Green engineering of biomolecule-coated metallic silver nanoparticles and their potential cytotoxic activity against cancer cell lines

Leaves of A. scholaris (Apocynaceae), A. paniculata (Acanthaceae), A. marmelos (Rutaceae) and C. asiatica (Apiaceae) were collected freshly from Biodiversity Garden, Periyar University, Salem, and they were washed thoroughly with copious amount of deionized water, shade dried for 2–3 weeks and powdered. Each individual plant sample (5 g) was boiled in 50 ml of deionized water at 60 °C for 15 min. After cooling the mixtures were filtered through whatman no.1 filter paper and the filtrates were used for further experiments.

Leaf extract mediated synthesis of silver nanoparticles (AgNPs) was carried out according to Velmurugan et al [21]. In brief, 90 ml of 1 mM aqueous silver nitrate (Sigma-Aldrich, St. Louis, MO, USA) solution was added to 10 ml of each prepared leaf extract individually by fine shaking and incubated overnight at room temperature in the dark. The initial AgNPs formation was confirmed by colour change. On completion of the synthesis, the reaction mixture was centrifuged at 8000  ×  g for 10 min and the nanoparticle pellet was dissolved in sterile distilled water and washed thrice by centrifugation to remove impurities.

A UV–Vis spectrophotometer (UV-1800-Shimadzu, Kyoto, Japan) was operated at 1 nm resolution within a working wavelength range of 300–700 nm using a dual beam. X-ray powder diffraction (XRD) of the samples was performed using a Rigaku x-ray diffractometer (Rigaku, Japan), with scanning done in the region of 2θ from 2θ  =  20 to 80° at 0.04° min⁻¹ with a time constant of 2 s. Fourier transform infrared spectroscopy (FTIR) spectra of the AgNPs were obtained with a Perkin-Elmer FTIR spectrophotometer (Norwalk, CT, USA) in diffuse reflectance mode at a resolution of 4 cm⁻¹ in KBr pellets. Scanning electron microscopy—energy dispersive spectroscopy (SEM-EDS) spectra was conducted (SEM, JEOL-JSM 6390, Japan) to examine the surface morphology and to record the spot-profile mode by focusing the electron beam and also to confirm the elemental composition of the samples. The particle size distribution of AgNPs was evaluated using dynamic light scattering (DLS) measurements and zeta potential analysis was conducted with a zetasizer nanoseries compact scattering spectrometer (Malvern Instruments Ltd, UK).

The cell lines namely HepG2 (human liver cancer), PC3 (human prostate cancer) and Vero (African monkey kidney) were obtained from the National Centre for Cell Sciences (NCCS), Pune, India. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and Eagle's minimal essential medium (EMEM), supplemented with 2 mM L-glutamine, and balanced salt solution (BSS) and adjusted to contain 1.5 g l⁻¹ sodium carbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 1.5 g l⁻¹ glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) and 10% fetal bovine serum (GIBCO, USA). Penicillin and streptomycin (1 IU µg⁻¹) were added at 1 ml l⁻¹. The cells were maintained in 25 cm² culture flask (Corning, USA) at 37 °C with 5% CO₂ in a humidified CO₂ incubator.

The 50% inhibitory concentration (IC₅₀) value was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay carried out according to the method of Priyadharshini et al [22]. Cells were cultured and ~1  ×  10⁴ cells/well were seeded into 96-well culture plates and incubated for 48 h. Cell lines viz., HepG2, PC3 and Vero cells were treated with series of concentrations (1–100 µg ml⁻¹) of A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs and 5-FU. The NPs treated cells were incubated for 48 h to study the cytotoxicity by using MTT assay. The MTT (Himedia) stock (5 µg ml⁻¹) was prepared and the culture medium was removed and MTT solution (100 µl) was added in each AgNPs treated and control well that were incubated for 4–6 h at 37 °C. Cell viability assay was performed by the conversion of the tetrazolium salt (MTT) to a purple colour formation by the mitochondrial dehydrogenases and these crystals were dissolved in 100 µl of dimethyl sulphoxide (DMSO) and absorbance was measured at 620 nm in a multi well ELISA plate reader (Thermo Multiskan EX, USA). The optical density (OD) value was used to calculate the percentage of cell viability by using the following formula

