptomycin, 50, 60, and 100 mg/L, respectively) under a relatively humid atmosphere (37°C, 5% CO2). Briefly, 1×105 cells/well were treated with diverse concentrations of V2O5NPs (10-100 μg/mL). After a 24 h incubation, the cells were washed two times by sterile phosphate-buffered saline (PBS), and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-triphenyltetrazolium bromide (MTT or thiazolyl blue; 0.5 mg/mL PBS) was added to the wells and incubated at 37°C for 4 h. The blue formazan crystals that formed were dissolved through adding dimethyl sulfoxide (100 μL/well), and the absorbance was read by a microplate scanning spectrophotometer at 570 nm (ELISA reader, Organon Teknika, Netherlands). The IC50 values of V2O5NPs on cancerous cells lines were then determined.


### 2.6.2. Cell Cytotoxicity Analysis by Optical Microscopy

Optical microscopic evaluation of the MCF-7 cells was performed to observe the morphological changes after exposure to V2O5NPs [35, 36]. MCF-7 cells were grown in a well plate and treated with V2O5NPs at the IC50 concentration for 24 h. The morphological changes of cells were observed under an invert optical microscope (Olympus, Japan).


### 2.6.3. Cell Cytotoxicity Analysis Using AO/EB Staining

Study of acridine orange (AO)/ethidium bromide (EB) fluorescent staining was performed according to the previous method with a few modifications [35]. Briefly, the MCF-7 cells (1×105/well) were deposited in a plate and treated by the IC50 concentration of V2O5NPs at 37°C for 24 h. The cells were collected, washed once with sterile PBS, and then suspended with sterile PBS. For AO/EB fluorescent staining, 1 μL of AO/EB (Sigma) dye mixture (10 mg/mL of AO/EB in PBS) was added to 9 μL of cell suspension and then covered with a cover slip on a clean slide. After incubation for 3 min, the cells were observed under a fluorescence microscope (Olympus, Japan).


### 2.6.4. Cell Cytotoxicity Analysis Using SEM and TEM

MCF-7 cells were prepared for scanning (SEM) and transmission electron microscopy (TEM) analysis according to the previous method with a few modifications [37]. Briefly, the cells were incubated for 24 h with mycosynthesized V2O5NPs at the IC50 concentration. The cells were harvested and fixed in 2.5% glutaraldehyde, washed with sodium cacodylate buffer, postfixed with 1% osmium tetroxide, dehydrated using gradual concentrations of ethanol, and dried using hexamethyldisilazane. Finally, the morphological changes of the cells were observed using SEM and TEM microscopy.


### 2.6.5. Cell Cytotoxicity Analysis by DNA Fragmentation

DNA fragmentation was performed according to a previous method [35]. An amount of 1×105 MCF-7 cells was exposed to V2O5NPs at IC50 concentrations at 37°C for 24 h. After slight harvesting with centrifugation, the cells were suspended in 10 mL of 10 mM TE buffer solution (pH 8.0, 10 mM EDTA, 10 mM Tris-HCl, 2% SDS, and 20 mg/mL proteinase K). The mixture was incubated for 3 h at 37°C, followed by DNA extraction. The DNA was treated with DNase-free RNase (20 mg/mL concentration, 4°C for 45 min) and precipitated with sodium acetate (100 mL, 2.5 M) and three volumes of absolute ethanol. A DNA fragmentation study was carried out with 10 μL of DNA prepared from treated cells by mycosynthesized V2O5NPs at the IC50 concentration and analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide for a period of 45 min at 100 V.


## 2.6.1. MTT Assay of Mycosynthesized V2O5NPs

The cytotoxicity effect of V2O5NPs was determined by the MTT assay [34]. The MCF-7 cell-lines were obtained from the National Cell Bank (NCBI), Pasteur Institute of Iran, Tehran, Iran. The cancerous MCF-7 cell-lines were cultured into Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (10%, FBS), glutamine (2 mM), and antibiotics (amphotericin B, penicillin G, and streptomycin, 50, 60, and 100 mg/L, respectively) under a relatively humid atmosphere (37°C, 5% CO2). Briefly, 1×105 cells/well were treated with diverse concentrations of V2O5NPs (10-100 μg/mL). After a 24 h incubation, the cells were washed two times by sterile phosphate-buffered saline (PBS), and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-triphenyltetrazolium bromide (MTT or thiazolyl blue; 0.5 mg/mL PBS) was added to the wells and incubated at 37°C for 4 h. The blue formazan crystals that formed were dissolved through adding dimethyl sulfoxide (100 μL/well), and the absorbance was read by a microplate scanning spectrophotometer at 570 nm (ELISA reader, Organon Teknika, Netherlands). The IC50 values of V2O5NPs on cancerous cells lines were then determined.


## 2.6.2. Cell Cytotoxicity Analysis by Optical Microscopy

Optical microscopic evaluation of the MCF-7 cells was performed to observe the morphological changes after exposure to V2O5NPs [35, 36]. MCF-7 cells were grown in a well plate and treated with V2O5NPs at the IC50 concentration for 24 h. The morphological changes of cells were observed under an invert optical microscope (Olympus, Japan).


## 2.6.3. Cell Cytotoxicity Analysis Using AO/EB Staining

Study of acridine orange (AO)/ethidium bromide (EB) fluorescent staining was performed according to the previous method with a few modifications [35]. Briefly, the MCF-7 cells (1×105/well) were deposited in a plate and treated by the IC50 concentration of V2O5NPs at 37°C for 24 h. The cells were collected, washed once with sterile PBS, and then suspended with sterile PBS. For AO/EB fluorescent staining, 1 μL of AO/EB (Sigma) dye mixture (10 mg/mL of AO/EB in PBS) was added to 9 μL of cell suspension and then covered with a cover slip on a clean slide. After incubation for 3 min, the cells were observed under a fluorescence microscope (Olympus, Japan).


