Biogenic silver/silver chloride nanoparticles inhibit human glioblastoma stem cells growth in vitro and Ehrlich ascites carcinoma cell growth in vivo

Abstract The importance of biogenic silver/silver chloride nanoparticles has become increasing day by day. In the present study, silver/silver chloride nanoparticles (Ag/AgCl‐NPs) were synthesized from Kaempferia rotunda tuberous rhizome extract to evaluate the antiproliferative activity against human glioblastoma stem cells (GSCs) in vitro and Ehrlich ascites carcinoma (EAC) cells in vivo in mice. Synthesis of nanoparticles was confirmed by colour change and UV‐visible spectrum and characterized by TEM, XRD, TGA, AFM and FTIR. K rotunda and recently synthesized Zizyphus mauritiana fruit extract‐mediated Ag/AgCl‐NPs inhibited 77.2% and 71% of GSCs growth at 32 µg/mL concentration with the IC50 values of 6.8 and 10.4 µg/mL, respectively. Cell morphological studies and caspase‐3 immunofluorescence assay revealed that both biogenic nanoparticles induced apoptosis in GSCs. Expression levels of several genes were checked by real‐time PCR after treatment with K rotunda tuberous rhizome‐mediated Ag/AgCl‐NPs. PARP, EGFR, NOTCH2 and STAT3 gene expression were decreased with the increase of NFκB, TLR9, IL1, TNFα, IKK and p21 gene that would be the cause of induction of apoptosis in GSCs. The cell cycle arrest at G2/M phase was confirmed by flow cytometric assay. Both nanoparticles were injected intraperitoneally to rapidly growing EAC cells for 5 consecutive days. Approximately, 32.3% and 55% EAC cells growth were inhibited by K rotunda tuberous rhizome‐mediated Ag/AgCl‐NPs at 6 and 12 mg/kg/day doses, respectively while only 20% cell growth inhibition was monitored at 12 mg/kg/day dose of Z mauritiana ‐mediated Ag/AgCl‐NPs. From the above results, it can be concluded that presently synthesized nanoparticles would be a potent anticancer agent.


| INTRODUC TI ON
Nanotechnology is widely used in sciences, medicine, biotechnology, electronics and other fields to create numerous novel materials with a variety of applications. 1 Recently, metal nanoparticles have become increasingly important as alternative antimicrobial and anticancer agents in the biomedical and pharmaceutical sector. 2 Noble metals such as silver, gold and platinum are used to make the most effective nanoparticles. 3 Due to the disinfecting nature and tremendous medicinal value, silver has been attracted to the researcher. Silver nanoparticles can be synthesized chemically, electrochemically, photochemically, biologically and by using radiation. Among the techniques, green synthesis of silver/ silver chloride NPs (Ag/AgCl-NPs) gained a lot of interest due to the usage of natural resources, rapidness and eco-friendliness. 4 Various biomolecules such as enzymes, proteins, flavonoids, terpenoids and cofactors are present in the fruit, bark, root, and leave of plant those act as reducing and capping agents of silver. [3][4][5][6][7][8] Some of the synthesized nanoparticles showed potent antiproliferative effect against different cancer cell lines. [9][10][11][12][13] It was reported that biologically synthesized metal nanoparticles showed genotoxicity that varies case-by-case and highly dependent on the synthesis parameters, biological source, applied assay, etc. 14 Several articles reported that biogenic silver nanoparticles are several times toxic for cancer cells than that of the normal cells 15 and few of them did not show any toxicity against normal cells. 16 Although green Ag/AgCl-NPs were subjected against different cancer cell lines but effects against glioblastoma stem cells (GSCs) in vitro and Ehrlich ascites carcinoma (EAC) cells in vivo were not reported.
Recently, Zizyphus mauritiana fruit extract-mediated Ag/AgCl-NPs were synthesized at our laboratory and characterized by different spectroscopic methods. 2 The Ag/AgCl-NPs exhibited antibacterial, antifungal and cytotoxic effects against EAC and human breast cancer cell (MCF-7). The cytotoxic effect against MCF-7 cell was due to the generation of ROS and induction of apoptosis in Fasmediated pathway. 2 Another silver nanoparticle was synthesized by using Kaempferia rotunda tuberous rhizome. K rotunda is a common medicinal herb in Bangladesh and India. Different phytochemical analysis of K rotunda demonstrated the content of many active phytoconstituents, include terpenoid, flavonoid, steroid, alkaloid, crotepoxide, chalcone, quercetin, flavanols, β-sitosterol, stigmasterol, syringic acid, protocatechuic acid and some hydrocarbons. 17,18 Two novel lectins with anticancer property were also isolated from K rotunda tuberous rhizome. 19,20 In the present study, we are first reporting biosynthesis of Ag/ AgCl-NPs from K rotunda tuberous rhizome extract with the morphological, structural, thermal, functional characterization and surface property. The cytotoxic effects of the K rotunda tuberous rhizome and Z mauritiana fruit extract-mediated Ag/AgCl-NPs were reported against human GSCs in vitro and against EAC cells in vivo in mice.

