Facile Synthesis of Silver Nanoparticles Using Asian Spider Flower and Its In Vitro Cytotoxic Activity Against Human Breast Carcinoma Cells

: Cancer is one of the most dangerous threats to human health and possibly the utmost task for current medicine. Currently, bio-based synthesis of nanoparticles from plants has gained much interest due to its potential medicinal applications. In the present study, a biological approach was employed for biogenic (green) synthesis of silver nanoparticles (AgNPs) using dried leaf extract of Asian spider ﬂower (Asf). The biogenic synthesis of Asf-AgNPs (Asian spider ﬂower-Silver nanoparticles) was established using ultra violet-visible (UV-vis) spectra which exhibited a wide superﬁcial plasmon resonance of AgNPs at 445 nm. These nanoparticles clearly showed the formation of poly-disperse crystalline solids (spherical shape) with particle size range of < 50 nm based on observation under a transmission electron microscope (TEM). Infrared spectroscopy (FTIR) revealed carboxylic acids (C = O stretch) known to act as a capping agent and a reductant in plant extracts. Elemental silver signal peak was observed in the graph obtained from energy-dispersive X-ray (EDX) analysis. Biocompatibility tests for Asf-AgNPs at di ﬀ erent doses were evaluated against human breast cancer cells (MCF7) for cell viability and apoptotic analysis. According to the evaluation, biosynthesized Asf-AgNPs could prevent the explosion of human breast tumor cells (MCF7) in IC 50 at a dose of 40 µ g / mL after 48 h of treatment. The results obtained in the IC 50 dosage treatments were statistically signiﬁcant ( p < 0.05) when compared with control. Nuclear damage of cells was further investigated using annexin V-FITC / PI dual staining and DAPI (4 (cid:48) ,6-diamidino-2-phenylindole) staining method. Bright blue ﬂuorescence with condensed and fragmented chromatin was observed. Western blot analysis showed increased expression levels of caspases-3 and 9 (apoptotic proteins). These results indicate that bio-approached AgNPs synthesized through Asf plant extract could be used as potential therapeutic medications for human cancer cells. treatments exhibiting the up-regulation in the production of caspases 3 and 9 (apoptotic proteins), which trigger the apoptotic pathway and cause signiﬁcant cell demise. The poly-potential activity of green synthesized AgNPs was due to their speciﬁc morphological (shape and size) features that enabled easy permeation into cells for targeted therapy. Our present investigation provides a green synthesis approach for the production of AgNPs in an ecofriendly environment, and also displays its potential applications in anticancer therapy.


Introduction
Nanotechnology, a branch of technology for the production of nanoscale materials including graphite, carbon nanotubes, and fullerenes with sizes of 1 to 100 nm, has gained attention due to its