## 2.6.4. Cell Cytotoxicity Analysis Using SEM and TEM

MCF-7 cells were prepared for scanning (SEM) and transmission electron microscopy (TEM) analysis according to the previous method with a few modifications [37]. Briefly, the cells were incubated for 24 h with mycosynthesized V2O5NPs at the IC50 concentration. The cells were harvested and fixed in 2.5% glutaraldehyde, washed with sodium cacodylate buffer, postfixed with 1% osmium tetroxide, dehydrated using gradual concentrations of ethanol, and dried using hexamethyldisilazane. Finally, the morphological changes of the cells were observed using SEM and TEM microscopy.


## 2.6.5. Cell Cytotoxicity Analysis by DNA Fragmentation

DNA fragmentation was performed according to a previous method [35]. An amount of 1×105 MCF-7 cells was exposed to V2O5NPs at IC50 concentrations at 37°C for 24 h. After slight harvesting with centrifugation, the cells were suspended in 10 mL of 10 mM TE buffer solution (pH 8.0, 10 mM EDTA, 10 mM Tris-HCl, 2% SDS, and 20 mg/mL proteinase K). The mixture was incubated for 3 h at 37°C, followed by DNA extraction. The DNA was treated with DNase-free RNase (20 mg/mL concentration, 4°C for 45 min) and precipitated with sodium acetate (100 mL, 2.5 M) and three volumes of absolute ethanol. A DNA fragmentation study was carried out with 10 μL of DNA prepared from treated cells by mycosynthesized V2O5NPs at the IC50 concentration and analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide for a period of 45 min at 100 V.


## 2.7. Statistical Analysis

The data were analyzed using the GraphPad Prism software Version 9.0, andP values under 0.05 were considered significant.


## 3. Results

### 3.1. Mycosynthesis of V2O5NPs

Exposure of the cell-free fungal culture filtrate ofF. oxysporum to NH4VO3 (1 mM final concentration) resulted in a time-dependent color change of the reaction mixture from light-yellowish to dark-brown, suggesting the successful biosynthesis of V2O5NPs. The change in color indicated the completion of reaction with vanadium ions. The intensity of color was correlated with the increase in the number of mycosynthesized V2O5NPs. At the same condition, the fungal culture filtrate (positive control) and the NH4VO3 solution (negative control) did not show any visual alteration.


### 3.2. Characterization of Mycosynthesized V2O5NPs

#### 3.2.1. UV-Visible Spectrophotometric Results

The absorption peak was obtained in the visible range at 410 nm after 96 h (Figure1(a)). The UV scan of mycosynthesized V2O5NPs revealed an absorbance peak of 410 nm, implying that V2O5NPs were successfully synthesized. Figure 1(b) shows the absorption peak of fungal cell-free filtrate and the NH4VO3 solution.Figure 1
(a) The UV-Vis spectrum of extracellular fungal filtrate reaction with 1 mM NH4VO3 solution (V2O5NPs). Mycosynthesized V2O5NPs showed the maximum absorption peak at around 410 nm after 96 h. (b) Extracellular fungal filtrate and 1 mM NH4VO3 solution.
(a)(b)


#### 3.2.2. DLS Observations

The DLS data has revealed that the size of mycosynthesized V2O5NPs was around 10-20 nm (Figure 2(a)). Further zeta potential of V2O5NPs was found to be −35.09 mV indicating its good stability of the bioreduced V2O5NPs (Figure 2(b)). The negative zeta potential value could be due to the capping of natural compounds present in the fungal cell-free filtrate. This negative value has indicated the strong repellent forces between particles due to high electrical charge on the surface of V2O5NPs, which in turn results in higher stability by preventing the aggregation of V2O5NPs.Figure 2
(a) DLS (particle size analysis) of mycosynthesized V2O5NPs. The size of mycosynthesized V2O5NPs was around 10-20 nm. (b) The zeta potential analysis of mycosynthesized V2O5NPs. The zeta potential was found to be −35.09 mV demonstrating good stability of the bioreduced V2O5NPs.
(a)(b)


#### 3.2.3. FESEM and TEM Observations

Figures3(a)–3(c) show the FESEM images of the mycosynthesized V2O5NPs in three magnifications implying that V2O5NPs were mostly spherical in shape with the estimated size ranging around 10-20 nm. Figure 3(d) shows the TEM analysis of the mycosynthesized V2O5NPs. TEM confirmed that the V2O5NPs were spherical in shape with the size ranging around 10-20 nm.Figure 3
(a–c) The FESEM image of mycosynthesized V2O5NPs in different magnifications. The image indicates that V2O5NPs were mostly spherical in shape with the estimated size ranging around 10-20 nm. (d) TEM image of V2O5NPs mycosynthesized by fungal cell-free filtrate confirmed the FESEM image.
(a)(b)(c)(d)


#### 3.2.4. XRD Results

The crystallographic nature of the mycosynthesized V2O5NPs was determined through XRD analysis, and the obtained results are demonstrated in Figure 4. The distinctive major diffraction peaks appeared at 2θ=15.2,20.1,21.6,26.1,30.9,32.2,33.1,34.2,41.0,41.8,45.3,47.2,48.6,51.1,51.9,55.4,and58.8 that corresponded to hkl, (200), (001), (101), (110), (310), (011), (111), (310), (002), (102), (411), (600), (302), (020), (601), (121), and (611) index planes. The obtained consequences were in good agreement with their standard JCPDS card No. 41-1426.Figure 4
XRD spectra of V2O5NPs mycosynthesized by extracellular fungal filtrate. The typical main diffraction peaks appeared at 2θ=15.2,20.1,21.6,26.1,30.9,32.2,33.1,34.2,41.0,41.8,45.3,47.2,48.6,51.1,51.9,55.4,and58.8 corresponding to hkl, (200), (001), (101), (110), (310), (011), (111), (310), (002), (102), (411), (600), (302), (020), (601), (121), and (611) index planes.


