An Eco-friendly Fabrication of Silver Chloride Nanoparticles (AgCLNPs) using Onopordum acanthium L extract Induces Apoptosis in Breast Cancer MDA-MB-232 Cells

In the current experimental work, silver chloride nanoparticles (AgClNPs) were fabricated using Onopordum acanthium L extract and their apoptotic and cytotoxicity properties on breast cancer MDA_MB232 and normal HEK293 cell lines were also evaluated. AgClNPs formation was determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) pro�le. Effect of fabricated AgClNPs on MDA_MB232 and HEK293 cells viability was performed using colorimetric MTT assay. Alterations in the mRNA expression levels of CAD and Bax genes in MDA-MB-232 cells were done using quantitative real-time reverse transcription-PCR (qRT-PCR) method. Subsequently, apoptotic properties were determined using �ow cytometry and �uorescence microscopy studies. MTT results investigated that AgCLNPs have a signi�cant dose-dependent lethal activity on MDA_MB232 compared to HEK293 cell lines. Quantitative real-time reverse transcription-PCR (qRT-PCR) results have also shown that AgCLNPs could up-regulate the apoptotic Bax and CAD gene expressions in the MDA_MB232 cells. Additionally, apoptotic assessment was performed by cell cycle analysis, annexin V/PI test, Hoescht 33258 dye, acridine orange and ethidium bromide (AO/EB) staining along with the detection of the reactive oxygen species (ROS) generation. Our results suggest that novel silver chloride nanoparticles fabricated by Onopordum acanthium L extract can display some promising cytotoxic properties through inducing apoptosis pathway.


Introduction
Nanotechnology, including the production and use of nanoparticle materials, is rapidly growing, which has a wide range of applications in various scienti c and industrial elds (Reddy et al. 2014).Advances in nanotechnology and an in-depth understanding of cellular processes have helped researchers in designing and synthesizing nanoparticles for therapeutic and diagnostic purposes (Umar et al. 2019).It should be noted that different preparation methods cause different structural properties in the synthesized nanoparticles, which in turn lead to different functional properties in nanoparticles (Rajeshkumar et al. 2018).Nanoparticles due to their wide applications in industry, medicine, and biotechnology, have attracted many scientistsʼ attention.Among these, silver nanoparticles (AgNPs) and silver chloride nanoparticles (AgCLNPs) were found as more effective products due to their promising biomedical applications (Dhas et al. 2014).
Currently, many approaches have been developed for the fabrication of metallic nanoparticles using physical, chemical, and biological methods (Chugh et al. 2018).Biological procedure of synthesis includes plants or the green synthesis that is well-known due to their simplicity, cost-effectiveness, biocompatibility, and feasibility for nanoparticle fabrication over the chemical and physical methods (Singh et al. 2018).Moreover, the green synthesis of nanoparticles from plant extracts is the greatest ideal strategy for cancer treatment due to its environmentally friendly approach (Popescu et al. 2010).
Traditionally, Onopordum acanthium L., called as Scotch thistle, is a widely distributed plant used in medical eld.Additionally, Onopordum acanthium L displayed different in vitro and in vivo pharmacological properties such as Antipyretic, Antihypertensive, anti-in ammatory, Antibacterial, and Antiradical (Garsiya et al. 2019).Thus, the phytochemicals of Onopordum acanthium L can act as a reducing and stabilizing agent in the preparation of green AgNPs, which can consequently lead to the enhanced cytotoxicity activity.
Previous studies implicated that green-route mediated fabrication of AgNPs inhibits the progression of tumor cell lines in experimental animals (Murugesan et al. 2019;Jeyaraj et al. 2013).The biologically fabricated AgNPs inhibit the A549 lung cancer cells by the caspase-dependent mitochondrial pathway (Kanipandian et al. 2019).George et al. in their study investigated the cytotoxicity of biosynthesized AgNPs, and showed that it is associated with the nuclear damage, dysfunction of cellular mitochondrial membrane, and intracellular reactive oxygen species (ROS) production for human breast cancer cells MCF-7 (George et al. 2018).
However, a limited number of investigations have been performed on the cytotoxic effect of silver chloride nanoparticles on breast cancer cell lines, so in this study, we aimed to investigate it.According to a previous research, the biosynthesis of AgClNPs using Onopordum acanthium L extract has not been reported.In our work, we investigated the simple and rapid biosynthesis of silver chloride nanoparticles using the Onopordum acanthium L extract, as well as its apoptotic effects on breast cancer MDA-MB232 and normal HEK-293 cell lines.

