Evaluation of antioxidant and anticancer properties of zinc oxide nanoparticles synthesized using Aspergillus niger extract

Microorganisms and plants have grabbed great attention as potential biological sources for ecofriendly synthesizing nanoparticles. In this study, zinc nitrate and Aspergillus niger were applied to synthesize stable spherical zinc oxide nanoparticles (ZnO-NPs). The FTIR, DLS, SEM, TEM and XRD methods were utilized to characterize the synthesized nanoparticles in terms of structure, morphology, and optic features. Electron microscopic images revealed poly dispersed nanoparticles with the length of 30 to 70 nm. Regarding morphology, the majority of the particles were spherical. Regarding antioxidant capacity, the synthesized ZnO-NPs showed the IC50 of about 1000 μg ml−1. The synthesized ZnO-NPs also induced apoptosis and inhibited cellular growth in neoplastic MCF-7 cells.


Introduction
Nanotechnology is the science of engineering nanoscale (1-100 nm) materials (i.e. nanoparticles-NPs), characterizing their physiochemical features, and finally divulging their effects on living cells [1][2][3][4]. Various medicinal diagnostic (e.g. imaging techniques, biosensing) and therapeutic applications (e.g. drug targeting, cancer therapy) have been noted for metal oxides as sources for synthesizing inorganic NPs [5][6][7][8]. Zinc oxide (ZnO) NPs are commonly utilized as solar cells, biosensors, and photocatalysts in various industrial and pharmaceutical fields [9][10][11][12][13]. Also, ZnO-NPs have been applied as safe antimicrobial agents in biomedicine [14][15][16][17]. In addition, ZnO-NPs are resistant against degradation by microorganisms delivering them as stable sources for developing antimicrobial agents [18]. The functional properties of ZnO-NPs largely depend on their structural (e.g. size, morphology, direction, and surface ratio) and physiochemical (e.g. electrical and thermal conductance) features [19,20]. Although physicochemical methods which are commonly used to synthesize NPs can deliver large volumes of these particles in a relatively short period of time, trace depositions of toxic elements on the surface of physiochemically synthesized materials limit their clinical application [21]. As alternatives to physicochemical biosynthesis methods, green-synthesis approaches have increasingly been used to safely and ecofriendly produce NPs [22,23].
Anti-apoptotic mechanisms of cancer cells are important for their proliferation and propagation [24]. Therefore, apoptosis induction is one of the main therapeutic goals of any cancer therapeutic agent [25]. In the present study, we aimed to ascertain the cytotoxicity effects of ZnO-NPs against MCF-7 breast cancer cell line. Furthermore, the antioxidant capacity of the biosynthesized ZnO-NPs was also studied.
Fungi have notable metal biding and bioaccumulating abilities and produce a wide variety of enzymes. They are also ease in the scale-up process, handling the biomass, and cost-effective to be grown [26,27]. In this study, we used Aspergillus niger to synthesize ZnO-NPs and evaluate their antioxidant and anticancer properties.

ZnO-NPs synthesis
In this study, A. niger (PTCC: 5012) was prepared from the Iranian Bank of industrial fungi and bacteria. After activation, 2 ml of the fungus suspension was transferred to plates containing Potato Dextrose Agar (PDA). The plates were incubated for 7 to 10 days at 25°C. The fungal biomass was cultured in a fluid culture medium while shaking. After that, 10 ml of 0.1 M sterile nitrate solution was prepared and added to 50 ml of the bacterial culture medium. The solution was heated in a hot water bath at 80°C for 10 to 5 min. The turning to white of the precipitate marked the beginning of the production of ZnO-NPs.

Characterizing of biosynthesized ZnO-NPs
The synthetized ZnONPs were characterized using particle size analyzer, as well as TEM, SEM, XRD, and FTIR analyses. The physical properties (i.e. size and shape) of the ZnO-NPs were characterized by TEM (JEOL, Japan) and FESEM (JEOL, Japan). The crystal structure and purity of the ZnO-NPs were also determined using Philips PW1800 x-ray diffractometer (XRD) (Almelo, Netherlands). Meanwhile, FTIR was carried out (Perkin Elmer, Walthman, MA, USA) to further characterize the NPs.

DPPH test
DPPH method was utilized to determine the radical scavenging capacity of the synthesized NPs. Equal volumes of various concentrations of ZnO-NPs were admixed with 0.1 mM methanolic DPPH solution. The solutions were then incubated at room temperature for 30 min. The absorbance of the sample was finally read at 517 nm. The butylated hydroxyanisole (BHA) was used as a reference antioxidant compound [28].
2.5. Apoptosis assay using flow-cytometry and AO/PI staining The MCF-7 cells were cultured in 6-well plates and then treated with different concentrations of ZnO-NPs for 48 h. After incubation and washing with Phosphate-buffered saline (PBS), 1 mg ml −1 PI was added to the wells. Finally, the plates were placed in incubator for 30 min, and then the cells were separated and analyzed using flow-cytometry. To perform acridine-orange (AO) test, cell suspensions (5 ml) were cultured and incubated for 24 h. At the next step, the culture medium was discarded, and the cells were treated with different concentrations of ZnO-NPs for 48 h. After trypsinization and washing cells with PBS, 10 μl acridine orange and 10 μl PI (Propodium Iodide) were added to 10 μl of the cell suspension. The mixture was then incubated for 5 min. In the next step, 20 μl of the mixture was placed on a slide to be photographed and examined by fluorescence microscopy [30].

Characterizing of the biosynthesized ZnO-NPs
The x-ray diffraction (XRD) pattern was acquired using a x-ray diffractometer (Panalytical X'PERT) equipped with a Ni filter applying Cu Kα (λ=1.540 56 Å) radiation as an x-ray source at room temperature. The crystalline structure of the ZnO-NPs has been shown in      representing flavonoid and phenolic (i.e. aromatic) groups in the structure of biosynthesized ZnO-NPs. Another study showed the peak at 1637.56 cm −1 showing −C=Caromatic structure [31]. Thus, the strong aromatic ring identified in FTIR analysis seems to participate in the biosynthesis of ZnO-NPs.

DPPH antioxidant assay
The capacity of ZnO-NPs to scavenge free radicals was investigated by the DPPH assay which is an easy, rapid, and amenable (requiring only an UV-vis spectrophotometer) method widely used to determine antioxidant activity of plant extracts [32]. The results showed that the synthesized ZnO-NPs dose-dependently scavenged free radicals ( figure 6, p<0.001). The IC 50 of the NPs was obtained as 1000 μg ml −1 . The BHA was used as a reference antioxidant.

Cytotoxicity activity of ZnO-NPs
The cytotoxicity of the synthesized ZnO-NPs against MCF-7 breast cancer cells was investigated by MTT assay at 0, 15.6, 31.

Conclusion
Fungi have gained attention as benefical sources for synthesizing nano-materials. We here utilized A. niger extract to introduce a simple and fast approach to synthesize ZnO-NPs. The synthesized ZnO-NPs were revealed as spehrical particles with an average length of 40 nm (the range of 30 to 70 nm). Various biological compounds in A. niger extract (e.g. phenols, flavonoids, and proteins) can contribute to the development of ZnO-NPs. The biosynthesized ZnO-NPs showed potent antioxidant activity and also inhibited the growth of MCF-7 cancer cells.