#### 3.2.5. FTIR Spectroscopy

The V2O5NPs mycosynthesized by the cell-free fungal filtrate of F. oxysporum were not in direct contact, signifying stabilization of the NPs by a capping agent which was confirmed using the FTIR analysis. Figure 5 shows the FTIR spectrum of F. oxysporum cell-free filtrate, NH4VO3, and mycosynthesized V2O5NPs. The FTIR spectrum of mycosynthesized V2O5NPs showed five distinct peaks, 1311, 1759, 2090, 2521, and 3403 cm−1.Figure 5
The FTIR spectrum of fungal cell-free filtrate, NH4VO3, and V2O5NPs mycosynthesized by extracellular fungal filtrate. For fungal cell-free filtrate, the peak at 3364 cm-1 was associated with N-H stretching, peak at 2521 cm-1 with S-H stretching, peaks at 2200 cm-1 with C≡C stretching, peaks at 1940 and 1797 cm-1 with C-H bending, and peaks at 1434 and 1316 cm-1 with O-H bending. For mycosynthesized V2O5NPs, the peak at 3403 cm-1 was associated with N-H stretching, peak at 2521 cm-1 with S-H stretching, peaks at 2090 cm-1 with C≡C stretching, peak at 1759 cm-1 with C-H bending, and peak at 1311 cm-1 with O-H bending. It was also confirmed that the carbonyl group from the protein and amino acid had stronger ability to bind with V2O5NPs or act as capping and stabilizing agents.


### 3.3. Effects of V2O5NPs on Fungal Radial Growth and Spore Germination

The results of antifungal activity of V2O5NPs are summarized in Table 1 which shows the inhibition of fungal growth in the range of 20.3 to 67.3%. As shown in Figure 6, the dose-dependent inhibition of mycelium growth was observed for all tested fungi compared with nontreated controls. The V2O5NPs were able to inhibit the spore germination of all selected fungi in the range of 64.8 to 89.9% with the maximum inhibition at the 100 μg/mL concentration (Table 1). The highest inhibition in spore germination was reported for Aspergillus fumigatus.Table 1
Effect of V2O5NPs on spore germination (%) and radial mycelium growth (% of inhibition) of pathogenic fungi in comparison with non-V2O5NP-treated control.
FungiV2O5NP concentration (μg/mL)5255010052550100Spore germination (%)Inhibition of mycelia growth (%)Fusarium oxysporum31.322.615.412.220.328.646.167.3Fusarium graminearum33.226.517.816.325.230.552.062.2Aspergillus fumigatus29.723.215.610.128.332.146.661.0Aspergillus niger27.221.417.119.726.230.443.355.7Aspergillus flavus34.731.225.520.628.133.440.548.6Alternaria alternata35.227.222.818.332.243.256.157.0Penicillium citrinum32.327.919.614.328.248.252.558.0Control: sterile distilled water.Figure 6
The radial growth of fungal mycelia of different pathogenic fungi exposed to various concentrations of V2O5NPs. A dose-dependent inhibition of fungal growth is evident for all tested fungi in comparison with nontreated controls. The highest inhibition of fungal growth is evident for 100 μg/mL of V2O5NP concentration.


### 3.4. Cytotoxic Effect of Mycosynthesized V2O5NPs

#### 3.4.1.In Vitro Cytotoxicity in MTT Assay

Figure7 indicates the results obtained by MTT on MCF-7 cells exposed to 1-100 μg/mL of V2O5NPs after 24 h. A concentration-dependent reduction in the viability of MCF-7 cancer cell-line was reported in the range of 10.74% to 85.45% which was significant compared to the nontreated control at all concentrations. The IC50 of the V2O5NPs for MCF-7 was calculated as 55.89 μg/mL.Figure 7
In vitro cytotoxicity of V2O5NPs for MCF-7 cell-lines examined by the MTT assay. All data were obtained from three independent (N=3) biological replicates in two separate experiments. Meaningful dose-dependent reduction in cell viability in the range of 10.74% to 85.45% is evident for all concentrations compared to nontreated control (∗∗P≤0.01, ∗∗∗P≤0.001, ∗∗∗∗P≤0.0001).


#### 3.4.2. Cell Cytotoxicity Results in Optical Microscopy

As indicated in Figure8, the chromatids of cancer cells were condensed in a sample treated with the IC50 concentration of V2O5NPs. Progressive structural modification and reduction of cancer cell populations were also evident.Figure 8
The optical, fluorescence, SEM, and TEM microscopy images of mycosynthesized V2O5NP-treated using IC50 concentration (55.89 μg/mL) and normal/untreated MCF-7 cells. The morphological changes and reduction of cancer cell populations were observed in treated MCF-7 cell-line exposed to V2O5NPs compared with normal/nontreated cells. Optical microscopy showed that chromatids of cancer cells were condensed, when treated with the IC50 concentration of V2O5NPs. AO/EB staining displayed that the nontreated proapoptotic cells and dead/necrotic cells were detected to be green, orange, or yellow, and because of the binding of AO into cell membranes, proapoptotic cells were detected as orange/yellow red color, respectively. TEM and SEM showed cell shrinkage, chromatin condensation, and integrity of plasma membrane after being treated with V2O5NPs.


