Hypnea musciformis-mediated Ag/AgCl-NPs inhibit pathogenic bacteria, HCT-116 and MCF-7 cells' growth in vitro and Ehrlich ascites carcinoma cells in vivo in mice.

In the present study, Ag/AgCl-NPs were biosynthesised using Hypnea musciformis seaweed extract; NPs synthesis was confirmed by a change of colour and observation of a razor-sharp peak at 424 nm by UV-visible spectroscopy. Synthesised nanoparticles were characterised by transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray powder diffraction and Fourier transform infrared spectroscopy. Bacterial cell growth inhibition proves that the Ag/AgCl-NPs have strong antibacterial activity and cell morphological alteration was observed in treated bacterial cells using propidium iodide (PI). Ag/AgCl-NPs inhibited Ehrlich ascites carcinoma (EAC) cells, colorectal cancer (HCT-116) and breast cancer (MCF-7) cell line in vitro with the IC50 values of 40.45, 24.08 and 36.95 μg/ml, respectively. Initiation of apoptosis in HCT-116 and MCF-7 cells was confirmed using PI, FITC-annexin V and Hoechst 33342 dye. No reaction oxygen species generation was observed in both treated and untreated cell lines. A significant increase of ATG-5 gene expression indicates the possibility of autophagy cell death besides apoptosis in MCF-7 cells. The initiation of apoptosis in EAC cells was confirmed by observing caspase-3 protein expression. Ag/AgCl-NPs inhibited 22.83% and 51% of the EAC cell growth in vivo in mice when administered 1.5 and 3.0 mg/kg/day (i.p.), respectively, for 5 consequent days.

INTRODUCTION

In recent periods, cancer is a serious disease, and globally, it is the second leading cause of death. According to a WHO report in 2018, globally, 9.6 million deaths occurred due to cancer. Cancer deaths are increasing in low‐ and middle‐income countries [1] and Bangladesh is a country where the number of cancer patients is gradually rising. According to cancer statistics in Bangladesh, about 0.2 million cancer patients are diagnosed each year [2]. Many synthetic drugs are available in the market for cancer eradication but are not satisfactory to patients. Serious side effects occur that lead to death of patients. Depending on patient demand, scientists are searching for a new drug for cancer eradication with reduced side effects.

As alternative anticancer and antimicrobial agents, metal nanoparticles are gradually gaining more importance in the biomedical area. Green synthesis of plant extract‐mediated silver nanoparticles (AgNPs) has attracted researchers for the use of natural resources and their eco‐friendliness, benignancy and rapid production, and they do not produce toxic by‐products. Various bioactive molecules are present in plants that participate in the formation of AgNPs.

Plant‐mediated AgNPs have become increasingly popular as antibiotic agents in textiles and wound dressings, medical devices and appliances such as refrigerators and washing machines. Many researchers already reported the antibacterial activity of plant‐mediated AgNPs [3, 4, 5]. Nowadays, AgNPs have gained increased attention due to their promising role as anticancer agents [6, 7]. Several scientists reported that AgNPs inhibited the L929 cells, DLA cells, Hep2 cells, MCF‐7 cells, HT29 cells and Ehrlich ascites carcinoma (EAC) cell growth in a dose‐dependent manner [8, 9, 10, 11]. Recently, more than 60% of anticancer drugs are being invented from plants, microbes and marine organisms' natural resources [12, 13]. Natural resource‐derived drugs have fewer side effects and act more specifically than synthetic drugs [14].