$$\begin{eqnarray}&&\text{Percentage}\,\text{of}\,\text{cell}\,\text{viability}\\ &&\,=\,\frac{\text{OD}\,\text{value}\,\text{of}\,\text{experimental}\,\text{AgNPs}\,\text{treated}\,\text{sample}}{\text{OD}\,\text{value}\,\text{of}\,\text{experimental}\,\text{untreated}\,\text{sample}} \times\text{100}\text{.}\end{eqnarray}$$

PC3 and HepG2 cells that were grown on cover slips (1  ×  10⁵ cells/cover slip) were incubated with A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs at IC₅₀ concentration, and they were fixed in ethanol:acetic acid mixture (3:1, v/v). The cover slips were gently mounted on glass slides for the morphological changes. The morphological changes of PC3 and HepG2 cells were analyzed using bright field inverted microscopy (Nikon, Japan) at 40×  magnification [23].

Both PC3 and HepG2 cells were seeded in 24 well plate (1  ×  10⁵ cells ml⁻¹) and treated with A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs at IC₅₀ concentration for 48 h at 37 °C. The AgNPs treated cancer cells were collected washed with phosphate buffered saline (PBS; pH 7.2). For staining, 1 µl of AO/EtBr dye mixture 10 mg ml⁻¹ of acridine orange (AO) and 10 mg ml⁻¹ ethidium bromide (EtBr) in PBS was added into 9 µl of cell suspension (1  ×  10⁵ cells ml⁻¹) on clean microscopic cover slips. After incubation for 2–3 min, the cells were visualized under a Fluorescence microscope (Nikon Eclipse, Inc., Japan) at 40×  magnification with an excitation filter set at 510–590 nm. Percentage of apoptotic cells was determined according to the following formula

$$\begin{eqnarray}&&\text{Percentage}\,\text{of}\,\text{apoptotic}\,\text{cells}\,\\ &&=\,\frac{\text{Total}\,\text{number}\,\text{of}\,\text{apoptotic}\,\text{cells}}{\text{Total}\,\text{number}\,\text{of}\,\text{normal}\,\text{and}\,\text{apoptotic}\,\text{cells}} \times\text{100}\text{.}\end{eqnarray}$$

Cancer cell lines (PC3 and HepG2) were seeded and treated with A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs for 48 h in a 6-well plate (Nunc), and then fixed with 500 µl of methanol and kept for drying, prior to washing with PBS and 500 µl of 4% (v/v) formaldehyde for 20 min. The washed cells were then stained with 100 µg ml⁻¹ of rhodamine-123 (Sigma, USA) for 20 min in the dark at room temperature. Stained images were captured using fluorescent microscope (Zeiss) with appropriate excitation filter.

Cell lines (PC3 and HepG2) were seeded in a 6-well plate (Nunc) and treated with A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs for 48 h. The cells were washed with PBS and then fixed in 500 µl of methanol and 500 µl of 4% (v/v) formaldehyde for 20 min and kept for drying at room temperature. Cells were then stained with 100 µg ml⁻¹ of HOECHST-33258 (Sigma, USA) for 10 min in the dark at room temperature. Hoechst-stained nuclei images were recorded under fluorescent microscope (ZEISS) using appropriate excitation filter.

Cell lines of PC3 and HepG2 (1  ×  10⁶ cells) were exposed to IC₅₀ concentration of AgNPs for 48 h and were gently scraped and harvested by centrifugation. Cells were then suspended in 10 ml of TE buffer solution (10 mM tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 2% sodium dodecyl sulfate (SDS), and 20 mg ml⁻¹ proteinase K). The mixture was incubated at 37 °C for 3 h, followed by DNA extraction with a phenol:chloroform:isoamyl alcohol (25:24:1) solution. The DNA was treated with DNase-free RNase at a concentration of 20 mg ml⁻¹ at 37 °C for 30 min and precipitated with 1/10 volume of 2.5 M sodium acetate (pH 7.0) and three volumes of 100% ethanol. DNA fragmentation analysis was carried out with 10 µl of DNA prepared from IC₅₀ concentration of AgNPs treated cells and analyzed using 1.5% (w/v) agarose gel containing ethidium bromide.