#### 3.4.3. Cell Cytotoxicity Observations by AO/EB Staining

As shown in Figure8, the morphology of the necrotic/dead, apoptotic, and normal/untreated cells of MCF-7 cells was recognized separately using fluorescence microscopy after staining by AO/EB based on the cell-membrane integrity. MCF-7 cells displayed a green fluorescence color that shows the presence of live cells in controls (untreated cells). Orange/yellow-colored cells contain proapoptotic bodies and red necrotic/dead cells found in the mycosynthesized V2O5NP-treated MCF-7 cell-lines (Figure 8). Microscopic observation indicated that the untreated viable cells were detected to be green because of the binding of AO into cell membranes, whereas proapoptotic cells were detected as orange/yellow-colored bodies due to the shrinkage of nuclei and nuclear blebbing because of the induction of EB into cells. However, the dead/necrotic cells were turned into a red color due to their loss of outer membrane integrity using mycosynthesized V2O5NPs.


#### 3.4.4. Cell Cytotoxicity Results by SEM and TEM

After treatment with mycosynthesized V2O5NPs, the morphological changes were observed in treated MCF-7 cell-lines and also compared with normal/untreated cells. Figure 8 shows the SEM and TEM micrographs of the MCF-7 cell-line. The SEM micrograph shows that normal MCF-7 cancer cells (untreated) are round in shape and are characterized using short lamellipodia, whereas V2O5NP-treated cells show a semiflattened surface structure containing microvilli with extending lamellipodia known as membrane ruffles. The TEM micrograph displays morphological change in apoptotic characters in the mycosynthesized V2O5NP-treated MCF-7 cells. The cells show shrinkage, chromatin condensation, and integrity of plasma membrane.


#### 3.4.5. Cell Cytotoxicity Observations by DNA Fragmentation

Study of DNA fragmentation/damage was carried out to detect whether the death of MCF-7 cells had occurred outstanding to the cytotoxic effect of mycosynthesized V2O5NP treatment. As shown in Figure 9, DNA ladder formation with low molecular weight was detected in the V2O5NP-treated cells, while an intact DNA band was noticed in the control (normal/untreated) cell with high molecular weight.Figure 9
DNA fragmentation analysis of MCF-7 cell-lines. First line: 100 bp DNA ladder, second line: DNA from untreated/normal MCF-7 cell-line, and third line: DNA from mycosynthesized V2O5NP-treated MCF-7 cell-line. It was clear when the DNA of MCF-7 cell-line was treated with mycosynthesized V2O5NPs, DNA fragmentation has occurred compared with nontreated cell-line.


## 3.1. Mycosynthesis of V2O5NPs

Exposure of the cell-free fungal culture filtrate ofF. oxysporum to NH4VO3 (1 mM final concentration) resulted in a time-dependent color change of the reaction mixture from light-yellowish to dark-brown, suggesting the successful biosynthesis of V2O5NPs. The change in color indicated the completion of reaction with vanadium ions. The intensity of color was correlated with the increase in the number of mycosynthesized V2O5NPs. At the same condition, the fungal culture filtrate (positive control) and the NH4VO3 solution (negative control) did not show any visual alteration.


## 3.2. Characterization of Mycosynthesized V2O5NPs

### 3.2.1. UV-Visible Spectrophotometric Results

The absorption peak was obtained in the visible range at 410 nm after 96 h (Figure1(a)). The UV scan of mycosynthesized V2O5NPs revealed an absorbance peak of 410 nm, implying that V2O5NPs were successfully synthesized. Figure 1(b) shows the absorption peak of fungal cell-free filtrate and the NH4VO3 solution.Figure 1
(a) The UV-Vis spectrum of extracellular fungal filtrate reaction with 1 mM NH4VO3 solution (V2O5NPs). Mycosynthesized V2O5NPs showed the maximum absorption peak at around 410 nm after 96 h. (b) Extracellular fungal filtrate and 1 mM NH4VO3 solution.
(a)(b)


### 3.2.2. DLS Observations

The DLS data has revealed that the size of mycosynthesized V2O5NPs was around 10-20 nm (Figure 2(a)). Further zeta potential of V2O5NPs was found to be −35.09 mV indicating its good stability of the bioreduced V2O5NPs (Figure 2(b)). The negative zeta potential value could be due to the capping of natural compounds present in the fungal cell-free filtrate. This negative value has indicated the strong repellent forces between particles due to high electrical charge on the surface of V2O5NPs, which in turn results in higher stability by preventing the aggregation of V2O5NPs.Figure 2
(a) DLS (particle size analysis) of mycosynthesized V2O5NPs. The size of mycosynthesized V2O5NPs was around 10-20 nm. (b) The zeta potential analysis of mycosynthesized V2O5NPs. The zeta potential was found to be −35.09 mV demonstrating good stability of the bioreduced V2O5NPs.
(a)(b)


### 3.2.3. FESEM and TEM Observations

Figures3(a)–3(c) show the FESEM images of the mycosynthesized V2O5NPs in three magnifications implying that V2O5NPs were mostly spherical in shape with the estimated size ranging around 10-20 nm. Figure 3(d) shows the TEM analysis of the mycosynthesized V2O5NPs. TEM confirmed that the V2O5NPs were spherical in shape with the size ranging around 10-20 nm.Figure 3
(a–c) The FESEM image of mycosynthesized V2O5NPs in different magnifications. The image indicates that V2O5NPs were mostly spherical in shape with the estimated size ranging around 10-20 nm. (d) TEM image of V2O5NPs mycosynthesized by fungal cell-free filtrate confirmed the FESEM image.
(a)(b)(c)(d)