Hypnea musciformis is red in colour, sometimes yellowish‐brown colour. The nutritive value of red seaweed is very high. It contains a lot of protein, vitamins, minerals, electrolytes, natural antioxidants, fibres and little amount of fat [15]. Marine sources have an important aspect as antibacterial and anticancer agents due to the presence of bioactive molecules. Different seaweeds have been reported as sources for the synthesis of AgNPs with antibacterial and anticancer properties [16, 17]. We have found three published articles on H. musciformis‐synthesised AgNPs. One involves only synthesis [18], the second one involves nanoparticle synthesis and activity against Aedes aegypti and Plutella xylostella [19] and the third one involves nanoparticle synthesis and activity against Xanthomonas campestris and Ralstonia solanacearum [20]. But till now, H. musciformis‐mediated Ag/AgCl‐NPs with oral antibacterial and anticancer properties have not been reported in the literature. Therefore, our research work's total point was to study the antibacterial and antitumour properties of H. musciformis‐mediated Ag/AgCl‐NPs. Green synthesised Ag/AgCl‐NPs was formed by H. musciformis extract and it was characterised by UV‐vis spectroscopy, transmission electron microscopy (TEM), energy‐dispersive X‐ray spectroscopy (EDX), X‐ray powder diffraction (XRD) spectra and Fourier transform infrared spectroscopy (FTIR). Additionally, the efficacy of Ag/AgCl‐NPs inhibition against two pathogenic bacteria was evaluated and cell morphological alteration was monitored by propidium iodide (PI). Furthermore, anticancer properties of Ag/AgCl‐NPs were checked in EAC cells in vivo and colorectal cancer (HCT‐116), and breast cancer (MCF‐7) cell line in vitro. Ag/AgCl‐NPs‐induced apoptotic cell death in EAC, HCT‐116 and MCF‐7 cells was detected by caspase‐3 protein expression, PI, FITC‐annexin V and Hoechst 33342 dye. Besides apoptosis, autophagy cell death in MCF‐7 cells was also evaluated by gene expressions.

MATERIALS AND METHODS

Sample preparation

Sample was prepared according to the modified method by Venkatesan et al. [21]. Firstly, seaweed (H. musciformis) was collected from the Cox's Bazar region of Bangladesh and was washed with tap water and dried in sunlight. Then, it was powdered by a blender machine and homogenised with distilled water at a 1:100 ratio (w/v) for 15 min at 90°C. The homogenate was filtered two times by a muslin cloth. Subsequently, it was centrifuged to get a clear solution at 6000 g for 15 min. Finally, a clear light brown‐coloured supernatant solution or extract was collected in a falcon tube and kept at 4°C.

Ag/AgCl‐NPs synthesis

Freshly prepared silver nitrate solution (1.0, 2.0 and 3.0 mM) was mixed with H. musciformis extract at a 1:1 ratio (v/v). The mixture solution was kept in sunlight at different reaction times (1, 2 and 4 h) for Ag/AgCl‐NPs synthesis.

Characterisation of the synthesised nanoparticles

The formation of nanoparticles was confirmed by UV–visible spectra analysis; morphological characterisation was carried out by TEM analysis; elemental characterisation was completed by EDX analysis; structural characterisation was carried out by XRD analysis and functional characterisation was carried out by FTIR analysis according to Kabir et al. [22] and the method is provided in supporting information S1.

Growth inhibition assay of HCT‐116, MCF‐7 and EAC cells in vitro

In vitro, EAC cell growth inhibition was performed following the method by Kabir et al. [22, 23] with a slight modification and provided in supporting information S1.

Propidium iodide and FITC‐Annexin V staining

Ag/AgCl‐NPs treated and untreated cells were performed using a FITC‐annexin V and PI detection kit (ebioscience). First, HCT‐116 and MCF‐7 cells (2 × 104/well) were seeded in a 96‐well culture plate, and after 24 h, three wells of cells were treated with 20 μg/ml concentration of H. musciformis extract‐mediated Ag/AgCl‐NPs, respectively, for 48 h. Untreated wells with cells were used as control. According to the manufacturer's (US Ever Bright) direction, cells were stained with PI and annexin V. Finally, cell morphological changes were observed by using a fluorescence microscope (Olympus IX71).

Study of cell nuclei change by Hoechst 33342 staining

Around 10,000 HCT‐116 and MCF‐7 cells were seeded in each well of a 96‐well cell culture plate and then treated with 20 μg/ml concentration of Ag/AgCl‐NPs for 48 h as described above. After that, the cells were rinsed with phosphate buffer saline and stained with Hoechst 33342, according to Kabir et al. [22]. Finally, cell morphology was observed under both dark and bright fields using a fluorescence microscope.