| Sample preparation
K rotunda tuberous rhizomes were collected from the local market.
After washing with deionized water, rhizomes were cut into small pieces and homogenized with deionized water at 1:5 ratios (w/v).
Then pH of the homogenized sample was adjusted around 7.0 with 1.0 M Tris-HCl buffer and centrifuged at 10 000 g. The clear supernatant was used for silver nanoparticles synthesis and lyophilized by freeze dryer for cytotoxicity test.

| Synthesis of Ag/AgCl-NPs and analysis of UVvisible spectra
For the synthesis of silver nanoparticles, 1.0 M of AgNO 3 was added to the clear supernatant of K rotunda tuberous rhizome extract to a final concentration of 1, 2, 3, 4 and 5 mM and kept in sun light for 3 hours. The sample colour changed to brown and deep brown and then analysed by UV-visible spectrophotometer (Hitachi U-1800, Japan) at the wavelength range from 200 to 700 nm. At the concentration of 5 mM of AgNO 3 best result was observed. For the large-scale synthesis, 250 mL of K rotunda transparent supernatant was taken in a 500 mL beaker and 0.5 M of AgNO 3 was added to reach the final concentration of 5 mM. After that the mixture was kept in sunlight for 3 hours and a deep brown colour was developed.
Then the mixture was centrifuged at 10 000 g for 30 minutes at 4•C. Afterwards deionized water was used to rinse the pellet several times. Lyophilized colloidal nanoparticles were used only for the concentration determination and some characterization. The colloidal solution was used for further characterization and biological application. Z mauritiana fruit extract-mediated Ag/AgCl-NPs were synthesized as described Kabir et al.

| Characterization of Ag/AgCl-NPs
Transmitted electron microscope (TEM, JEOL, Japan) was used to observe the morphology of synthesized Ag/AgCl-NPs. 'ImageJ' software was used to detect the size of synthesized Ag/AgCl-NPs. X-ray powder diffraction (XRD) was used to know the structure of the synthesized Ag/AgCl-NPs by PANalytical Empyrean X-ray diffractometer with a Cu-Kα (1.5418A) radiation source and the data were analysed by QualX2.0 software. The scanning was performed in the region of 10° to 80°. Thermal stability of Ag/AgCl-NPs was performed by using thermal gravimetric analysis (TGA, PerkinElmer STA 8000, USA) at a heating rate of 20°C/min under nitrogen atmosphere from 30 to 835°C. Colloidal Ag/AgCl-NPs were dried in a glass slide and the surface properties were analysed by an atomic force microscope (AFM, Park system XE 70, Korea) where titanium coated nitrate tips were in tapping mode.
For Fourier transform infrared spectroscopy (FTIR, Perkin Elmer, Spectrum 100, USA) band, lyophilized Ag/AgCl-NPs and crude extract were mixed with potassium bromide (KBr) separately and measured at the frequency range from 4000 to 225 cm −1 with a resolution of cm −1 .

| Culture of GSCs and EAC cells
The established human glioblastoma stem cells-3 (GSCs) was used in this study. 21,22 Briefly, laminin in PBS (1:100) was added to the cell culture petridish or 96 well flat bottom plate and incubated at 37°C for 2.0 hours. Then cells in DMEM/F12 culture medium were seeded and grown in a CO 2 incubator at 37°C. EAC cells were propagated biweekly at our laboratory as described by Kabir et al. 23

| Cytotoxicity study by MTS assay
GSCs in DMEM/F12 medium were seeded in a 96 well culture plate (2 × 10 4 cells/well) which was previously treated with laminin and incubated in a CO 2 incubator. After 24 hours of initial seeding, cells were treated with 2-32 µg/mL concentration of K rotunda tuberous rhizome and Z mauritiana fruit extract-mediated Ag/AgCl-NPs for 48 hours. EAC cells were collected from mice as described earlier. 23  was added to each well and incubated in the dark for 2 hours. After colour development, absorbance was measured at 490 nm using a plate reader. Cells without Ag/AgCl-NPs were used as control.