### 3.2.4. XRD Results

The crystallographic nature of the mycosynthesized V2O5NPs was determined through XRD analysis, and the obtained results are demonstrated in Figure 4. The distinctive major diffraction peaks appeared at 2θ=15.2,20.1,21.6,26.1,30.9,32.2,33.1,34.2,41.0,41.8,45.3,47.2,48.6,51.1,51.9,55.4,and58.8 that corresponded to hkl, (200), (001), (101), (110), (310), (011), (111), (310), (002), (102), (411), (600), (302), (020), (601), (121), and (611) index planes. The obtained consequences were in good agreement with their standard JCPDS card No. 41-1426.Figure 4
XRD spectra of V2O5NPs mycosynthesized by extracellular fungal filtrate. The typical main diffraction peaks appeared at 2θ=15.2,20.1,21.6,26.1,30.9,32.2,33.1,34.2,41.0,41.8,45.3,47.2,48.6,51.1,51.9,55.4,and58.8 corresponding to hkl, (200), (001), (101), (110), (310), (011), (111), (310), (002), (102), (411), (600), (302), (020), (601), (121), and (611) index planes.


### 3.2.5. FTIR Spectroscopy

The V2O5NPs mycosynthesized by the cell-free fungal filtrate of F. oxysporum were not in direct contact, signifying stabilization of the NPs by a capping agent which was confirmed using the FTIR analysis. Figure 5 shows the FTIR spectrum of F. oxysporum cell-free filtrate, NH4VO3, and mycosynthesized V2O5NPs. The FTIR spectrum of mycosynthesized V2O5NPs showed five distinct peaks, 1311, 1759, 2090, 2521, and 3403 cm−1.Figure 5
The FTIR spectrum of fungal cell-free filtrate, NH4VO3, and V2O5NPs mycosynthesized by extracellular fungal filtrate. For fungal cell-free filtrate, the peak at 3364 cm-1 was associated with N-H stretching, peak at 2521 cm-1 with S-H stretching, peaks at 2200 cm-1 with C≡C stretching, peaks at 1940 and 1797 cm-1 with C-H bending, and peaks at 1434 and 1316 cm-1 with O-H bending. For mycosynthesized V2O5NPs, the peak at 3403 cm-1 was associated with N-H stretching, peak at 2521 cm-1 with S-H stretching, peaks at 2090 cm-1 with C≡C stretching, peak at 1759 cm-1 with C-H bending, and peak at 1311 cm-1 with O-H bending. It was also confirmed that the carbonyl group from the protein and amino acid had stronger ability to bind with V2O5NPs or act as capping and stabilizing agents.


## 3.2.1. UV-Visible Spectrophotometric Results

The absorption peak was obtained in the visible range at 410 nm after 96 h (Figure1(a)). The UV scan of mycosynthesized V2O5NPs revealed an absorbance peak of 410 nm, implying that V2O5NPs were successfully synthesized. Figure 1(b) shows the absorption peak of fungal cell-free filtrate and the NH4VO3 solution.Figure 1
(a) The UV-Vis spectrum of extracellular fungal filtrate reaction with 1 mM NH4VO3 solution (V2O5NPs). Mycosynthesized V2O5NPs showed the maximum absorption peak at around 410 nm after 96 h. (b) Extracellular fungal filtrate and 1 mM NH4VO3 solution.
(a)(b)


## 3.2.2. DLS Observations

The DLS data has revealed that the size of mycosynthesized V2O5NPs was around 10-20 nm (Figure 2(a)). Further zeta potential of V2O5NPs was found to be −35.09 mV indicating its good stability of the bioreduced V2O5NPs (Figure 2(b)). The negative zeta potential value could be due to the capping of natural compounds present in the fungal cell-free filtrate. This negative value has indicated the strong repellent forces between particles due to high electrical charge on the surface of V2O5NPs, which in turn results in higher stability by preventing the aggregation of V2O5NPs.Figure 2
(a) DLS (particle size analysis) of mycosynthesized V2O5NPs. The size of mycosynthesized V2O5NPs was around 10-20 nm. (b) The zeta potential analysis of mycosynthesized V2O5NPs. The zeta potential was found to be −35.09 mV demonstrating good stability of the bioreduced V2O5NPs.
(a)(b)


## 3.2.3. FESEM and TEM Observations

Figures3(a)–3(c) show the FESEM images of the mycosynthesized V2O5NPs in three magnifications implying that V2O5NPs were mostly spherical in shape with the estimated size ranging around 10-20 nm. Figure 3(d) shows the TEM analysis of the mycosynthesized V2O5NPs. TEM confirmed that the V2O5NPs were spherical in shape with the size ranging around 10-20 nm.Figure 3
(a–c) The FESEM image of mycosynthesized V2O5NPs in different magnifications. The image indicates that V2O5NPs were mostly spherical in shape with the estimated size ranging around 10-20 nm. (d) TEM image of V2O5NPs mycosynthesized by fungal cell-free filtrate confirmed the FESEM image.
(a)(b)(c)(d)


## 3.2.4. XRD Results

The crystallographic nature of the mycosynthesized V2O5NPs was determined through XRD analysis, and the obtained results are demonstrated in Figure 4. The distinctive major diffraction peaks appeared at 2θ=15.2,20.1,21.6,26.1,30.9,32.2,33.1,34.2,41.0,41.8,45.3,47.2,48.6,51.1,51.9,55.4,and58.8 that corresponded to hkl, (200), (001), (101), (110), (310), (011), (111), (310), (002), (102), (411), (600), (302), (020), (601), (121), and (611) index planes. The obtained consequences were in good agreement with their standard JCPDS card No. 41-1426.Figure 4
XRD spectra of V2O5NPs mycosynthesized by extracellular fungal filtrate. The typical main diffraction peaks appeared at 2θ=15.2,20.1,21.6,26.1,30.9,32.2,33.1,34.2,41.0,41.8,45.3,47.2,48.6,51.1,51.9,55.4,and58.8 corresponding to hkl, (200), (001), (101), (110), (310), (011), (111), (310), (002), (102), (411), (600), (302), (020), (601), (121), and (611) index planes.