Observation of changes of the reactive oxygen species

After treatment with 20 μg/ml of Ag/AgCl‐NPs, changes of the reaction oxygen species (ROS) level in the HCT‐116 and MCF‐7 cells were detected using 2′,7′‐dichlorofluorescein‐diacetate (DCFH‐DA) staining. Cells were cultured in a 96‐well plate and treated with Ag/AgCl‐NPs for 48 h as described above. Cells were washed once by the serum‐free medium and incubated with diluted DCFH‐DA (1:1000) at 37°C for 20 min, and finally, the cells were examined by a fluorescence microscope.

mRNA expression by real‐time polymer chain reaction

For RNA isolation, MCF‐7 cells (16 × 104/well) were seeded in a 6‐well cell culture plate. After that, it was treated with 64 μg/ml concentration of Ag/AgCl‐NPs. Untreated cells were used as control. After the 48‐h incubation period, the next procedure was followed according to the method by Kabir et al. [24]. Forward and reverse primers were used for the reaction stated in Table S1.

EAC cells growth inhibition assay in vivo in mice

EAC cell growth inhibition was determined according to Kabir et al. [25] and provided in supporting information S1.

Caspase‐3 protein expression assay

After cell separation from mice, the treated (3.0 mg/kg/day) and untreated EAC cells were incubated with the US Ever Bright super view‐488 caspase‐3 substrate for 30 min according to the manufacturer's guideline. Finally, cell morphology was examined using a fluorescence microscope described by Kabir et al. [26].

Bacterial cell growth inhibition by MTT colourimetric assay

Bacterial cell growth inhibition was performed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay according to Kabir et al. [22] and provided in supporting information S1.

Zone of bacterial growth inhibition by disc diffusion assay

Antibacterial activity of the synthesised Ag/AgCl‐NPs was determined using the agar well diffusion assay method and furnished in supporting information S1.

Antibiofilm assay

Antibiofilm assay was performed to observe film formation on top of the bacterial nutrient broth in test tubes with and without Ag/AgCl‐NPs according to the method by Hasan et al. [27] and provided in supporting information S1.

Bacterial cell morphological study by propidium iodide

Bacterial cells were treated with 32 μg/ml of Ag/AgCl‐NPs to observe the morphological change. Details are given in supporting information S1.

RESULTS

Ag/AgCl‐NPs formation

For Ag/AgCl‐NPs formation, we added 1.0 ∼ 3.0 mM of the silver nitrate solution in H. musciformis extract at a 1:1 ratio (v/v). After that, it was kept in sunlight for reaction at different times. The intensity of the solution colour increased from light red to deep brown with increased AgNO3 concentrations for each reaction time, which is shown in Figure 1a–c. UV‐Visible spectra were measured from 285 to 700 nm to confirm the formation of Ag/AgCl‐NPs. A sharp peak around 424 nm was observed in the test tube containing 2 mM AgNO3 at 2 h incubation as shown in Figure 1c–e. At that condition, nanoparticles were synthesised for further studies.

FIGURE 1

The formation of Ag/AgCl‐NPs was identified by a colour change and UV–visible spectra: (a) Hypnea musciformis powder; (b) H. musciformis extract; (c) after reaction between silver nitrate and H. musciformis extract. (1) 1 mM AgNO3; (2) 2 mM AgNO3; (3) 3 mM AgNO3. UV–visible spectra of the reaction mixture at different concentrations of AgNO3 and reaction time during the synthesis of H. musciformis extract‐mediated Ag/AgCl‐NPs; (d) 1–3 mM AgNO3, 1 h; (e) 1–3 mM AgNO3, 2 h; (f) 1–3 mM AgNO3, 4 h

The spherical shape observed in synthesised Ag/AgCl‐NPs using TEM that was well monodispersed is shown in Figure 2a. The average particle size was estimated as 11 nm calculated by image J software shown in Figure 2b. The 3 KeV is a typical silver peak region. Metallic silver nanocrystalline absorption occurs at this region due to surface plasmon resonance. The presence of elemental Ag, C and Cl in the synthesised Ag/AgCl‐NPs was confirmed by EDX as shown in Figure 2c. Different XRD reflection peaks 27.72°, 32.14°, 46.24°, 54.76°, 57.32° and 67.26° indicate the following crystallographic planes (111), (200), (220), (311), (222) and (400), respectively. The above‐mentioned peak points out the formation of AgCl‐NPs compared to card no. 00‐901‐1666. In contrast, we also identified other XRD reflection peaks 38.10° and 44.32° that indicate the crystallographic planes (111) and (200), respectively. Compare to card no. 00‐150‐9146, the above two peaks identified the formation of AgNPs (Figure 2d). H. musciformis extract shows different major peaks at 3427.88, 1639.74, 1384.42, 1097.33 and 618.04 cm−1. On the contrary, the FTIR spectrum of synthesised Ag/AgCl‐NPs shows the major peaks at 3411.45, 2920.32, 1631.03, 1384.13, 1101.1 and 618.34 cm−1 (Figure 2e,f).