| FITC labelled Annexin V and PI staining
Apoptosis assays of Ag/AgCl-NPs treated and control cells were performed using an FITC labelled annexin V/PI detection kit (ebioscience, USA). At first GSCs (2 × 10 4 /well) were seeded in a 96 well culture plate and after 24 hours, three wells of cells were treated with 6.8 and 10.4 μg/mL concentration of K rotunda tuberous rhizome and Z mauritiana fruit extract-mediated Ag/AgCl-NPs, respectively for 48 hours. Untreated wells with cells were used as control.
Cells were stained with Annexin V and PI according to the manufacturer direction. Finally, early and late apoptosis was observed by using a fluorescence microscope (Olympus IX71).
Three wells were treated for 48 hours with 6.8 and 10.4 μg/mL concentrations of K rotunda tuberous rhizome and Z mauritiana fruit extract-mediated Ag/AgCl-NPs, respectively as described above.
Remaining three wells without Ag/AgCl-NPs were used as control.
After that, cells were fixed by 4% paraformaldehyde for 1 hour at room temperature and incubated with 0.1% triton X-100 in PBS for 5 minutes at room temperature and washed thrice with PBS.
Subsequently, 200 µL of 10% goat serum in PBS was added to each well and incubated for 1 hour at room temperature. After removal of the serum, cells were washed by PBS and 100 µL of the caspase-3 primary antibody (400 times diluted by 0.5% tween-20 and 1% BSA in PBS) was added and incubated for 1 hour at room temperature.
Then cells were washed three times with 0.1% tween-20 in PBS at room temperature. After 10 minutes tween-20 was removed and 100 µL of Cy3 goat-anti Rabbit IgG antibody (500 times diluted in PBS) was added to each well in the dark and incubated for 2 hours at room temperature. Finally, cells were washed thrice by 0.1% tween-20 in PBS for 5 minutes and stained with DAPI in PBS and observed in a fluorescence microscope.

| Genes expression analysis by real-time PCR
For the isolation of RNA, GSCs (3.2 × 10 5 /well) were seeded in 6-well culture plate and 24 hours later treated with 6.8 μg/mL of K rotunda tuberous rhizome-mediated Ag/AgCl-NPs as described above. Cells incubated without Ag/AgCl-NPs were used as control.
For the isolation of RNA, cells were washed by PBS and dissolved in Tri-reagent (Sigma, USA). Then by using trichloromethane, isopropanol and 70% ethanol in different steps RNA was isolated and collected in DNases and RNases free water. After purification of RNA, 18s gene was used for checking the quality and as references to normalize the qPCR data. cDNA was synthesized from an equal amount of Ag/AgCl-NPs treated and untreated RNA by reverse transcriptase enzyme as described by the manufacturer (Thermo scientific). Samples were prepared by the addition of cDNA, forward and reverse primers (Supplementary Table-1), water and 2 × Syber master mix as described by the manufacturer (Applied Biosystems). Bio Rad thermal cycler was used for qPCR and the condition was set to 50 and 95°C for 2 minutes each and for 40 cycles, 95°C for 15 seconds and 60°C for 1 minute. Finally, real-time PCR data were analysed by double delta CT methods using Excel software where 18s was used as reference gene to normalize the data.

| Cell cycle analysis
For cell cycle analysis, GSCs were treated for 48 hours with K rotunda tuberous rhizome-mediated Ag/AgCl-NPs as described above and sample was prepared according to Kabir et al. 24 Finally, 10 μL of propidium iodide solution (1 mg/mL) was added to the cell suspension and cell cycle was analysed by flow cytometry (BD FACS Calibur, BD Biosciences).