Biological Synthesis and Silver Nanoparticles Characterization
Plant extracts encompass numerous primary and secondary functional metabolites. They might effectively reduce silver ions to silver nanoparticles with stable condition. Plant extracts contain equally polar and nonpolar compounds that can improve nanoparticle synthesis. The procedure of reduction of an aqueous solution of silver nitrate is henceforth the most broadly used technique for the production of silver nano colloids [35]. In a previous study, a phytochemical screening of Asf extracts has revealed the presence of terpenes, flavonoids, phenol carboxylic acid, and polyphenols [36]. In this work, nanoparticles were manufactured by mixing Asf leaves extract (1 mL from stock) with 1 mM aqueous AgNO 3 solution for 2 h to form AgNPs (yellowish-brown precipitation) (Figure 1). In the colloidal solution of nanoparticles, alteration in color was observed owing to the excitation of superficial plasmon vibrations (fundamentally the vibration of the group leading electrons) [37]. Ultra violet-visible (UV-vis) absorption spectra at 445 nm showed a wide range of surface plasmon resonance. In vitro constancy of Asf-AgNPs (Asian spider flower-Silver nanoparticles) was examined by observing plasmon wavelength and plasmon bandwidth at different time points (12,24,48,72, and 96 h) ( Figure 2). As stated previously, the test was set up with variable intervals (viz., 5, 15, 25, 35, 45, 60, 90 min). Testers were then scanned in the range of 400-600 nm to notice the development of extensive absorbance peak and color alteration implication [38]. Similar observations reported earlier in viewing a stable upsurge in the strength of SPR without any alteration in peak location is demonstrated in the synthesis of AgNPs with Carica papaya latex at different reaction time (0 to 72 h) [39]. Additionally, in the same study, using Euphorbia antiquorum latex extract, SPR intensity is increased with a decrease of silver ions after 24 h of incubation at room temperature from manufactured AgNPs [40]. Moreover, UV-Vis investigation ascertained the formation of stable metal nanoparticles exhibiting novel optical properties [41]. Synthesis of Size-controlled nanoparticles for stable systems have been prepared through combined action of solvated metal atom dispersion technique [42].    The size and morphology of prepared silver nanoparticles were analyzed using transmission electron microscopy (TEM) (Figure 3), which clearly exhibited the formation of polydisperse spherical and different magnitudes of crystalline AgNPs ranging from 20 to 50 nm in diameter, consistent with results of previous studies [43,44]. Elementary compositions of these biosynthesized silver nanoparticles were determined by energy-dispersive X-ray (EDX) spectrum analysis ( Figure 4). Results showed a distinct signal at 0.2 and 3 KeV regions representing the development of AgNPs from the leaf extract of Asf. The spectrum also designates that peaks designed are owing to the occurrence of copper from carbon-coated copper grids. Likewise, the third extreme peak positioned on the spectrum at 3 KeV designates the attendance of elemental silver from manufactured silver nanoparticles arbitrated compound [34]. The information provided by the infrared spectroscopy (FTIR) spectra was able to recognize biomolecules accountable for plugging and stabilizing these silver nanoparticles. Treatments with the Asf extract showed intense peaks at 2996, 1750, 1375, 1250, 1065, 870, 671, and 481 cm −1 . These synthesized particles (Asf-AgNPs) showed bands at 2996, 1747, 1375, 1249, 1065, 862, and 6650 cm −1 , 1747 cm −1 (C = O stretch), 1375 cm −1 (N = O stretch), 1249 cm −1 (C-O stretch) ( Figure 5). These results indicate that carboxylic acids present in the plant extract can act as a capping and reducing mediator for the development of functionalized AgNPs. Comparable explanations have also been suggested for silver nanoparticles synthesized with Desmodium gangeticum [45]. The action of plugging and stabilization on AgNPs brings a well-organized biosynthesis technique with less toxicity [46][47][48].
To determine the size distribution of synthesized Asf-AgNPs, dynamic light scattering of particles was measured. Figure 6A showed the size distribution of particles with a maximum intensity at 19.59 nm. Figure 6B showed the zeta potential of stable dispersion of AgNPs at 8.30 mV. Such green synthesized particles showed good stability and well dispersibility mainly due to the presence of plant based functional metabolites known to have less toxic effects on human cells [49,50]. Green manufactured silver nanoparticles using Oscillatoria limnetica aqueous extract has known to act as a dipping and stabilizing agent have also been reported [51].

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay
The present investigation the inhibitory effects of different concentrations (5, 10, 20, 40, 80, and 160 µg/mL) of silver nitrate (AgNO 3 ) and synthesized Asf-AgNPs under in vitro conditions on MCF7 breast cancer cell line and normal green monkey kidney cell line was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test (Figures 7 and 8). MCF7 cells preserved through silver nanoparticles (40 µg/mL) after 48 h of development with 50% hang-up of cell propagation. In contrast, while the same concentrations of Asf-AgNPs were used to treat normal Vero cells, showing the inhibition of cell viability was observed only at maximum concentrations (85.77% inhibition after 48 h incubation). The activity of Asf-AgNPs on Vero cell line was constantly less at experimented dilutions as compared with cancer cell line. Furthermore, in the same concentration of AgNO 3 were used to both MCF7 and Vero cells showed highly significant (p < 0.05) reduce the cell viability when compared to the control. The cell viability assay made in the present attempt indicated that synthesized Asf-AgNPs are less toxic than AgNO 3 and the biocompatibility of Asf-AgNPs was gradually increased at all concentrations by dose and time dependent. C. viscosa extract mediated AgNPs and AgNO 3 alone used to treated and untreated MCF7 and Vero cells at 48 hrs were observed under inverted microscope. Figure 9A,D untreated (control) Vero and MCF7 showed irregular confluent aggregates with round polygonal cells appeared normal. Figure 9B,E showed AgNO 3 treated Vero and MCF7 cells appeared to have shrinkage, become spherical in shape, and detached dead cells. In contrast, Vero and MCF7 cells treated with AgNPs shows persuade cell reduction lead to the changes in membrane integrity and cell shrinkage due to physiognomies of apoptotic cell demise ( Figure 9C,F). These results were similar to previous reports [52,53], which was mainly based on the agents that cause reduction and capping silver nitrate into silver. Cell uptake and internalization of nanomaterials are key factors determined by the surface property of AgNPs [54]. This permeabilization activity is based on surface charges of AgNPs and cell membranes [55][56][57][58]. Based on the results of inhibitory activity and cell morphology observation, the IC 50 concentration of Asf-AgNPs and control alone were used for further apoptotic experiments.

