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Biogenic synthesis and cytotoxic effects of silver nanoparticles mediated by white rot fungi

Silver nanoparticles (AgNPs) were successfully synthesized using silver nitrate via the biological route using the culture filtrate of Ganoderma enigmaticum as well as Trametes ljubarskyi white rot fungi materials at room temperature. The proposed synthetic technique was applied for the first time for AgNPs preparation via the biological route through a low-cost pathway, which considered as an adequate direction of preparation compared to the commercial methods. This study reports the in vitro cytotoxic effect of biologically synthesized AgNPs in disposing of the human lung cancer cell line (A549) and human breast cancer cell (MCF-7) by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, the viability of the tested cell lines was tested after treatment for 24 h in the presence of the prepared nanoparticles. The obtained results indicated the reduced viability of cancer cell lines with improving concentrations of AgNPs (40–120 μg/mL) at 24 h. Furthermore, at 120 μg/mL concentration, the fungal nanoparticles showed substantial cytotoxic effects toward the treated cells. Consequently, the results designated that the biologically synthesized silver nanoparticles have effective behavior for treating A549 and MCF-7 cancer cells from the laboratory experiment approach; however, additional studies are required to validate these results in vivo models as anticancer agents depending on their cytotoxic activity.


Introduction
Nanobiotechnology as a branch of nanoscience can use several nanobased systems for a diversity of biomedical applications, including treatment pathways of human cancers [1,2,3,4,5]. Furthermore, the reason for using nanoparticles for disease therapy depends on their structure and dimension compared to the conventional small-molecule medicines [6,7,8]. Amongst the metal nanoparticles, silver nanoparticles (AgNPs) are a good candidate for infection control owing to their contact with the mitochondrial respiratory chain, which shows compatibility and bioavailability toward the treatment of various persistent illnesses, including cancer [9,10,11]. In addition, AgNPs have the advantage of disrupting mitochondrial function with the aid of prompting the generation of reactive oxygen species and the advantage of facilitating the preparation of ATP [12]. Metallic silver with ultrafine particles in nanometer (nm) size have distinct morphologies and characteristics that pave the way toward using it in different engineering technologies [13,14]. Moreover, the purpose of using AgNPs for nanomedicine applications may come from their special features and clear therapeutic prospects in considering a range of diseases, such as retinal neovascularization as well as developed immunodeficiency syndrome because of the human immunodeficiency virus [15,16,17,18]. Additionally, the antimicrobial perspective of AgNPs against numerous other viruses, such as hepatitis B, respiratory syncytial virus, and herpes simplex virus type make AgNPs possess intrinsic cytotoxic action toward diseases [19]. Currently, cancer is a leading cause of death across the world, which directs scientists worldwide to find several therapies for treating numerous kinds of cancer cells [20]. Consequently, chemotherapy involving the use of cytotoxic agents is the further most normally applied treatment to handle several kinds of cancer cells [21]. Alternatively, these therapeutic methods are related to serious side-effect results, particularly multidrug resistance (MDR). Therefore, to overcome the undesirable side effects results of chemotherapy, numerous medically associated nanoparticles like those that AgNPs were considered alone or in blend owing to their in vitro cytotoxicity aspect for drug therapy and radiation treatment [22]. Further, several researches have been accomplished to use silver nanoparticles as an anti-cancer agent, and they have all shown positive results, which paves the way for different new directions in the field of medication [23]. To the best of our knowledge, only a few studies on the effect of biosynthesized AgNPs toward tumor control via their cytotoxic effects. One research work detected the antiproliferative activity of AgNPs against different cancer cells including the   [24]. Another study assessed the silver nanoparticles synthesized from different biological sources such as Penicillium brevicompactum, Trichoderma viride, and Croton Bonplandianum and indicated their potent cytotoxicity against different cancer cells via MTT assay through in vitro analysis. Silver nanoparticles showed promising effects on cancer cells up to a certain concentration. Using different concentrations of nanoparticles, the IC50 value showed different values of (% cell viability) and (% cell inhibition) for the different cells' lines depending on the time of incubation [25,26,27]. There are several biological bases of AgNPs synthesis, and the difference between the current prepared materials compared to the others is the source of the biological route [28,29,30,31,32]. Here, the difference between the current work and previous ones is that we intended to prepare AgNPs via the biological route using Ganoderma enigmaticum as well as Trametes ljubarskyi white rot fungus materials. The proposed synthetic pathway is used for the first time to prepare AgNPs via the biological route in a low-cost approach; this is considered to be an adequate direction compared to the commercial and conventional methods. Thus, the current study intended to estimate the anti-cancer influence of the biologically attained AgNPs depending on their cytotoxic activity.