All data were used for statistical analysis with three replicates and expressed as mean  ±  S.D. A probability level of P  <  0.05 was used for the significance of differences between mean values. Data were analyzed using GraphPad InStat and Prism version 3.00 for Windows, GraphPad Software, San Diego, CA, USA.

The reduction of pure Ag⁺ ions to Ag⁰ was monitored based on a visual colour change (from light yellow to brown), and the UV–visible absorbance of the reaction mixture show a strong intense peaks for A. marmelos (375 nm), A. scholaris (380 nm), A. paniculata (420 nm) and C. asiatica (380 nm) (figure 1) during 3 h incubation period. The colour change of the reaction mixture occurs due to the excitation of surface plasmon vibrations. Mukherjee et al [24] and Velmurugan et al [25] reported that the reduction process of Ag⁺ to Ag⁰ nanoparticles occurred possibly due to the presence of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent dehydrogenase enzyme and phytochemical compounds including phenolic groups in the plant extracts. The presence of various phytochemicals in the plant extracts could be responsible for the reduction of Ag⁺.

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The FTIR spectra of A. marmelos, A. paniculata, A. scholaris and C. asiatica aqueous leaf extracts and their respective AgNPs are depicted in figure 2. The major peaks of aqueous leaf extracts exhibit various wave numbers that correspond to different functional groups and were observed between 3353–616 cm⁻¹, 3407–653 cm⁻¹, 3404–654 cm⁻¹ and 3342–697 cm⁻¹ for A. marmelos, A. scholaris, A. paniculata and C. asiatica, respectively (figure 2(a)). A strong peaks at 3407 and 3404 cm⁻¹ represents N–H stretching amines group, 2935 cm⁻¹ corresponds to C–H stretching alkanes group, 2368 and 2363 cm⁻¹ represents primary amines, 2155 and 2136 cm⁻¹ corresponds to terminal alkynes of (C  ≡  C) C–C bond groups, 1574, 1591 and 1590 cm⁻¹ corresponds to N–H bends of amines groups, 1399 cm⁻¹ represents C–H in-plane bend with alkenes group, 1124 and 1122 cm⁻¹ corresponds to C–C stretching anhydrides group, 826 and 830 cm⁻¹ corresponds to P–H bend phosphines group, 697 and 700 cm⁻¹ corresponds to the C–H bend of mono aromatics ring groups, 616 cm⁻¹ represents acetylenic C–H bend with alkynes groups respectively. The synthesized AgNPs show intense peaks at 3427–668 cm⁻¹ A. marmelos, 3446–607 cm⁻¹ A. paniculata, 3441–602 cm⁻¹ A. scholaris and 3424–669 cm⁻¹ C. asiatica (figure 2(b)). Peaks at 3446 and 3427 cm⁻¹ corresponds to N–H stretch (amines), 2921 and 2914 cm⁻¹ associated with C–H stretch (alkanes), 2852 cm⁻¹ represents to C–H stretch (aldehydes), 2339 cm⁻¹ corresponds to P–H stretch (phosphines), 1633 cm⁻¹ indicates the C  =  C stretch (alkenes), 1629 cm⁻¹ linked to nitro groups (aliphatic), 1383 cm⁻¹ corresponds to C–H in plane bend stretching alkenes group, 1265 and 1255 cm⁻¹ corresponds to acetyl group (ethers), 1236 cm⁻¹ represents to C–O stretch (carboxylic acids), 1092 cm⁻¹ corresponds to ethers (C–O–C dialkyl), 1030 cm⁻¹ represents C–N of aliphatic amines, 803 cm⁻¹ indicates the aromatics (C–H bend), 668 cm⁻¹ corresponds to alkenes group, 607 cm⁻¹ represents alkyl halides.