## 3.2.5. FTIR Spectroscopy

The V2O5NPs mycosynthesized by the cell-free fungal filtrate of F. oxysporum were not in direct contact, signifying stabilization of the NPs by a capping agent which was confirmed using the FTIR analysis. Figure 5 shows the FTIR spectrum of F. oxysporum cell-free filtrate, NH4VO3, and mycosynthesized V2O5NPs. The FTIR spectrum of mycosynthesized V2O5NPs showed five distinct peaks, 1311, 1759, 2090, 2521, and 3403 cm−1.Figure 5
The FTIR spectrum of fungal cell-free filtrate, NH4VO3, and V2O5NPs mycosynthesized by extracellular fungal filtrate. For fungal cell-free filtrate, the peak at 3364 cm-1 was associated with N-H stretching, peak at 2521 cm-1 with S-H stretching, peaks at 2200 cm-1 with C≡C stretching, peaks at 1940 and 1797 cm-1 with C-H bending, and peaks at 1434 and 1316 cm-1 with O-H bending. For mycosynthesized V2O5NPs, the peak at 3403 cm-1 was associated with N-H stretching, peak at 2521 cm-1 with S-H stretching, peaks at 2090 cm-1 with C≡C stretching, peak at 1759 cm-1 with C-H bending, and peak at 1311 cm-1 with O-H bending. It was also confirmed that the carbonyl group from the protein and amino acid had stronger ability to bind with V2O5NPs or act as capping and stabilizing agents.


## 3.3. Effects of V2O5NPs on Fungal Radial Growth and Spore Germination

The results of antifungal activity of V2O5NPs are summarized in Table 1 which shows the inhibition of fungal growth in the range of 20.3 to 67.3%. As shown in Figure 6, the dose-dependent inhibition of mycelium growth was observed for all tested fungi compared with nontreated controls. The V2O5NPs were able to inhibit the spore germination of all selected fungi in the range of 64.8 to 89.9% with the maximum inhibition at the 100 μg/mL concentration (Table 1). The highest inhibition in spore germination was reported for Aspergillus fumigatus.Table 1
Effect of V2O5NPs on spore germination (%) and radial mycelium growth (% of inhibition) of pathogenic fungi in comparison with non-V2O5NP-treated control.
FungiV2O5NP concentration (μg/mL)5255010052550100Spore germination (%)Inhibition of mycelia growth (%)Fusarium oxysporum31.322.615.412.220.328.646.167.3Fusarium graminearum33.226.517.816.325.230.552.062.2Aspergillus fumigatus29.723.215.610.128.332.146.661.0Aspergillus niger27.221.417.119.726.230.443.355.7Aspergillus flavus34.731.225.520.628.133.440.548.6Alternaria alternata35.227.222.818.332.243.256.157.0Penicillium citrinum32.327.919.614.328.248.252.558.0Control: sterile distilled water.Figure 6
The radial growth of fungal mycelia of different pathogenic fungi exposed to various concentrations of V2O5NPs. A dose-dependent inhibition of fungal growth is evident for all tested fungi in comparison with nontreated controls. The highest inhibition of fungal growth is evident for 100 μg/mL of V2O5NP concentration.


## 3.4. Cytotoxic Effect of Mycosynthesized V2O5NPs

### 3.4.1.In Vitro Cytotoxicity in MTT Assay

Figure7 indicates the results obtained by MTT on MCF-7 cells exposed to 1-100 μg/mL of V2O5NPs after 24 h. A concentration-dependent reduction in the viability of MCF-7 cancer cell-line was reported in the range of 10.74% to 85.45% which was significant compared to the nontreated control at all concentrations. The IC50 of the V2O5NPs for MCF-7 was calculated as 55.89 μg/mL.Figure 7
In vitro cytotoxicity of V2O5NPs for MCF-7 cell-lines examined by the MTT assay. All data were obtained from three independent (N=3) biological replicates in two separate experiments. Meaningful dose-dependent reduction in cell viability in the range of 10.74% to 85.45% is evident for all concentrations compared to nontreated control (∗∗P≤0.01, ∗∗∗P≤0.001, ∗∗∗∗P≤0.0001).


### 3.4.2. Cell Cytotoxicity Results in Optical Microscopy

As indicated in Figure8, the chromatids of cancer cells were condensed in a sample treated with the IC50 concentration of V2O5NPs. Progressive structural modification and reduction of cancer cell populations were also evident.Figure 8
The optical, fluorescence, SEM, and TEM microscopy images of mycosynthesized V2O5NP-treated using IC50 concentration (55.89 μg/mL) and normal/untreated MCF-7 cells. The morphological changes and reduction of cancer cell populations were observed in treated MCF-7 cell-line exposed to V2O5NPs compared with normal/nontreated cells. Optical microscopy showed that chromatids of cancer cells were condensed, when treated with the IC50 concentration of V2O5NPs. AO/EB staining displayed that the nontreated proapoptotic cells and dead/necrotic cells were detected to be green, orange, or yellow, and because of the binding of AO into cell membranes, proapoptotic cells were detected as orange/yellow red color, respectively. TEM and SEM showed cell shrinkage, chromatin condensation, and integrity of plasma membrane after being treated with V2O5NPs.