| Determination of EAC cells growth inhibition in vivo in mice
EAC cells growth inhibition was examined according to Kabir et al. 24 In shortly, EAC cells were collected from EAC bearing mice and 1 × 10 6 cells in 0.1 mL of saline were injected intraperitoneally to 30 Swiss albino mice and kept at room temperature for tumour inoculation for 24 hours. After randomly distributed the mice into five groups (six mice per group), four groups were treated intraperitonially with K rotunda tuberous rhizome and Z mauritiana fruit extractmediated Ag/AgCl-NPs separately at the doses of 6.0 and 12.0 mg/ kg/day for five consecutive days. The remaining fifth group was used as control. On the seventh day of the EAC inoculation each of the mice was sacrificed to harvest a total number of EAC cells in normal saline. Then the cells were counted by a light microscope. Finally, the following formula was used to calculate the percent of growth inhibition.

| Synthesis and characterization of Ag/AgCl-NPs from K rotunda tuberous rhizome
Intensity of the deep brown colour formation was checked after incubating of K rotunda tuberous rhizome extract with AgNO 3 at different concentrations. The deepness of brown colour was raised with the increase of AgNO 3 concentration that indicated the formation of Ag/AgCl-NPs as shown in Figure 1A. The maximum absorbance peak was observed at 437 nm when the extract was incubated with 5 mM of AgNO 3 for 3 hours as shown in Figure 1A. About 1.5 g of silver nanoparticles was synthesized from 1.0 kg of K rotunda tuberous rhizome with the final concentration of 10 mg/mL.

| Morphological characterization Ag/AgCl-NPs
Highly monodispersed spherical particles were found with a diameter of 3 to 35.0 nm. Maximum nanoparticles were below 10 nm and the average size was obtained 17 nm ( Figure 1B).

| Structural characterization of Ag/AgCl-NPs
The XRD pattern of the synthesized Ag/AgCl-NPs was recorded and While reflection peaks 38.04° and 64.36° corresponding to planes (111) and (220), respectively for AgNPs (card no. 00-150-9146) as shown in Figure 1C. In both cases crystal systems were cubic.

| Thermal characterization of Ag/AgCl-NPs
The synthesized Ag/AgCl-NPs lost the weight in three steps that was represented in the TGA plot/profile ( Figure 1D). First, second and third weight lost were performed in the temperature range from 30 to 100°C, 100.1 to 350°C and 350.1 to 676°C corresponding to 3.9%, 24.74% and 29.98% weight loss, respectively.

| Surface properties of the Ag/AgCl-NPs
AFM was used to determine the dimensions of the synthesized nanoparticles. In the AFM image, the bright spots indicate the presence of Ag/AgCl-NPs. Whereas, size and dispersion of the nanoparticles represented by the dark spots. The sharp peaks in the 3D image confirm the lower to higher sizes of nanoparticles (Figure 2A).

| Functional characterization of Ag/AgCl-NPs
FTIR spectra of K rotunda tuberous rhizome crude extract and Ag/ AgCl-NPs were presented in Figure 2B. The major peaks obtained

| Study of Ag/AgCl-NPs induced apoptosis by Annexin V/PI and immunofluorescence assay
After treatment of GSCs with Ag/AgCl-NPs, cells were stained with FITC labelled annexin V and propidium iodide (PI). A significant number of early apoptotic and late apoptotic cells were observed for K rotunda tuberous rhizome and Z mauritiana fruit extract-mediated Ag/AgCl-NPs by fluorescence microscope (Figure 4A-C). In the immunofluorescence assay, expression of caspase-3 was increased due to the induction of apoptosis in the cells as presented in Figure 4D.

| Cell cycle analysis
The percentages of G 0 /G 1 , S and G 2 /M phases in control GSCs were calculated to be 60.9, 16.1 and 22.94, respectively. After treatment with K rotunda tuberous rhizome-mediated Ag/AgCl-NPs, percent of G 0 /G 1 and S decreased to 58.9, and 13.73 respectively, consequently with the increased of G 2 /M phase to 27.37%.
The above results revealed the G 2 /M phase cell cycle arrest in GSCs ( Figure 6A-C).

| Antitumour activity against EAC cells in vivo in mice
At the 6 mg/kg/day dose, 32.3% of growth inhibition was observed for K rotunda tuberous rhizome-mediated Ag/AgCl-NPs and no inhibition was found for Z mauritiana fruit extract-mediated Ag/AgCl-NPs. When the dose level was increased to 12 mg/kg/day, 55% and 20% of EAC cells growth were inhibited by K rotunda tuberous rhizome and Z mauritiana fruit extract-mediated Ag/AgCl-NPs, respectively ( Figure 7A).