Preparation of green AgCLNP
At rst, to prepare the hydroalcoholic extract of plant, we added 50 grams of powdered dried plant Onopordum acanthium L into 200 cc of 50% ethanol, followed by centrifugation at 14000 rpm for 20 minutes.The obtained solution was then passed through a lter paper, dried, and stored in the refrigerator for further testing.For nanoparticlesʼ fabrication, the rst 100 mL of a solution of 1 mM silver chloride was prepared in distilled water.Thereafter, 5 ml of 4 mg/mL solution of Onopordum acanthium extract and 1 mM KCL salt were drop wise added to the stirred silver nitrate AgNO 3 solution.Next, the resulted solution was stirred for 24 hours at room temperature.This solution was then centrifuged at 13,000 rpm for 10 minutes; the supernatant was discarded and then centrifuged again with distilled water.To investigate the puri cation of silver chloride nanoparticles, the remained pellets were dissolved in 500 ml of deionized water, incubated it for 1 minute, and then centrifuged for 10 minutes at 3000 rpm.
The fabrication of AgClNPs was monitored by the calculation of the phase identi cation using X-ray diffraction (XRD) technique.The size and surface morphological properties of the synthesized AgClNPs were investigated using FESEM image by utilizing the Philips XL30 instrument.Moreover, the chemical composition elements of AgClNPs were identi ed by the energy-dispersive spectroscopy (EDS) analysis.

Cell culture
The MDA-MB232 and HEK293 cell lines were cultured in RPMI 1640 medium with 10% FBS serum and 1% streptomycin-penicillin solution.The cells were then stored at 37 ° C in an about 70% humid atmosphere with a concentration of 5% CO 2 .

Cell viability assay
To exhibit the in vitro cytotoxicity, the colorimetric MTT method was employed for investigating the lethal properties of silver chloride nanoparticles.Afterward,different concentrations (0,0.3125,0.625,12.5,25,50,and 100mg/mL) of AgClNPs were added to the cells (1 × 10 4 cell /well) seeded on 96-well plate, which were incubated overnight.Thereafter, 100 μl of 0.05mg/well MTT solution was added to the plate at 37˚C.To solubilize the viable cells formazan crystals production, we added 100 μl/well of dimethyl sulfoxide (DMSO) to them.Finally, the optical density of the measurement was evaluated at 570 nm.

Real time PCR reaction analysis
To evaluate the qRT-PCR method, total RNA extraction was performed using the RNA extraction kit in terms of the manufacturer's protocols (Qiagen, Valencia, CA).PrimeScript TM RT Kit (Takara, Japan) was utilized for the fabrication of complementary DNA (cDNA).Brie y, 100 ng cDNA (1 micro Liter) was used for real time PCR reaction, followed by the addition of 10 μL of SYBR green master mix in a total volume of 20 μl per reaction.Bax and CAD primers were then developed using NCBI primer blast in this study (Table 1).Thereafter, qRT-PCR was performed for 10 min at 95°C as an initial denaturation and 40 cycles for 15 s at 95°C were also done, followed by annealing for 1 min at 60°C.Additionally, the result was evaluated by ABI StepOne using the Applied Biosystems qRT-PCR thermo cycler device.

