Biomimetic synthesis of silver nanoparticles using Matricaria chamomilla extract and their potential anticancer activity against human lung cancer cells

Accepted Manuscript Biomimetic synthesis of silver nanoparticles using Matricaria chamomilla extract and their potential anticancer activity against human lung cancer cells

Mehdi Dadashpour, Akram Firouzi-Amandi, Mohammad Pourhassan-Moghaddam, Mohammad Jafar Maleki, Narges Soozangar, Farhad Jeddi, Mohammad Nouri, Nosratollah Zarghami, Younes Pilehvar-Soltanahmadi PII: DOI: Reference:

S0928-4931(17)34187-5 doi:10.1016/j.msec.2018.07.053 MSC 8765

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

21 October 2017 29 June 2018 20 July 2018

Please cite this article as: Mehdi Dadashpour, Akram Firouzi-Amandi, Mohammad Pourhassan-Moghaddam, Mohammad Jafar Maleki, Narges Soozangar, Farhad Jeddi, Mohammad Nouri, Nosratollah Zarghami, Younes Pilehvar-Soltanahmadi , Biomimetic synthesis of silver nanoparticles using Matricaria chamomilla extract and their potential anticancer activity against human lung cancer cells. Msc (2018), doi:10.1016/ j.msec.2018.07.053

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Biomimetic synthesis of silver nanoparticles using Matricaria chamomilla extract and their potential anticancer activity against human lung cancer cells

Mehdi Dadashpour1,2,3#, Akram Firouzi-Amandi4#, Mohammad Pourhassan-Moghaddam5, Mohammad Jafar Maleki5, Narges Soozangar6, Farhad Jeddi6, Mohammad Nouri3, Nosratollah

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Zarghami5,7, Younes Pilehvar-Soltanahmadi2,3,5,7*

Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran

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Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

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Stem Cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences,

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Tabriz, Iran

Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences,

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Tabriz, Iran

Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz,

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Iran

Research Laboratory for Embryology and Stem cells, Department of Anatomy and Pathology,

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School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

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Abadan School of Medical Sciences, Abadan, Iran

# Co-first Authors (these authors contributed equally to this work) * Corresponding author: Younes Pilehvar-Soltanahmadi, PhD, Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. +989104423137, E-mail: [email protected]

Tel:

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Abstract Herbs having various natural substances can be utilized for the biosynthesis of Silver nanoparticles (AgNPs) and act as a stable, reliable and biocompatible alternative instead of the current physical and chemical approaches. It has been reported that Matricaria chamomilla

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possesses unique properties, especially anti-cancerous effects. The objective of the current

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work was to assess the chemical characteristics and anticancer effects of biosynthesized AgNPs

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applying aqueous extracts of M. chamomilla against A549 lung cancer cells. UV–visible spectrum showed the maximum absorption of the biosynthesized AgNPs at 430 nm. The

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crystalline structure of biosynthesized AgNPs in optimal conditions was confirmed by XRD. Moreover, the presence of Ag as the ingredient element was exhibited via EDX analysis. FT-

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IR results also verified the AgNPs synthesis using a plant extract. The spherical shapes of the AgNPs with an average diameter size around 45.12 nm and a zeta potential value of -34 mV

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were characterized using DLS, and confirmed through FE-SEM and TEM. In vitro cytotoxicity

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assay using MTT revealed that the biosynthesized AgNPs exhibited a dose- and timedependent cytotoxic effect against A549 lung cancer cells. Moreover, the apoptotic effects of

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the AgNPs were demonstrated using DAPI staining, real-time PCR and flow cytometry. According to these findings, using M. chamomilla in combination with AgNPs via green-

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synthesis approach may be an efficient strategy for effective treatment of lung cancer. Keywords: Matricaria chamomilla, AgNPs, Green synthesis, Lung cancer

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1. Introduction Nanotechnology, as an interdisciplinary approach, is the most interesting area for generating novel nanoproducts in biotechnology and biocompatible medicine (1, 2). One of the most important and well-known nanoproducts applied for medical purposes is silver nanoparticles (AgNPs) (3). AgNPs have attracted great attention as an antibiotic agent in various

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commercially available products, such as textiles, wound caring and implantable medical

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devices (4). It has been described that AgNPs have antifungal, antimicrobial, antioxidant, and

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anti-inflammatory activities (4, 5).

Bionanotechnology is the most hopeful area for producing innovative types of nanomaterials

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for application in biomedicine (6-8). Numerous noble metal NPs including gold, silver and copper were broadly and commonly synthesized using physical and chemical methods (9). The

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chemical and physical synthesis of metal NPs have numerous disadvantages such as extremely costly, the use of hazardous and toxic chemicals, and potential biological and environmental

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stakes (10).