| D ISCUSS I ON
In the present study, Ag/AgCl-NPs were synthesized by treating of AgNO 3 with K rotunda tuberous rhizome extract. Formation of K rotunda tuberous rhizome extract-mediated Ag/AgCl-NPs was confirmed by the change of colour from transparent to deep brown and absorbance peak between 400 and 450 nm of UVvisible spectroscopy. The highest absorbance peak at the wavelength of 437 nm was due to the surface plasmon resonance. 25 No such type of peak was observed for K rotunda tuberous rhizome extract. The intensity of the absorbance was increased with the rise of AgNO 3 concentration that could be due to the number of nanoparticle formation.
Size of the nanoparticles was observed to be spherical and highly tuberous rhizome extract were then used to stabilize the AgCl as AgCl-NPs. In the earlier investigation, we had also reported Z mauritiana fruit extract-mediated Ag/AgCl-NPs. 2 From the TGA data, it was found that the nanoparticles lost their weight in three stages.
The first weight loss happened due to the removal of water whereas second and third weight loss occurred for the decomposition or elimination of capping biomolecules. 27 That designate the synthesized nanoparticles possessed high thermal stability. AFM image was used to determine the size and shape of the nanoparticles. 28,29 In the present study, it was not possible due to the agglutination of the particles on the glass surface. The topography of the agglutinated Ag/AgCl-NPs was observed in the AFM image. Such type of agglutination is common and was observed previously. 2,30 The presence of various functional groups in K rotunda rhizome Targeting these signalling pathways and their receptors in GSCs holds promise for glioblastoma therapy.
In mammals, four homologous proteins function as cytoplasmic receptors are known as Notch1, Notch2, Notch3 and Notch4. 37 Notch signalling pathway is important for cell proliferation, apoptosis, stem cell maintenance and tissue homeostasis. [38][39][40] In Glioblastoma tissue, astrocyte fate genes, stemness genes and anti-apoptotic proteins are correlated with the expression level of Notch2 gene. 41,42 It was reported that lower levels of miR-34c-3p and miR-34c-5p correlate with a higher glioma grade. The over expression of miR-34c-3p (miRNAs) strongly inhibits glioma invasion and promotes the S-phase arrest, increases cell apoptosis and reduces Notch2 expression. 43 Ruan et al reported that another miRNA (miR-181c) reduces cell invasion, cell proliferation and self-renewal capacities of glioblastoma cell through the down-regulation of Notch2 gene. 44 In the present study, Notch2 gene was down-regulated after treatment with the synthesized nanoparticles that may be a possible reason of apoptosis in GSCs.

The signal transducer and activator of transcription 3 (STAT3)
is involved in the central nervous system development, immune response, stem cell maintenance and tumorigenesis. Activation of STAT3 plays important role in glioma biology. 45  EGFR expression was also down-regulated in the present study which might be also another reason for GSCs growth inhibition.
Members of the NF-κB family regulated cell invasion, cell proliferation, cell cycle progression and apoptosis as important transcription factors in tumours. [53][54][55] NF-κB (p65) pathway typically activated by phosphorylation of IκBα, which is the inhibitory subunit of NF-κB. 56 IκBα is phosphorylated by IKKα/β, which leads to IκBα degradation as a result p65 and p50 are released and trafficked into the nucleus where they evoke gene transcription. 56,57 TNFα is known to increase phosphorylation of IKK causing a decrease of IKBα levels and increase of nuclear p65. 58  very few were performed in vivo in mice. 9 To check the in vivo antitumour activity of K rotunda tuberous rhizome and Z mauritiana fruit extract-mediated Ag/AgCl-NPs, EAC cells were selected for its rapid proliferate in mice due to lack of H2 histocompatibility antigen. 63 In vivo experiments stated that Z mauritiana fruit extract-mediated Ag/ AgCl-NPs showed low antitumour activity against EAC cells. While, K rotunda tuberous rhizome-mediated Ag/AgCl-NPs retained strong antitumour activity. The in vivo results revealed that antitumour properties of biogenic nanoparticles vary from sample to sample for different factors those including size distribution, morphology, surface charge, surface chemistry and capping agents. 13,16 In conclusion, Ag/AgCl-NPs were synthesized from the K rotunda

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.