Chemicals
All the required chemicals were bought from HiMedia (India). Furthermore, distilled water was used during the proposed experiments.

Collection and molecular identification of white rot fungi
Fungi, in the form of fruiting bodies, were obtained and sterilized with ethyl alcohol disinfectants, and approximately 3 Â 3 mm was placed on Malt extract agar medium in slants. After the growth of mycelium, it was transferred into the slants. This was repeatedly conducted until pure culture was obtained. Molecular identification on ribo-typing of 18S rRNA was conducted at Eurofins Laboratories Bangalore, India, and the sequence was deposited in the NCBI database for the accession number.

Preparation of silver nanoparticles (AgNPs)
In order to synthesize silver nanoparticles (AgNPs), T. ljubarskyi and G. enigmaticum were developed aerobically in liquid medium containing glucose, 15.0 gm; KH 2 PO 4 , 7.0 gm; K 2 HPO 4 , 2.0 gm; (NH 2 )SO 4 , 1.0 gm; MgSO 4 ⋅7H 2 O, 0.1 gm; and yeast extract, 1.0 gm. Furthermore, the Erlenmeyer flasks were protected with the fungal culture and were incubated on an incubator shaker at 30 C (120 rpm). Then, the collection of biomasses was completed after 3 days through filtration using a filter paper (Whatman No. 1). Additionally, the culture filtrate was tested with an equal amount of 1 mm silver nitrate solution, which is arranged in deionized water and incubated at 32 C (120 rpm). Finally, the control samples were obtained using the filtrates without silver ions. In addition, all the prepared and used materials were purified to avoid any kind of toxicity. For the reproducibility course, all the experimental tests were checked in triplicate to confirm the obtained results.

Investigation of anticancer activity
The cytotoxicity potential of compounds was tested on human breast carcinoma cells via MCF 7 and lungs carcinoma cells using MTT-based  cell proliferation assay [33]. Human breast carcinoma and lung carcinoma cell lines were achieved from the National Center for Cell Science (NCCS), Pune (India), and cultivated in Dulbecco's modified Eagle's red medium (DMEM) (Sigma Life Science, USA) containing 10% fetal bovine serum (FBS). Additionally, yellow-colored MTT is transformed into a blue formazan invention via metabolically active mitochondria, and the absorbance is straight proportional to the number of viable cells. Thereafter, A549, HeLa, and MCF-7 cells were plated in 96-well flat-bottom tissue culture plates at the density of approximately 1.2 Â 10 4 cells/well and allowed to ascribe overnight at 37 C. In addition, AgNPs with altered concentration were added to the medium to discard it and to enable the incubation of the cells, and the process was continued for 24 h. Consequently, the medium was discarded after the incubation process, and 100 μL fresh medium was added to 10-μL MTT (5 mg/mL).
Additionally, 4 h later, the medium was discarded again and another 100-μL DMSO was added to dissolve the formazan crystals. We found that  the absorbance was checked at 570 nm in a microliter plate reader. Finally, cell survival was calculated via the following Eqs. (1) and (2):

Principle of cell viability detection
With the same procedure as discussed in our previous related studies, NAD-dependent mitochondrial dehydrogenase of viable cells is used to cleave the MTT (3-(4, 5-dimethyl thiazol-2yl)À2, 5-diphenyl tetrazolium bromide) to form a quantifiable purple formazan product [34,35,36,37,  38]. We can observe the proportional relationship between the attained formazan production and viable cell number.