Fluorescence microscopy study
The apoptotic and necrotic patterns in cells after the treatment of AgClNPs were ascertained using a dual acridine orange and ethidium bromide (AO/EB) staining.The MDA-MB232 cells were exposed to nanoparticles, xed by the addition of 4% formaldehyde, and AO/EB solution (10 μL), followed by 5 min incubation.For Hoechst nuclear staining, the cells were exposed to AgClNPs and then washed with PBS.A Hoechst 33258 solution was added for 5 min in the dark followed by washing thrice with PBS.The cells were nally visualized by uorescence microscope.
Annexin V/PI staining by ow cytometery Apoptotic and necrotic percentages were assessed using the uorescein isothiocyanate (FITC)-Annexin-V/PI staining kit (Roch, Switzerland).Subsequently, the MDA-MB232 cells, at a density of 3 × 10 5 cells per well, were allowed to be treated by 58.61 mg/mL of AgClNPs.After the incubation for 24 h, the cells were washed, followed by re-suspending in binding buffer, and staining with in Annexin V/PI in terms of the kit's Protocol, which were then acquired using a ow cytometer.

Detection of reactive oxygen species
To assess the effect of the produced AgClNPs on intracellular reactive oxygen species (ROS) generation, 2',7'-dichloro dihydro uorescein diacetate (DCFH-DA) assay was evaluated by ow cytometry.Firstly, MDA-MB232 cells were seeded in 6-well plates, which were then allowed to grow overnight in the CO 2 incubator.For ROS activity, the cells were washed with PBS and 20 μM of DCFH-DA dye was pretreated for 30 min followed by employing ow cytometry.

Cell cycle evaluation
The cell cycle assay was further done via ow cytometry for the determination of the properties of AgClNPs on cell cycle distribution for 24 h.Afterward, the MDA-MB232 cells were treated with 58.61 mg/mL of the AgClNPs and were then washed, collected, and xed in cold ice 70% ethanol.The cells were washed again, followed by staining with PI and 100 µg/ml of RNase treatment for 1 hour at 25°C.Finally, the cell DNA content was monitored the cell cycle distribution using ow cytometer.

Synthesis of AgClNPs
The application of nanomaterials in biomedical has been growing in recent years (Greque de Morais et al. 2014).Many research groups reported the roles of different metal nanoparticles such as cancer therapy, molecular imaging, hyperthermal therapy, and drug delivery properties in this eld (Mukherjee et al. 2020;Chenthamara et al. 2019).
Previous investigations revealed the anticancer properties exhibited by this plant as well as bacterialderived nanoparticles.
The bio-fabrication of AgClNPs using cell-free supernatant of Escherichia coli culture has been reported by Bigdeli et al. (Bigdeli et al. 2019).They investigated the anti-cancer properties of AgClNPs in human MCF-7 breast cancer cells.In our work, for the rst time, the anti-cancer properties of AgCLNPs fabricated by the Onopordum acanthium extract were con rmed.This plant-mediated green route was found to be cost-effective, fast, simple, and non-toxic for environment (Salem et al 2020).Different studies have also investigated that the Onopordum acanthium L are rich in different chemical agents such as Lignans (Lajter et al. 2015), Flavonoids (Habibatni et al. 2017), Coumarins (Bogs et al. 1965), Terpenoids (Ivanova et al. 2010), Tocols, and Steroids (Zhelev et al. 2014), so it has been employed in traditional medicine as a cardiotonic and antitumor agent.Fabrication of Onopordum acanthium extract-mediated AgCLNPs was characterized by a change in the mixture from colorless to purple color.
In our study, the silver and chloride were the maximum constituent elements in the fabricated AgClNPs as observed by EDS pro le.According to gure 1, further peaks such as Silisium, Aluminium, Magnesium, Cupper, and Carbon ascertain the existence of compounds, which are biomolecules attached to the NPs.
The XRD pattern's analysis was performed to describe the crystalline nature of AgClNPs according to the cubic structure (FCC) (Figure 2).Notably, the identi cation of AgClNPs was consistent with the Joint Committee on Powder Diffusion Standards (JCPDS) database ( le no.85-1355).
The morphological determination of nanoparticles was established by SEM image.The fabricated AgClNPs appear to have spherical nature with a particle diameter ranged from 92.53 to 157.7 nm (Figure 3).