Characterisation of Hypnea musciformis extract‐mediated Ag/AgCl‐NPs. (a) Transmission electron microscopy micrograph showing the formation of Ag/AgCl‐NPs. Inset: black solid bar indicating 100 nm. (b) Frequency distribution of synthesised Ag/Agcl‐NPs. (c) The energy dispersive X‐ray spectrum of Ag/AgCl‐NPs. (d) X‐ray powder diffraction (XRD) pattern of synthesised Ag/Agcl‐NPs (+) and (*) indicating AgCl‐NPs and silver nanoparticles (AgNPs), respectively. (e) Fourier transform infrared (FTIR) spectrum of H. musciformis extract. (f) FTIR spectrum of synthesised Ag/AgCl‐NPs

Antibacterial activity

MTT assay was used to investigate the effect of Ag/AgCl‐NPs on oral bacterial cell growth inhibition. The Ag/AgCl‐NP‐induced bacterial cell growth inhibition was found to dose reliant focussed in Figure 3a,b. At 32 μg/ml, the inhibitory effect was 89.20% and 82.71% for Staphylococcus aureus and Streptococcus mutans, respectively, and it progressively declined to 57.46% and 51.66% at 4 μg/ml. Additionally, the significant zone of bacterial growth inhibition was observed against both bacteria shown in Table 1.

FIGURE 3

Antibacterial activity and detection of possible apoptosis by fluorometric assay. (a) Staphylococcus aureus cell growth inhibition in vitro; (b) Streptococcus mutans cell growth inhibition in vitro; (c) Ag/AgCl‐NPs untreated (‘1’ and ‘2’) and treated (‘3’ and ‘4’) bacterial cells were incubated with propidium iodide (PI). ‘a’ and ‘b’ indicating S. mutans and S. aureus bacteria where ‘1’ and ‘3’ representing optical images; ‘2’ and ‘4’ representing fluorometric images after stained with PI

TABLE 1

Zone of bacterial growth inhibition by Hypnea musciformis extract‐mediated Ag/AgCl‐NPs

Zone of bacterial growth inhibition
Name of bacteria	Hypnea musciformis‐mediated Ag/AgCl‐NPs (μg/ml)				Streptomycin (unit)
	10	20	40	80	15
Staphylococcus aureus	19.30 ± 1.00	20.50 ± 1.20	21.50 ± 1.00	22.00 ± 1.00	12.00 ± 0.50
Streptococcus mutans	19.00 ± 1.00	20.30 ± 0.75	21.40 ± 1.50	21.70 ± 1.00	11.60 ± 0.25

Identification of apoptosis by propidium iodide

After using PI staining in Ag/AgCl‐NP‐treated S. aureus and S. mutans bacterial cells, a considerable number of red colour containing apoptotic bacterial cells were observed by a fluorescence microscope (Figure 3c).

Antibiofilm activity

H. musciformis extract‐mediated Ag/AgCl‐NPs inhibited biofilm formation against S. mutans and S. aureus at different concentrations shown in Table S2. There was no visualised biofilm formation up to 96 h in S. mutans and S. aureus by using 20 μg/ml of Ag/AgCl‐NPs.

Cell growth inhibition in vitro and in vivo

Ag/AgCl‐NPs inhibited the EAC, HCT‐116 and MCF‐7 cells growth inhibition in a dose‐dependent manner (Figure 4a,b). The IC50 values were calculated to be 40.45 μg/ml, 24.08 and 36.95 μg/ml, respectively. Besides the EAC cell growth inhibition in vitro, Ag/AgCl‐NPs had effectively inhibited EAC cell growth inhibition in vivo in mice. We found 22.83% of EAC cells growth inhibition at the dose of 1.5 mg/kg/day. But when the dose was increased to 3.0 mg/kg/day, the cell growth inhibition rose to 51% (Figure 4c).