The cytotoxicity effect of silver nanoparticles (AgNPs) based on G. enigmaticum through the MTT assay
To check the cytotoxic effect of the biosynthesized silver nanoparticles on tumors, the cytotoxic activity of the attained silver nanoparticles based on G. enigmaticum was detected (in vitro) and considered at different concentrations against human lung cancer (A549) and human breast cancer cell lines (MCF7). We observed that the prepared AgNPs demonstrated a strong cytotoxicity effect for the tested cell lines in comparison with the untreated cells. Furthermore, the acquired results indicated that higher cell mortality occurs at higher concentrations of the used nanoparticles. To check the inhibition effect, the reaction was kept and incubated for 24 h (Figure 1). displays the variations in the percentage of inhibition in nanoparticles that is used to treat (A549) lung cancer cells. In addition, the cytotoxicity activity effect of G. enigmaticum-AgNPs materials against (A549) cells were detected by treatment of different concentrations of the prepared nanoparticles (Control, 80, 100 and 120 μg/mL) to check the inhibition of the tumor cells (as illustrated in Figure 2). The results indicated that the maximum cell growth inhibition (84.36%) was perceived for the sample with a concentration of 120 μg/mL (Figure 1). Additionally, the dose-dependent approach (MTT assay) on A549 cells after treatment with altered concentrations of the prepared silver nanoparticles of G. enigmaticum is shown in Table 1. The results showed a relationship between the (% cell viability) and (% cell inhibition) for A549 cell lines with the concentration of the used nanoparticles. As the concentration of the nanoparticles decreased, the viability of the A549 cell lines dose-dependently reduced ( Table 1). The detected decrease in cell viability indicates the anticancer properties of AgNPs [39].
Similarly, the same sequence of measurements was performed for the (MCF-7) cancer cell lines using AgNPs based on G. enigmaticum in vitro,   and the results specified that, the reaction was incubated for 24 h to check the inhibitory effect. The MTT assay on MCF-7 cancer cells after treatment with different concentrations of the prepared silver nanoparticles based on G. enigmaticum is found in Table 2. The results show that there is a relation between the % cell viability and % cell inhibition for the MCF-7 cancer cell lines with the concentration of the used nanoparticles [40]. As the concentration of the nanoparticles decreased, the viability of the (MCF-7) cells dose-dependently reduced ( Table 2). Furthermore (Figure 3), displays the variations in the percentage of inhibition in nanoparticles that is used to treat MCF-7 breast cancer cells. In addition, the cytotoxicity activity effect of AgNPs materials based on G. enigmaticum against MCF-7 cells was detected via the treatment of different concentrations of the prepared nanoparticles (Control, 80, 100, and 120 μg/mL) to check the inhibition of the tumor cells (as illustrated in Figure 4). The results indicated that the maximum cell growth inhibition (75.64%) was perceived for the sample with a concentration of 120 μg/mL (Figure 3). Additionally, no cytotoxicity was detected against standard untreated cells [41]. The result of the prepared silver nanoparticles was the same as obtained in our previous research work, and it shows that the gained particles were spherical in shape with particle size distribution of 5-40 nm [42,43].