Evaluation of cell viability assay
To determine the effect of IC 50 dose of nanoparticles on cell lines, we examined MDA_MB232 and HEK293 cells treated with 0.3125, 0.625, 12.5, 25, 50, and 100mg/mL of AgClNPs for overnight using MTT assay.In this regard, AgClNPs were able to decrease cell viability in a dose-dependent manner, as shown in Figure 4.Moreover, IC 50 concentration of AgClNPs toward MDA-MB232 and HEK293 cells was 58.61 and 139.6 μg/mL, respectively.
Different types of reports are available on anti-tumor effect of green silver nanoparticles toward various cell lines such as MCF-7, A549 (Venugopal et al. 2017), PC-3 (He et al. 2016), and HTC-116 cells (Kuppusamy et al. 2016).AgNPs fabricated using the culture supernatant of Bacillus funiculus showed the reduced cell viability against MDA-MB-231 breast cancer cells using 10 μg/mL and higher amounts (Gurunathan et al. 2013).
However, a limited number of investigations have showen the cytotoxicity effect of the biologically fabricated silver chloride nanoparticles toward cell lines in vitro.Sattari et al. (2020) using Levisticum O cinale extract, reported that Ag and Ag chloride nanoparticles were toxic to MDA-MB-468 breast cancer cells (Sattari et al. 2020). Moreover, charelli et al. (2018) in their study revealed that silver chloride nanoparticles from B. megaterium have a little effect on toxicity of human adipose tissue stem cells (ASCs) in spheroid 3D culture.Furthermore, they showed that sub-lethal AgCLNP doses trigger the ROS production at day 7 after the exposure (Charelli et al. 2018).