Apoptosis Study
In bio-cellular systems, cell death (apoptosis or involuntary cell demise) is a well-documented spectacle that has been clearly witnessed. Experimental topographies of apoptosis comprise the following: reduced cell extent and chromatin reduction, and nuclear disintegration owing to endonuclease breaking of DNA. Membrane phospholipid phosphatidylserine (PS) is changing location from internal to the external leaflet of plasma membrane in the initial stage of cell demise, revealing PS to the outside of cellular atmosphere. Annexin V is a 35-36 kDa calcium-dependent phospholipid-required protein connected with a high empathy for PS. It binds to unprotected apoptotic cell's superficial PS to facilitate the initial of apoptosis. The PI is a red glowing dye that enters a cell depending on the penetrability of the membrane [30]. Results of annexin V-FITC/PI dual staining are shown in Figure 10A,B. The control cells of MCF7 showed negative staining in reaction with annexin V-FITC and PI. However, MCF7 cells treated with Asf extract arbitrated AgNPs at IC 50 (40 µg/mL) showed positive staining with annexin V and PI, which lead the indication sign on the progress of apoptosis. The apoptotic effect of Asf-AgNPs on MCF7 cells was observed through DAPI (4 ,6-diamidino-2-phenylindole) staining ( Figure 11). DAPI is a nuclear stain that stains injured DNA in the central nucleus. The contact of AgNPs with MCF7 cancer cells produced a bright blue fluorescence with shortened and disjointed chromatin compared to less effective fluorescence for control cells (untreated). Earlier reports have suggested that plant formulated syntheses of AgNPs are responsible for the generation of oxidative stress, caspase dependent apoptosis, and mitochondrial dysfunction [59]. The cytotoxic effect of AgNPs formulated from the extracts of Kleinia grandiflora [60] or Punica granatum on DNA damage in cancer cells also provides evidence that such NPs might have anticancer activities [61]. revealing PS to the outside of cellular atmosphere. Annexin V is a 35-36 kDa calcium-dependent phospholipid-required protein connected with a high empathy for PS. It binds to unprotected apoptotic cell's superficial PS to facilitate the initial of apoptosis. The PI is a red glowing dye that enters a cell depending on the penetrability of the membrane [30]. Results of annexin V-FITC/PI dual staining are shown in Figure 10A, B. The control cells of MCF7 showed negative staining in reaction with annexin V-FITC and PI. However, MCF7 cells treated with Asf extract arbitrated AgNPs at IC50 (40 µg/mL) showed positive staining with annexin V and PI, which lead the indication sign on the progress of apoptosis. The apoptotic effect of Asf-AgNPs on MCF7 cells was observed through DAPI (4′,6-diamidino-2-phenylindole) staining ( Figure 11). DAPI is a nuclear stain that stains injured DNA in the central nucleus. The contact of AgNPs with MCF7 cancer cells produced a bright blue fluorescence with shortened and disjointed chromatin compared to less effective fluorescence for control cells (untreated). Earlier reports have suggested that plant formulated syntheses of AgNPs are responsible for the generation of oxidative stress, caspase dependent apoptosis, and mitochondrial dysfunction [59]. The cytotoxic effect of AgNPs formulated from the extracts of Kleinia grandiflora [60] or Punica granatum on DNA damage in cancer cells also provides evidence that such NPs might have anticancer activities [61].