### 3.4.3. Cell Cytotoxicity Observations by AO/EB Staining

As shown in Figure8, the morphology of the necrotic/dead, apoptotic, and normal/untreated cells of MCF-7 cells was recognized separately using fluorescence microscopy after staining by AO/EB based on the cell-membrane integrity. MCF-7 cells displayed a green fluorescence color that shows the presence of live cells in controls (untreated cells). Orange/yellow-colored cells contain proapoptotic bodies and red necrotic/dead cells found in the mycosynthesized V2O5NP-treated MCF-7 cell-lines (Figure 8). Microscopic observation indicated that the untreated viable cells were detected to be green because of the binding of AO into cell membranes, whereas proapoptotic cells were detected as orange/yellow-colored bodies due to the shrinkage of nuclei and nuclear blebbing because of the induction of EB into cells. However, the dead/necrotic cells were turned into a red color due to their loss of outer membrane integrity using mycosynthesized V2O5NPs.


### 3.4.4. Cell Cytotoxicity Results by SEM and TEM

After treatment with mycosynthesized V2O5NPs, the morphological changes were observed in treated MCF-7 cell-lines and also compared with normal/untreated cells. Figure 8 shows the SEM and TEM micrographs of the MCF-7 cell-line. The SEM micrograph shows that normal MCF-7 cancer cells (untreated) are round in shape and are characterized using short lamellipodia, whereas V2O5NP-treated cells show a semiflattened surface structure containing microvilli with extending lamellipodia known as membrane ruffles. The TEM micrograph displays morphological change in apoptotic characters in the mycosynthesized V2O5NP-treated MCF-7 cells. The cells show shrinkage, chromatin condensation, and integrity of plasma membrane.


### 3.4.5. Cell Cytotoxicity Observations by DNA Fragmentation

Study of DNA fragmentation/damage was carried out to detect whether the death of MCF-7 cells had occurred outstanding to the cytotoxic effect of mycosynthesized V2O5NP treatment. As shown in Figure 9, DNA ladder formation with low molecular weight was detected in the V2O5NP-treated cells, while an intact DNA band was noticed in the control (normal/untreated) cell with high molecular weight.Figure 9
DNA fragmentation analysis of MCF-7 cell-lines. First line: 100 bp DNA ladder, second line: DNA from untreated/normal MCF-7 cell-line, and third line: DNA from mycosynthesized V2O5NP-treated MCF-7 cell-line. It was clear when the DNA of MCF-7 cell-line was treated with mycosynthesized V2O5NPs, DNA fragmentation has occurred compared with nontreated cell-line.


## 3.4.1.In Vitro Cytotoxicity in MTT Assay

Figure7 indicates the results obtained by MTT on MCF-7 cells exposed to 1-100 μg/mL of V2O5NPs after 24 h. A concentration-dependent reduction in the viability of MCF-7 cancer cell-line was reported in the range of 10.74% to 85.45% which was significant compared to the nontreated control at all concentrations. The IC50 of the V2O5NPs for MCF-7 was calculated as 55.89 μg/mL.Figure 7
In vitro cytotoxicity of V2O5NPs for MCF-7 cell-lines examined by the MTT assay. All data were obtained from three independent (N=3) biological replicates in two separate experiments. Meaningful dose-dependent reduction in cell viability in the range of 10.74% to 85.45% is evident for all concentrations compared to nontreated control (∗∗P≤0.01, ∗∗∗P≤0.001, ∗∗∗∗P≤0.0001).


## 3.4.2. Cell Cytotoxicity Results in Optical Microscopy

As indicated in Figure8, the chromatids of cancer cells were condensed in a sample treated with the IC50 concentration of V2O5NPs. Progressive structural modification and reduction of cancer cell populations were also evident.Figure 8
The optical, fluorescence, SEM, and TEM microscopy images of mycosynthesized V2O5NP-treated using IC50 concentration (55.89 μg/mL) and normal/untreated MCF-7 cells. The morphological changes and reduction of cancer cell populations were observed in treated MCF-7 cell-line exposed to V2O5NPs compared with normal/nontreated cells. Optical microscopy showed that chromatids of cancer cells were condensed, when treated with the IC50 concentration of V2O5NPs. AO/EB staining displayed that the nontreated proapoptotic cells and dead/necrotic cells were detected to be green, orange, or yellow, and because of the binding of AO into cell membranes, proapoptotic cells were detected as orange/yellow red color, respectively. TEM and SEM showed cell shrinkage, chromatin condensation, and integrity of plasma membrane after being treated with V2O5NPs.


## 3.4.3. Cell Cytotoxicity Observations by AO/EB Staining

As shown in Figure8, the morphology of the necrotic/dead, apoptotic, and normal/untreated cells of MCF-7 cells was recognized separately using fluorescence microscopy after staining by AO/EB based on the cell-membrane integrity. MCF-7 cells displayed a green fluorescence color that shows the presence of live cells in controls (untreated cells). Orange/yellow-colored cells contain proapoptotic bodies and red necrotic/dead cells found in the mycosynthesized V2O5NP-treated MCF-7 cell-lines (Figure 8). Microscopic observation indicated that the untreated viable cells were detected to be green because of the binding of AO into cell membranes, whereas proapoptotic cells were detected as orange/yellow-colored bodies due to the shrinkage of nuclei and nuclear blebbing because of the induction of EB into cells. However, the dead/necrotic cells were turned into a red color due to their loss of outer membrane integrity using mycosynthesized V2O5NPs.


## 3.4.4. Cell Cytotoxicity Results by SEM and TEM

After treatment with mycosynthesized V2O5NPs, the morphological changes were observed in treated MCF-7 cell-lines and also compared with normal/untreated cells. Figure 8 shows the SEM and TEM micrographs of the MCF-7 cell-line. The SEM micrograph shows that normal MCF-7 cancer cells (untreated) are round in shape and are characterized using short lamellipodia, whereas V2O5NP-treated cells show a semiflattened surface structure containing microvilli with extending lamellipodia known as membrane ruffles. The TEM micrograph displays morphological change in apoptotic characters in the mycosynthesized V2O5NP-treated MCF-7 cells. The cells show shrinkage, chromatin condensation, and integrity of plasma membrane.