Estimation of AgClNP apoptotic properties
To further examine the effect of AgCLNP on cell cycle phase's distribution, ow cytometry methods were performed.Therefore, cell cycle progression was arrested in MDA_MB232 cells, followed by the treatment with AgClNP.
In addition, Ag nanoparticles mediated cell cycle arrest in all phases.Interestingly, a previously performed investigation in Ehrlich ascites carcinoma (EAC) cells in vivo and human glioblastoma stem cells (GSCs) in vitro found that silver/silver chloride nanoparticles fabricated from Kaempferia rotunda fruit extract increased the population of cells in the G2/M phase.Accordingly, the increase in IL1, p21, TNFa, TLR9, IKK, and NFкB genesʼ expressions were also observed that were related to apoptosis in GSCs (Kabir et al. 2020).Another investigation showed that the AgNPs-mediated extract of Nepeta de ersiana plant up-regulated the ROS and lipid peroxidation (LPO) in cervical cancer cells (HeLa), which consequently caused an increase in apoptotic SubG1 peak (Al-Sheddi et al. 2018).The triggering apoptosis due to the existence of SubG1 peak in the process of cell cycle con rmed the involvement of apoptotic cell death pathway (Pumiputavon et al. 2017).According to cell cycle analysis, an increase was observed in the number of cell population in sub-G1 phase (implicates apoptotic pathway cell death) compared to the control cells (Figure 5).Notably, several investigations were conducted on the potentials of different nanoparticles in cell's ROS production.ROS in tumoral cells can act in the regulation of energy metabolism, cell growth, motility, autophagy, and cell death (Liou et al. 2010).
The production of ROS induced by nanoparticles lead to lipid peroxidation, nucleic acid cleavage, biomoleculesʼ destruction, membrane and organelle structuresʼ destruction, and mitochondrial membrane potentials (MMP) increase, which further resulted in apoptosis and necrosis (Yu et al. 2020).
Mao et al. suggested that lethal and sublethal doses of the fabricated citrate-coated AgNPs can induce ROS-mediated stress responses such as DNA damage, apoptosis, and autophagy on in vivo platform Drosophila melanogaster (Mao et al. 2018).Similar investigations performed on the activity of the biologically synthesiszed Ag/AgCl NPs by leafy vegetable of Rumex.acetosa demonstrated high antioxidant and cytotoxicity activities against the human osteosarcoma (HOS) cell lines (Kota et al. 2017).
In this regard, ROS levels induced by AgClNP were validated using ow cytometric analysis.Importantly, ow cytometric analysis followed by staining with DCFH-DA dye was done to explain the role of oxidative stress in nanoparticles-treated cell-death.The results reveal that AgClNPs has signi cantly increased ROS levels in MDA_MB232 cells compared to those untreated cells (Figure 6).These results suggest that ROS productions in MDA-MB-232 cells play signi cant roles in triggering cells death.
Alterations in the mRNA expression levels of CAD and Bax genes in MDA-MB-232 cells were done using qRT-PCR method followed by the exposure to nanoparticle (as shown in Figure 7).Furthermore, the Caspase-activated DNase (CAD) and Bax genes were shown to be involved in the apoptosis pathway.The Bax gene is known as pro-apoptotic member of the B cell lymphoma-2 (Bcl-2) gene family, which is the main regulator of this pathway.Thus, the dysfunction in Bcl-2 gene family activities has been associated with many diseases (Siddiqui et al. 2015).
Within the activation of apoptosis pathway, DNA fragmentation has occurred, which is responsible for triggering apoptotic bodies.The Caspase-activated DNase (CAD), as a nuclease enzyme, cleaves dsDNA, which helps chromatin condensation and apoptotic DNA fragmentation (Miles et al. 2017).Bigdeli et al. have reported that AgClNPs-mediated apoptosis is dependent on the increased p53, bax, caspase 3, caspase 8, and caspase 9 genesʼ expressions (Bigdeli et al. 2019).According to our study, the results of real time PCR displayed that the expression of CAD was up-regulated by a 5.5-fold, while mRNA level of Bax was down-regulated by 0.53-fold when compared to the untreated cells, proposing that AgClNPs trigger apoptosis via an intrinsic (mitochondrial mediated) pathway.
To determine the mechanism of cell death in the cancer cells, annexin V/PI ow cytometry was employed.
In the treatment of MDA-MB-232 cells by IC 50 of AgClNPs (58.61 g/mL), 35.5%, and, 2.89% of early and late stages' apoptosis were observed, respectively (Figure 8).While the untreated cells revealed 0.3% and 0.1 % of early and late stages' apoptosis, respectively.
Considering that the apoptosis/necrosis induction causes change in cell's morphology, we explored AO/EB double staining to discriminate the apoptotic, necrotic pathway, and integrity of membrane in MDA-MB-232 cells after the exposure to nanoparticles overnight.The Hoescht 33258 results reveal that AgClNPs has increased apoptosis through nuclear fragmentation, cytoplasmic and chromatin condensation (Figure9).Chromatin condensation, nuclear fragmentation, cell membrane blebbing, and cell shrinkage are usual apoptotic properties (Curčić et al. 2012).
Viable cells with intact DNA display a green color in their nuclei.Apoptotic cells with the condensed DNA show orange nuclei, while red nuclei represent necrotic cells.According to ndings shown in Figure 10 the increased number of apoptotic cells can be observed in comparison with untreated cells, con rming ow cytometric results.

Conclusion
In summary, AgClNPs were fabricated from the Onopordum acanthium extract, which were con rmed by XRD, SEM, and EDS pro le.Anti-cancer properties of Onopordum acanthium extract-mediated AgClNPs are due to the increased ROS level, decreased cell viability, modulation of pro-apoptotic Bax gene, sub-G1 cell cycle arrest, and the inhibited cell growth signi cantly due to the apoptosis of MDA-MB232 cells.For the breast cancer therapy, further in vivo studies are required to understand the e cacy of the Onopordum acanthium extract-mediated AgClNPs.

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