Western Blot Investigation
The maintenance of homeostasis through the regulation of cell death and inflammation is governed by a family of caspases. Caspase stimulation is generally considered to be an important marker of apoptosis led by several apoptotic mediators [62]. Among them, cysteine proteases serve as primary mediators of apoptosis. In the mammalian system, apoptotic caspase-3 is not an initiator caspase but acts an executioner which leads to the degradation of cellular components for apoptosis. In general it has three functional groups: caspases 3, 8, and 9. Involuntary cell death is commenced by the stimulation of caspase cascade (caspases 3, 6, and 9) followed by the capture of G2/M phase in the cell cycle [63]. Caspase-3 is an important killer of apoptosis. Its active form is vital for flouting cellular modules associated with DNA overhaul and regulation. Caspase-3 has also been used in numerous circumstances such as for anticancer drug handling [64]. In this work, MCF7 cells treated with Asf-AgNPs triggered apoptosis showing an up-regulation on the active forms of caspases 3 and 9 ( Figure 12). MCF7 cells treated with Asf-AgNPs showed enhanced expression levels of caspases-3 and 9 compared to MCF7 cells without Asf-AgNPs. B-actin was used as an internal control. The statistical significance in the in the over expression of apoptotic protein was observed in the treatments (MCF7 treated with Asf-AgNPs (IC 50 at 40 µg/mL) when compared with the control at p value < 0.05. Manikandan et al. [65] have reported that Solanum trilobatum extract can decide the AgNPs-influenced ROS generation and trigger the inherent apoptotic pathway. Modulation of anti-apoptotic (Bax and Bcl-2) and apoptotic (caspase-3 and 9) protein countenance in mitochondria can affect cell death through a caspase-dependent pathway. Previous reports have revealed that AgNPs have shown acceptable potential anticancer activities [66][67][68][69] in human cancer.

Plant Selection and Extract Preparation
Asian spider flower (Asf) (Cleome viscosa L.) was collected, identified, and authenticated by botanists. Leaves of the Asf were dehydrated under a shade condition at 30 • C and then milled to powder. The powder sample (5 g) was then heated in 100 mL of distilled water for 20 min and clarified thrice using a Whatman number 1 sieve paper. The filtrate was stored at 4 • C in a refrigerator until analysis.

Silver Nanoparticles Synthesis
Silver nitrate (1 mM) aqueous solution was utilized to produce nano silver. To 45 mL of silver nitrate solution (1 mM), 5 mL of Asf extract was added and mixed. The mixture was then incubated at 30 • C in the dark for 12 h to reduce silver nitrate to silver ions.

Characterization of Silver Nanoparticles
The bio-reduction of sliver nitrates to ions of silver was observed with a UV-Visible spectrophotometer (Shimadzu-1800, Kyoto, Japan). Measurements were performed at room temperature using silver nitrate (1 mM) as a blank at wavelength of 300 to 700 nm with a resolution of 1 nm. These synthesized nanoparticles were then analyzed with a transmission electron microscope (TEM, FEI, TECNAI T30). The TEM samples were prepared by dropping aqueous dispersions onto carbon-coated copper grids and then dehydrated it at 30 • C. Various sizes of NPs obtained from a random field of TEM images were measured for determination of general morphology of nanoparticles. The biomolecules associated with the NPs were further identified using Fourier Transform Infrared (FTIR) spectroscopy (Perkin-Elmer, Waltham, MA, USA) with diffuse reflectance method at a resolution of 4 cm −1 in KBr pellets. Element compositions of nanoparticles were determined with Energy Dispersive X-ray Spectroscopy (EDX) analysis. Particle size and zeta potential were measured with a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK).

Cell Culture and Maintenance
Breast cancer cell lines (MCF-7) were tested in this investigation. These cells were cultured with DMEM (Dulbecco's Modified Eagle Media) medium supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%). Cells were cultured at 37 • C with 5% CO 2 water-saturated atmosphere. For further investigation, cells passaged at pre-confluent concentrations were used.