FIGURE 4

Antiproliferative activity of Hypnea musciformis extracts mediated Ag/AgCl‐NPs. (a) Ehrlich ascites carcinoma (EAC) cell growth inhibition in vitro; (b) colorectal cancer (HCT‐116) and MCF‐7 cell growth inhibition in vitro; (c) effect of Ag/AgCl‐NPs on EAC cells growth in vivo in mice. Data are expressed in mean ± SD (n = 6)

Induction of apoptosis by different fluorometric assays

After treatment with the Ag/AgCl‐NPs, morphological changes in cells, early and late apoptotic features and no ROS production in the microscopic image of HCT‐116 and MCF‐7 cells were detected as presented in Figure 5a,b, respectively.

FIGURE 5

Induction of apoptosis and generation of reaction oxygen species by different fluorometric assays (a) and (b) detection of early and late apoptosis in colorectal cancer (HCT‐116) and breast cancer (MCF‐7) cells, respectively, where ‘a’ and ‘b’ indicates control and treated cells. ‘1’ indicating optical image and ‘2’ and ‘3’ indicating fluorometric images for early and late apoptosis, respectively. ‘c’ and ‘d’ indicate control and treated cells stained with Hoechst 33342 dye, where ‘1’ indicating optical image and ‘2’ indicating fluorometric image. Finally, e1 and e2 indicate fluorometric images for control and treated cells after staining with DCFH‐DA. The image was captured at 20× magnification

Detection of caspase‐3 protein and gene expressions

We used an optical and fluorescence microscope to observe the morphological examination of Ag/AgCl‐NPs treated and untreated EAC cells. A round shape was observed in untreated cells, whereas a morphological variation was observed in treated cells using an optical microscope focussed in Figure 6a. By using a fluorescence microscope, the green colour cell image was not visualised in the untreated EAC cell surface. However, the image of the cells was green in treated EAC cells (Figure 6a). It might have happened due to substrate bound with caspase‐3 that indicates the induction of apoptosis. We treated H. musciformis‐mediated Ag/AgCl‐NPs on MCF‐7 cells to determine the expression level of apoptosis‐related genes by real‐time polymer chain reaction. In this study, ATG‐5 gene expression level was increased by several times, whereas FAS, BAX and Bcl‐2 gene expression level was decreased. A little rise in NFκB gene expression was also found (Figure 6b).

FIGURE 6

Gene and protein expressions. (a) and (b) representing control and treated cells, where ‘1’ and ‘2’ indicate optical and fluorometric views; (b) percentages of relative mRNA expression after treatment of MCF7 cells with Ag/AgCl‐NPs

DISCUSSION

Seaweeds have many bioactive compounds [28, 29] and possibly they are used as reducing and capping agents to synthesise biogenic nanoparticles. In the present study, we have green synthesised Ag/AgCl‐NPs from the H. musciformis extract, and it was characterised by UV–vis spectra, TEM, EDX, XRD and FTIR. The change of solution colour after the reaction is the primary indication of the formation of Ag/AgCl‐NPs. Our synthesised Ag/AgCl‐NPs solution colour changed from light red to deep brown after the reaction. The absorbance peak of all reaction solutions was between 400 and 450 nm determined by UV–visible spectroscopy. A sharp absorbance peak at the wavelength of 425 nm was due to the surface plasmon resonance [30]. The intensity of the solution colour increased with the increase in AgNO3 concentrations for each reaction time that might have happened due to the formation of different sizes of nanoparticles. The highly monodispersed and spherical shape of the majority of particles was observed by TEM. The sizes of the nanoparticles diameter were within the range of 10–80 nm, where an average diameter was 11 nm estimated by image J software. Roni et al. [19] and Vadlapudi and Amanchy [20] reported that H. musciformis‐mediated Ag/AgCl‐NPs were spherical, and the size of the diameter was within the range of 40–65 nm and 16–42 nm, respectively. Different cultivated samples, the sample extraction process and reaction conditions might be responsible for forming different sizes of nanoparticles. Several researchers reported that most of the plant‐mediated AgNPs were spherical in shape and the average diameters were between 25 and 50 nm [31]. EDX exhibited elemental silver and chlorine ions and signals of carbon and oxygen in Ag/AgCl‐NPs. The weak signals can be formed due to the binding of bioactive molecules to the surface of Ag/AgCl‐NPs [32]. XRD proved the crystalline nature of the synthesised nanoparticles and showed the peaks for Ag and AgCl. Many scientists reported similar observations [22, 24, 33]. H. musciformis extract showed the major peaks at 3427.88, 1639.74, 1384.42, 1097.33 and 618.04 cm−1, whereas 3411.45, 2920.32, 1631.03, 1384.13, 1101.1 and 618.34 cm−1 peaks were obtained in synthesised Ag/AgCl‐NPs. The peaks at 3427.88 and 3411.45 cm−1 indicated the characteristics of the hydroxyl functional group in phenol and alcohol compounds [34]. The peak at 2920.32 cm−1 indicated CH stretching of alkanes [30]. The peaks at 1639.74 and 1631.03 indicated the bending vibration of the (NH) C=O group [35]. The peaks at 1384.42 and 1384.13 cm−1 indicated stretching for the (N–O) functional group [35].