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SEM images of A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs are depicted in figures 3(a)–(d), respectively; and their EDS spectra are shown in figures 4(a)–(d), respectively. The SEM images reveal that all types of NPs were found to be various shapes such as fibrous, rectangle and spherical with smooth edges (figures 3(a)–(d)), and the size of the nanocomplex was ranged from 37–79 nm, 36–65 nm, 53–94 nm and 49–97 nm for A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs, respectively. The EDS image of AgNPs showed strong silver metal peaks in the range between 2–4 keV and it showed weak signals of silicon, chlorine and oxygen which confirmed the pure elemental form of the synthesized AgNPs (figures 4(a)–(d)). Similar findings on SEM and EDS spectrum analysis were reported for synthesized AgNPs using leaf extracts of Artemisia nilagirica [26], Ficus benghalensis [27], Rauvolfia tetraphylla [28], Caesalpinia coriaria [29] and Naringi crenulata [30].

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The XRD patterns of the A. marmelos, A. paniculata, A. scholaris and C. asiatica AgNPs are shown in figures 5(a)–(d), respectively. The diffraction peaks at 2θ values of 32.12°, 38.41°, 44.22°, 46.22°, 54.94°, 64.38° and 77.36° corresponding to the (1 2 2), (1 1 1), (2 0 0), (2 3 1), (1 4 2), (2 2 0) and (3 1 1) planes; for A. marmelos, A. paniculata peaks at 2θ  =  38.31°, 44.60°, 64.75° and 77.49° corresponding to (1 1 1), (2 0 0), (2 2 0), (3 1 1) planes, C. asiatica peaks at 2θ  =  32.28°, 38.25°, 46.28°, 54.83°, 64.56°, 77.69° corresponding to (1 2 2), (1 1 1), (2 3 1), (1 4 2), (2 2 0), (3 1 1) planes, and A. scholaris peaks at 2θ  =  38.25°, 44.62°, 64.56°, 77.26° corresponding to the (1 1 1), (2 0 0), (2 2 0), (3 1 1) sets of lattice planes and it indicates the face-centred cubic (fcc) structure of AgNPs, which are closely linked with Joint committee on powder diffraction standards (JCPDS file no. 04-0783) for silver. Our XRD diffraction is consistent with Jeeva et al [29], and Jayaseelan and Rahuman [31] for synthesis of AgNPs using aqueous leaf extracts of C. coriaria and Ocimum canum. Similar results were reported by Selvi et al [19] using Padina tetrastromatica seaweed extract.

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AgNPs dispersed in millipore water were highly stable with a zeta (ζ) potential value of  −14.9 mV, –20.9 mV, –3.93 mV and –29.8 mV for A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs, respectively (table 1). Kumar et al [32] and Sukirtha et al [14] reported similar zeta potential values for AgNPs synthesized using G. corticata and Melia azedarach extracts in a well accordance with present results. DLS analysis showed the size distribution of silver nanoparticles with maximum intensity at 70, 80, 60 and 90 nm for A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs, respectively (table 1).