Analysis of Cell Viability
MTT dye uptake method [70] was adopted to determine cell viability. DMEM added with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), FBS (10%) was used as a carbon source for MCF-7 cell line. Cell culture flasks of 25 cm 2 were incubated at 37 • C in 5% CO 2 atmosphere. For further dosage studies, cells were seeded into 96-well plates (1×10 6 cells in each well) and incubated at 37 • C for 24 h. MCF 7 breast cancer cells and normal Vero cells were treated with Asf-AgNPs and AgNO 3 alone at various dosages (5,10,20,40,80, and 160 µg/mL). Plates were incubated for 24 and 48 h with additional treatment followed by cell viability test using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). After MTT solution (10 µL) was added to each well at concentration of 5 mg/mL, plates were incubated at 37 • C for 4 h. Formazan crystals of purple color appeared were dissolved in 100 µL of dimethyl sulfoxide (DMSO). Using a multi-well ELISA plate reader, the optical density was measured at 570 nm. With the following method, the % of cell viability was calculated.
% of cell viability = OD of Sample/OD of Control × 100

Morphological Analysis
In 6-well plates, MCF7 and Vero cells (1×10 5 ) were treated with IC 50 concentrations of Asf-AgNPs (40 µg/mL) at 48 hrs and AgNO 3 (40 µg/mL) at 48 hrs along with control (untreated). Further, the plates were washed twice with phosphate-buffered saline (PBS) and the cell morphology was observed using a radical inverted microscope along with a camera.

Apoptosis Assay
Apoptotic cells were determined with a dual staining method following the manufacturer's protocol [71]. Cells (1 × 10 6 /well) were cultured in 6-well plates and allowed to attach overnight with Asf-AgNPs (40 µg/mL) The IC 50 concentrations also observed with the overnight treatment. Control cells were also maintained independently for 48 h. These cells were then collected, washed twice with PBS, and resuspended in 1x binding buffer (500 µL). Cells were then stained with annexin V-FITC conjugate (5 µL) and propidium iodide (PI) solution (10 µL) in dark at 30 • C for 15 min. Using a fluorescent microscope (EVOS ® FLoid ® Cell Imaging Station), stained cells were analyzed to determine the cell viability (with annexin V-FITC and PI negative) and also to detect the early apoptosis (with annexin V-FITC positive and PI negative), or late apoptosis (with annexin V-FITC and PI positive). The % of annexin V positive cells was used to quantify the degree of apoptosis after treatments.

Western Blot Analysis
Six-well dishes were used for MCF 7 cell were culture and then treated with Asf-AgNPs 48 h. Control cells were also maintained for 48 h. Cells were harvested using buffer containing Tris-HCl (50 mM, pH 8.0), NaCl (150 mm), sodium azide (0.02%), phenylmethanesulfonyl fluoride, aprotinin, and Triton X (1%). Cell lysates were then centrifuged (12,000× g for 30 min at 4 • C) to harvest the supernatant as protein sample. Electrophoresis was performed for protein samples obtained from control and treated cell lines using 12% SDS-PAGE gel slabs. Protein bands obtained in the gel was transferred onto a nitrocellulose membrane. Immunoblotting was then carried out using primary antibodies against caspase 9 (mouse monoclonal antibody), caspase 3 (mouse monoclonal antibody), β-actin (mouse monoclonal antibody) at the manufacturer's recommended dilution after blocking with non-fat dry milk powder. Later, the membrane was incubated with mouse anti-goat peroxidase-tagged antibodies. Immune complexes were then detected using DAB (0.01%) and H 2 O 2 . The blot was further analyzed with the densitometry method of quantitation.

Conclusions
In this investigation, a biological approach was employed for the green synthesis of nanosilver particles (AgNPs) using dried leaves extract of Asian spider flower (Asf). The green chemistry approach in the synthesis AgNPs using plant extract was shown to be a simple, economical, efficient, and eco-friendly approach for medicinal applications. Asf leaves could be developed as promising bio-reductants to formulate functionalized silver nanoparticles. Formulated AgNPs were characterized through various spectral and microscopic analyses that clearly indicated the shape and size of crystalline nanoparticles. Such simple, rapid, and stable bio-based production of nanosilver nanoparticles displayed potential inhibitory cytotoxic effects on human breast carcinoma cells (MCF7). The Asf-AgNPs treatments exhibiting the up-regulation in the production of caspases 3 and 9 (apoptotic proteins), which trigger the apoptotic pathway and cause significant cell demise. The poly-potential activity of green synthesized AgNPs was due to their specific morphological (shape and size) features that enabled easy permeation into cells for targeted therapy. Our present investigation provides a green synthesis approach for the production of AgNPs in an ecofriendly environment, and also displays its potential applications in anticancer therapy.