The peaks at 1097.33 and 1101.1 cm−1 indicated a vibration of the (C=O) functional group [30]. The peaks at 618.04 and 618.34 cm−1 indicated a vibration of (C–Cl) groups [36]. As compared, we can say that the H. musciformis extract and the synthesised Ag/AgCl‐NPs share certain common functional groups. Probably, –OH groups might be responsible for reducing Ag+ to Ag° [30]. The band shift in hydroxyl and carbonyl groups in FTIR spectra confirm the oxidation of amines, phenols, alcohols, carboxylic acid and other secondary metabolites of seaweed extract and this shifting is also involved in stabilising/capping the nanoparticles as reported in many studies [37]. Thus, these functional groups may perform a significant role in the reduction of Ag+ into Ag/AgCl‐NPs. The above‐mentioned shift was observed during the nanoparticle formation from various sources of extracts [16, 38].

Inhibition of bacterial growth is a common biological characterisation of AgNPs. In the present study, we have also checked the antibacterial activities of the newly synthesised Ag/AgCl‐NPs against pathogenic bacteria by MTT assay, disc diffusion assay and antibiofilm assay. In all cases, synthesised Ag/AgCl‐NPs showed good inhibition to both S. aureus and S. mutans. The synthesised Ag/AgCl‐NPs were more effective against both bacteria compared to streptomycin. The exact mechanism of Ag/AgCl‐NPs against different bacteria is still unknown. Many scientists reported that AgNPs generate pore/pit in the bacterial cell wall and cause permeability on bacterial cell surfaces, and it increases for small‐size nanoparticles. Next, nanoparticles bind the functional groups of DNA and proteins and cellular alteration occurs [39, 40, 41]. Bacterial cells were stained with PI to find the molecular mechanism of the antiproliferative activity of Ag/AgCl‐NPs. Treated bacterial cells exhibited the induction of apoptosis. Induction of apoptosis in bacterial cells was reported by other scientists as well [41, 42].

Cancer is an uncontrolled proliferation of cells with tremendous lethal effects on patients. For the past few years, the outbreak of cancer has increased worldwide as a most important cause of death. At this point, annually, more than seven million people are being affected by cancer diseases worldwide. However, we have developed cancer diagnosis and treatment, but till now, it is an enormous trouble for society. Seaweeds contain plentiful bioactive compounds, and it has important therapeutic values that have become evident [43]. Some bioactive compounds assist in nanoparticle formation as reducing and capping agents can be used to develop new drugs against cancer cells. It was reported in the literature that marine plant‐mediated nanoparticles have antitumour property [10]. In the present study, we used EAC, HCT‐116, and MCF‐7 cells to evaluate the antiproliferative activity of H. musciformis‐mediated Ag/AgCl‐NPs and identified strong antiproliferative activity against EAC and MCF‐7 cells. The anticancer potential of plant extract‐mediated Ag/AgCl‐NPs against different cancer cell lines were different. Zizyphus mauritiana‐assisted Ag/AgCl‐NPs showed antiproliferation against EAC cells with the IC50 values of 84 μg/ml [22]. Ulva lactuca seaweed‐assisted AgNPs showed antiproliferation against MCF‐7 cells with the IC50 values of 37 μg/ml [10]. The IC50 values of oscillatoria‐mediated AgNPs and GM‐mediated AgNPs on HCT‐116 cells were 5.369 and 40.2 μg/ml, respectively [43, 44].