The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was carried out to determine the in vitro cytotoxic effect of the synthesized A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs. Both HepG2 and PC3 cancer cell lines were treated with different concentrations ranging from 1–100 µg ml⁻¹ for 48 h with A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs, whereas, the Vero cell lines were treated with various doses ranging from 10–100 µg ml⁻¹ for 48 h. The percent of growth inhibition by cell viability of NPs treated cancer cells along with untreated control cells are depicted in figure 6. The cell line cytotoxicity was found to be NPs concentration dependent. The cell viability was also decreased with increasing the concentrations of the four biosynthesized NPs. The IC₅₀ value was calculated using the MTT assay. The inhibitory concentration (IC₅₀) values of the HepG2 cells and PC3 cells were 75.68, 27.01, 43.76 and 40.32 µg ml⁻¹; and 54.68, 32.15, 53.16 and 52.99 µg ml⁻¹ for A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs, respectively (figures 6(a) and (b)). In this study A. paniculata AgNPs and C. asiatica AgNPs showed higher cytotoxicity at 27.01, 40.32 and 32.15, 52.99 µg ml⁻¹ for HepG2 and PC3 cell lines, respectively. A. marmelos AgNPs exhibited significant anticancer activity at 75.68 and 54.68 µg ml⁻¹ for HepG2 and PC3 cell lines, respectively. A. scholaris AgNPs showed efficient cytotoxicity against HepG2 and PC3 cells for 43.76 and 53.16 µg ml⁻¹ doses, respectively. Chavan et al [8] reported that methanolic leaf extracts of A. paniculata and ethanolic leaf extracts of A. marmelos showed anticancer activity against A549 cell lines at 60 µg ml⁻¹ and 30 µg ml⁻¹ doses, respectively. Pittella et al [9] reported that methanolic extracts of C. asiatica leaves showed cytotoxic effect on MDA-MB-231 cells at 648 µg ml⁻¹ dose. Thankamani et al [10] reported that methanolic extracts of A. scholaris roots showed anticancer activity on L929 cells. In the present study, four types of silver nanoparticles namely A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs were tested for their cytotoxic activity against two cancer cell lines (HepG2 and PC3). It is interesting to note that due to the presence of diterpenoids in A. paniculata AgNPs, it showed effective cytotoxic activity against HepG2 and PC3 cell lines followed by C. asiatica AgNPs, A. paniculata AgNPs and A. marmelos AgNPs. The IC₅₀ values for Vero cell lines were found to be 91.20, 69.99, 55.23, 54.87 µg ml⁻¹ for A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs, respectively (figure 6(c)). The AgNPs at lower doses had less cytotoxicity on the growth of normal cell lines (Vero), but enhanced cytotoxicity was observed with increasing the doses. The IC₅₀ values of PC3, HepG2 and Vero cell lines were found to be 3.756, 2.521 and 18.15 µg ml⁻¹ for using positive drug 5-fluorouracil (5-FU). The mortality data obtained in this study allowed us to predict that the green synthesised AgNPs are potential anticancer molecules/drugs to act as cytotoxic agents. The cytotoxic effects of AgNPs had indicated the occurrence of active physicochemical interaction of silver atoms with the functional groups of intracellular proteins, as well as with the nitrogen bases and phosphate groups in DNA [33].

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The morphological changes were observed in A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs treated HepG2 and PC3 cells over untreated control cells. The most recognizable morphological changes due to the AgNPs exposure were the cytoplasmic condensation, cell shrinkage, production of numerous cell surface bulged at the plasma membrane and aggregation of the nuclear chromatin into dense masses under the nuclear membrane against HepG2 (figures 7(a)–(e)) and PC3 (figures 8(a)–(e)) cells. Their membrane also became warped and vesicle shaped. AgNPs treatment resulted in swelling and rounded morphology of both cells with condensed chromatin. Progressive structural modification and reduction of HepG2 and PC3 cell populations were also observed. Similarly, Vivek et al [34] also observed cytotoxicity of silver nanoparticles synthesized using Annona squamosa leaf extracts against MCF-7 cells.