The cytotoxicity effect of silver nanoparticles (AgNPs) based on T. ljubarskyi through the MTT assay
To detect the cytotoxic effect of the prepared nanoparticles on the tumors, the cytotoxic activity of the attained AgNPs based on T. ljubarskyi was distinguished (in vitro) at different concentrations, and reflected the effect in contradiction of (A549) as well as (MCF7) cancer cells. The variations in the inhibition percentage in nanoparticles that are used to treat (A549) lung cancer cells and breast cancer cells (MCF7) are displayed in Figure 5 and Figure 6, respectively. In addition, the cytotoxicity activity effect of AgNPs based on T. ljubarskyi against both (A549) lung as well as (MCF7) cancer cells was detected through the treatment of different concentrations of the prepared nanoparticles (control, 80, 100, and 120 μg/mL) to check the inhibition of tumor cells as illustrated in Figures 7 and 8, respectively. Further, the MTT assay on A549 and MCF7 cells after treatment with different concentrations of the prepared silver nanoparticles based on T. ljubarskyi is found in Table 3 & Table 4, respectively. The results show that there is a relationship between the % cell viability and % cell inhibition for both A549 and MCF7 cell lines with the concentration of the used nanoparticles [44]. Hence, from the obtained results in the cases, the prepared nanoparticle materials were found to be effectively potent against lung and breast cancer cell lines. AgNPs based on G. enigmaticum showed more potency against the A549 cell line compared to the effect gained against MCF-7. Additionally, in case of AgNPs based on T. ljubarskyi, the opposite effect was obtained as the effect on MCF7 cell line was better than the effect obtained against A549 cell line.
The dose-dependent method (MTT assay) was used for the evaluation of the toxicity gained from the attained nanoparticles on both A549 and MCF-7. The mortality data obtained confirmed that the biosynthesized AgNPs materials attained via the white rot fungus sources (G. enigmaticum & T. ljubarskyi) have potential anticancer activity [45,46,47].
Conversely, besides the effect of prepared nanoparticles on cell viability, we can recognize that the obtained silver nanoparticles may have another effect, which called "nanomaterial induced endothelial cells leakiness" (NanoEL). This effect can be produced via inducing endothelial leakiness in endothelial cells. Different studies gave a wide description of the phenomenon, and based on their studies, we can conclude that changing the nanoscale dimensions of the prepared silver nanoparticles facilitates the movement of these nanoparticles inside the nanosized gaps between microvascular capillary endothelial cells and dislocated the protein connections that embrace neighboring cells. We can confirm that the NanoEL effect of the prepared nanoparticles has momentous implications toward nanomedicine and nanotoxicology. In addition, we can similarly predict the mechanism that explicates the silver nanoparticles connection with the cells characteristic state of absorptivity resistance [48,49,50].

Conclusion
The anticancer activity of the biosynthesized silver nanoparticles (AgNPs) materials using two white rot fungi (G. enigmaticum & T. ljubarskyi) displayed extensive cytotoxic effects in contradiction of different human cancer cell lines (A549) and (MCF7) cancer cells. The difference between the current work and previous ones was that we intended to prepare AgNPs via the biological route using white rot fungus materials. The proposed synthetic pathway is used for the first time to prepare AgNPs via the biological route through a low-cost pathway, which considered as an adequate direction of preparation compared to the commercial methods. As a result, the prepared AgNPs is considered as active anticancer agents. The dose-dependent method (MTT assay) was used for the evaluation of the toxicity gained from the attained nanoparticles on both A549 and MCF-7. The mortality data acquired confirm that the biosynthesized AgNPs materials attained via the white rot fungus sources (G. enigmaticum & T. ljubarskyi) have potential anticancer activity. However, an exhaustive study concerning the relations of microbial prepared AgNPs with cancer cell lines is required before their use in medical applications. In this regard, additional cytotoxicity studies and animal trials must be conducted before the product enters the market.

Declarations
Author contribution statement Gudikandula Krishna: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.
V. Srileka: Performed the experiments; Analyzed and interpreted the data. M. A. Singara Charya: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.
Esraa Samy Abu Serea: Analyzed and interpreted the data; Wrote the paper.
Ahmed Esmail Shalan: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of interests statement
The authors declare no conflict of interest.

Additional information
No additional information is available for this paper.