## 3.4.5. Cell Cytotoxicity Observations by DNA Fragmentation

Study of DNA fragmentation/damage was carried out to detect whether the death of MCF-7 cells had occurred outstanding to the cytotoxic effect of mycosynthesized V2O5NP treatment. As shown in Figure 9, DNA ladder formation with low molecular weight was detected in the V2O5NP-treated cells, while an intact DNA band was noticed in the control (normal/untreated) cell with high molecular weight.Figure 9
DNA fragmentation analysis of MCF-7 cell-lines. First line: 100 bp DNA ladder, second line: DNA from untreated/normal MCF-7 cell-line, and third line: DNA from mycosynthesized V2O5NP-treated MCF-7 cell-line. It was clear when the DNA of MCF-7 cell-line was treated with mycosynthesized V2O5NPs, DNA fragmentation has occurred compared with nontreated cell-line.


## 4. Discussion

Like many other transition metals, vanadium possesses useful optical, electronic, magnetic, and catalytic properties. Vanadium nanoparticles have received major consideration in recent years due to use in developing of compounds with novel biological applications and pharmacological properties [23].In the present study, V2O5NPs were successfully synthesized by a green approach using F. oxysporum as a fungal cell factory. Synthesized V2O5NPs showed maximum absorption peak at around 410 nm. They were mostly spherical with size around 10-20 nm with a zeta potential value of -35.09 mV, indicating that capped biomolecules have a highly negatively charged surface. FTIR confirmed that the carbonyl group from the protein(s) and amino acid has a stronger ability to bind with V2O5NPs or acts as capping and stabilizing agents. Synthesized V2O5NPs showed strong antifungal properties against a wide variety of pathogenic fungi, cell cytotoxicity toward MCF-7 cancer cell-line which showed shrinkage, chromatin condensation, integrity of plasma membrane, and DNA fragmentation indicating possible cancer cell apoptosis.Although biosynthesis of various metal nanoparticles byF. oxysporum has been reported and confirmed by different researchers [38–41], little has been documented about green synthesis of V2O5NPs using fungi. Likewise, in most cases, the biological activity of synthesized V2O5NPs has not been studied.Aliyu et al. [25] reported green synthesis of vanadium nanoparticles with spherical shape by leaf extract of Moringa oleifera [25]. Synthesized NPs showed antibacterial activity toward Escherichia coli and Salmonella typhi, but they did not show any obvious antifungal activity against Candida tropicalis and Candia albicans. Prasad et al. [42] reported green synthesis of vanadium oxide (10-60 nm) nanorods using Phyllanthus amarus [42]. They found that synthesized NPs had antibreast cancer properties. Farahmandjou and Abaeiyan [23] synthesized vanadium oxide NPs by a simple chemical technique using sodium metavanadate as a precursor and cetyltrimethylammonium bromide as the surfactant [23]. Synthesized NPs were in the range size of 5-10 nm.Jabbar et al. [24] synthesized V2O5NPs using the Foeniculum vulgare stem. Synthesized NPs were around 78 nm with cubic-like agglomerated morphology. The maximum absorbance of the NPs was 452 nm at 50 minutes. The FTIR of their NPs showed three peaks at 940 cm-1, 835 cm-1, and 718 cm-1. NPs showed 2 peaks at 2θ=20°and26° with the structural planes (001) and (101) in XRD [24].Wasmi et al. [43] synthesized vanadium pentoxide NPs by the solvothermal synthesis process that has almost perfect rods with 60-90 nm in size [43]. Taylor et al. [44] synthesized vanadium pentoxide NPs using pulsed laser ablation. Synthesized NPs have nearly spherical and flower-like morphologies. The colloidal solution of NPs had a zeta potential of −51 mV. Indeed, solutions with zeta potential smaller than −30 mV or larger than +30 mV were considered stable [44].Pinto et al. [45] synthesized vanadium NPs with size around 7 nm on activated carbon by the reduction of VCl3 salt. Rasheed et al. [46] green-synthesized vanadium oxide-zirconium oxide nanocomposite using Daphne alpine leaf extracts. The size of their NPs was 41.74 nm with different shapes. The diffraction bands appeared at the 2θ position with corresponding hkl value positions of 21.40 (200), 28.16 (103), 34.04 (004), 36.67 (213), 42.80 (303), 43.68 (400), and 56.43 (501). The FTIR of their NPs was showed stretching vibration of the OH group at around 3499 cm−1. The peaks at around 3000 and 2942 cm−1 are for C-H bending, whereas the band at 1725 cm−1 may be due to the carbonyl group of ester and carboxylic acid. The bands at 1433 and 1220 cm−1 are attributed to the N=O bending mode and carbonyl stretching, respectively [46].


## 5. Conclusions

In the present study, we successfully synthesized biologically active V2O5NPs by a green approach using an extracellular fungal filtrate from a hyaline hyphomycete, F. oxysporum, in a single-step bioreduction of vanadium ions for the first time. Our green-synthesized V2O5NPs displayed strong inhibitory activity toward the growth and spore germination of a wide variety of pathogenic fungi indicating that they may be considered as potential therapeutic agents in drug development against life-threatening fungal pathogens. Furthermore, cytotoxic effects of V2O5NPs against the breast cancer MCF-7 cell-line make them suitable in the line of anticancer studies. Further works on in vivo cytotoxicity and antifungal activity of green-synthesized V2O5NPs in animal experimental models are recommended.



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*Source: 7532660-2021-10-22.xml*