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The induction of apoptosis plays an important role in the drug development. Commercially available anticancer drugs have shown to induce apoptosis in susceptible (cancer) cells. The morphology of the necrotic, apoptotic and normal cells of both HepG2 and PC3 cells were identified separately using fluorescence microscopy after staining with acridine orange/ethidium bromide (AO/EtBr) based on the cell membrane integrity. The HepG2 and PC3 cells exhibited green fluorescence colour that expresses the presence of viable cells in untreated controls. After exposure to nanoparticles, the percent of apoptotic cells was calculated with their respective control cells. Red necrotic cells, as well as orange apoptotic cells containing apoptotic bodies were also noticed in the AgNPs treated HepG2 (figures 7(f)–(j)) and PC3 (figures 8(f)–(j)) cells. Microscopic observation showed that the normal viable cells were appeared to be green fluorescence because of the diffusion of acridine orange (AO) into cell membrane, whereas, apoptotic cells were observed as orange colored bodies due to the nuclear shrinkage and blebbing. However, the necrotic cells were turned into red color fluorescence due to their loss of membrane integrity by AgNPs induced cytotoxicity. It was observed that the total number of apoptotic cells increased when the cells were treated with their IC₅₀ concentrations of AgNPs compared to the respective controls. The percent of apoptotic bodies recorded was 58, 82, 68, and 73% against HepG2 cells, and 60, 76, 62 and 66% against PC3 cells for A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs, respectively, over control cells (figure 6(d)). Similarly, Kalaiarasi et al [17] reported that bio-fabricated silver nanoparticles using leaf extracts of bamboo had induced apoptosis in human PC3 cell lines. Most recently, Selvi et al [19] also reported that AgNPs synthesized using P. tetrastromatica seaweed extracts could induce apoptosis in human breast cancer (MCF-7) cell lines.

In order to confirm the apoptosis, rhodamine 123 staining was performed on HepG2 and PC3 cell lines that were treated with the A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs for 48 h. Silver nanoparticles treated cancer cell lines exhibited bright pink colour emission indicted the mitochondrial fragmentation which leads to the initiation of apoptosis. Interestingly, the control/untreated cells had shown intact mitochondria with normal morphology which emitted a weak pink fluorescence for HepG2 (figures 7(k)–(o)) and PC3 (figures 8(k)–(o)) cells. Wenjuan et al [35] reported that the exposure of nanoparticles had increased the levels of reactive oxygen species (ROS) generation which ultimately decreased the mitochondrial membrane potential leads to cell death.

As seen in figures 7 and 8, the Hoechst staining of untreated control cells had normal morphology with intact round nucleus which emitted by a weak blue fluorescence. It is interesting to note that both the HepG2 and PC3 cells were treated with A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs for 48 h and exposed cancer cells showed bright blue colour emission results in nuclear fragmentation with increased condensation of chromatins leading to initiation of apoptosis. Number of apoptotic cells was increased at 75.68, 27.01, 43.76, 40.32 µg ml⁻¹ and 54.68, 32.15, 53.16, 52.99 µg ml⁻¹ for A. marmelos AgNPs, A. paniculata AgNPs, A. scholaris AgNPs and C. asiatica AgNPs against HepG2 and PC3 cancer cell lines, respectively and the synthesized NPs could influence cell apoptosis in the HepG2 (figures 7(p)–(t)) and PC3 (figures 8(p)–(t)) cell lines. It has been reported that tamoxifen loaded chitosan nanoparticles showed antitumor activity in breast cancer cells [36].

The DNA fragmentation assay was carried out to determine whether the death of both PC3 and HepG2 cells had occurred due to the cytotoxic effect of AgNPs treatment or not. The DNA fragmentation experiment was performed using DNA samples obtained from AgNPs treated cell lines for 48 h along with control. Results showed that DNA ladder formation with low molecular size was observed in the treated group, while high molecular weight intact DNA band was noticed in the untreated cancer cell lines. Hence, both the cancer cell lines were found to be sensitive to biosynthesized silver nanoparticles at the IC₅₀ dose. Thus, the cancer cell growth inhibitory effect of AgNPs was closely associated with DNA-fragmenting mechanisms due to the occurrence of apoptosis (figure 9). However, the untreated PC3 and HepG2 cells did not show any DNA fragmentation. Similar results were also reported previously in Citrullus colocynthis mediated silver nanoparticles [33] and on Cassia auriculata leaf extracts [37]. It is reported that many chemotherapeutic drugs, including Vp16 and mitomicin C, induce cells to undergo apoptosis through damage of nuclear DNA [38]. Apoptotic cells are generally assumed that the toxicity of antitumor drugs is the consequences of their ability to cause genomic DNA damage in cancer cells [39].

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