After cancer cell growth inhibition, we used the dye PI in treated and untreated HCT‐116 and MCF‐7 cells to know the molecular mechanism of Ag/AgCl‐NPs. The induction of apoptosis in treated HCT‐116 and MCF‐7 cells was exhibited. A similar observation was confirmed when we used the dye FITC‐Annexin V. The induction of apoptosis in cells was further verified by using Hoechst 33342 dye. The irregular shape and condense nuclei were visualised in treated cells. Many scientists reported that AgNPs induce apoptosis in a different cancer cell lines [45, 46, 47, 48, 49]. Several studies stated that cellular uptake of AgNPs leads to the generation of ROS in cancer cells, which provokes oxidative stress causing apoptosis [22] but we did not find ROS generation in treated HCT‐116 and MCF‐7 cells. Our study revealed that the synthesised Ag/AgCl‐NPs could promote cell death without ROS generation.

In our previous experiments, it was found that after the treatment of MCF‐7 cells with the Zizyphus mauritiana fruit extract‐mediated Ag/AgCl‐NPs, the expression of Fas increased that activated the caspase‐3 expression, sequentially activating FADD and caspase‐8. Finally, PARP was cleaved, and apoptosis was induced in the Fas‐mediated pathway [22]. But in this experiment, after the treatment of MCF‐7 cells with the H. musciformis seaweed extract‐mediated Ag/AgCl‐NPs, the expression of Fas decreased indicating that the Fas pathway was not involved in this apoptosis process. In another experiment, Kaempferia rotunda caused a significant over‐expression of NFκB gene in glioblastoma stem cells by activating several genes. Finally, the NFκB entered into the nucleus and broke down DNA, and apoptosis occurred [24]. In the present study, NFκB was not responsible for apoptosis as the expression level of NFκB was not altered. The mitochondrial pathway may not be responsible for this apoptosis process as the expression level of Bcl‐2 and BAX decreased. A significant increase in autophagy cell death‐related ATG5 gene expression was observed. It indicates that autophagy cell death may also occur in the MCF‐7 cells besides apoptosis. After cancer study in vitro using Ag/AgCl‐NPs, we used the experiment in vivo on EAC cells. Ag/AgCl‐NPs inhibited 22.83% and 51% of the EAC cell growth in vivo in mice when administered 1.5 and 3.0 mg/kg/day (i.p.), respectively, for five consequent days. In our earlier experiments, the antitumour activity of K. rotunda tuberous rhizome and Z. mauritiana fruit extract‐mediated Ag/AgCl‐NPs was checked against EAC cells in vivo in mice. 20% cells growth inhibition was observed for Z. mauritiana fruit extract‐mediated Ag/AgCl‐NPs at the dose of 12 mg/kg/day. On the other hand, at the doses of 6 and 12 mg/kg/day, 32.3% and 55% EAC growth inhibition was monitored. From the results, it is clear that H. musciformis seaweed extract‐mediated Ag/AgCl‐NPs is a more effective anticancer agent than K. rotunda and Z. mauritiana fruit extract‐mediated Ag/AgCl‐NPs. Treated and untreated EAC cells were stained with the caspase‐3 substrate and a significant level of the caspase‐3 protein expression was found in the treated cells that indicated the induction of apoptosis in EAC cells.

CONCLUSIONS

By a cost‐effective, simple and eco‐friendly approach, Ag/AgCl‐NPs were successfully synthesised using H. musciformis extract, cultivated in Bangladesh, as a capping and reducing agent. The characteristics of H. musciformis‐mediated Ag/AgCl‐NPs were determined by UV, TEM, EDX, XRD and FTIR. These procedures proved that the synthesised Ag/AgCl‐NPs were of spherical shape with an average size of 11 nm and crystalline nature. Remarkable antibacterial activity of synthesised Ag/AgCl‐NPs was detected against oral pathogenic bacteria S. aureus and S. mutans. It has potent anticancer activity against EAC, HCT‐116 and MCF‐7 cells. Initiation of apoptosis was induced in EAC, HCT‐116 and MCF‐7 cells using synthesised Ag/AgCl‐NPs. Besides, the initiation of apoptosis autophagy cell death occurred in MCF‐7 cells.

CONFLICT OF INTEREST

The authors of this manuscript have declared no conflict of interest.

Supporting Information S1

Click here for additional data file.