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In pursuit of a novel cost-effective and eco-friendly approach, plants having various natural substances can be utilized as a crucial source for the biosynthesis of NPs and act as a stable,

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reliable and biocompatible alternative for the current physical and chemical approaches (11). Moreover, as the control of particle size and morphology are the essential features for

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application in biomedicine, the biological approach has the capability to better control of particle size in relative to the chemical and physical synthesis methods of metal NPs (4). In recent years, broad range of plants including Carica Papaya (12), Heterotheca inuloides (13), Pongamia pinnata (14), Artemisia marschalliana Sprengel (15) and Borago officinalis (16) have been used for synthesis of AgNPs. In addition, some recent studies revealed that AgNPs synthesized by the biological method using Annona squamosal, Albizia adianthifolia, Phyllanthus emblica and Anthemis atropatana, had antiproliferative effects against numerous

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cell lines (17, 18). Extracts of medicinal herb, M. chamomilla, has been widely used in traditional medicine since ancient times. It has been reported that M. chamomilla possesses antimicrobial, antifungal, antioxidant and anti-inflammatory effects (19). Nowadays, cancer is one of the main causes of mortality in the world (20, 21). Lung cancer is the first leading cause of cancer death among both men and women (22, 23). The vast majority

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(85%) of lung cancer cases are due to cigarette smoking and exposure to tobacco smoke (24,

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25). The current therapeutic agents used for lung cancer treatment are very costly and

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inefficient due to severe side effects and toxicity on non-cancerous tissues. Hence, it is required to explore a new cost-effective and biocompatible therapeutic approach against lung

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cancer.

So, the current study was designed to prepare and characterize the biosynthesized AgNPs using

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a bioreduction procedure with aqueous extract of M. chamomilla, as a bioreducing agent and

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2. Materials and methods

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evaluate its anti-cancer and apoptotic effects on A549 human lung cancer cell line.

2.1 Materials

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A549 (human lung adenocarcinoma cell line) was supplied by the National Cell Bank of Pasture Institute, Tehran, Iran. AgNO3, 4′, 6-diamidino-2-phenylindole (DAPI ), 3-[4, 5-

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dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and Dimethyl Sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640, Fetal Bovine Serum (FBS), Trypsin and Penicillin-Streptomycin solution were purchased from Gibco (Invitrogen, NY, USA). Annexin V/PI double staining kit was purchased from eBioscience (San Diego, CA, USA). All other reagents were of the highest grade commercially available.

2.2 Preparation of leaves extract

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The M. chamomilla were collected from Ahar, Horand and Tabriz regions, northwest of Iran. The fresh, healthy, and disease–free M. chamomilla leaves were selected and washed repeatedly with distilled water and dried for five days in shadow. The air-dried leaves were grounded and powdered finely. For preparation of aqueous leaf extract, 15 g of leaf powder was boiled in 100 mL sterile distilled water for 20 min and filtered using whatman Grade 2

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filter paper. Then, the resulting solution dried at 50 °C during three days and applied for this

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study.

2.3 Biological synthesis of AgNPs

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The extracellular synthesis of AgNPs was done according to Song et al (26) technique with slight modifications. Briefly, 10 mL of aqueous leaf extract was mixed with 150 mL of 1 mM

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aqueous solution of silver nitrate (AgNO3) for the reduction of Ag+ ions. The reduction process of Ag+ ions to Ag0 takes place completely within the period of 5 min and was observed visually

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by varying the initial colour of the reaction mixture from colourless to dark brown. In order to

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acquire the uniform and monodispersed sized AgNPs, the kinetics of the biosynthesized AgNPs were monitored and several parameter reaction conditions were optimized including

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concentrations of plant extract (15, 25, 35 and 45 g/L), pH (5, 7 ,9 and 11) and temperatures (22, 37, 45 and 60 ◦C). The acquired biosynthesized AgNPs were purified by repeating the

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centrifugation twice at 9000 rpm for 20 min, followed by redispersion in deionized water. It was then lyophilized and stored in screw-capped vials under ambient conditions for further characterization and application.

2.4 Characterization of AgNPs The color change of the biosynthesized AgNPs solution from colorless to yellowish brown solution was observed and characterized using a double beam spectrophotometer (Shimazdu,

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model UV-1800, Kyoto, Japan) in 200–800 nm wavelength. The particle size and zeta potential analysis of biosynthesized AgNPs were assessed using Transmission Electron Microscopy (TEM), Field Emission Scanning Electron microscopy (FE-SEM) (MIRA3 TESCAN, Czech) at 25 KV and Dynamic Light Scattering (DLS) (Malvern Instruments Ltd., Malvern, UK) equipped with a helium–neon laser beam at a wavelength of 633 nm and a fixed scattering

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angle of 90. Fourier-transform infrared spectra of M. chamomilla leaf extract powder and the

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dried AgNPs were acquired in the range 4,000 to 500 cm−1 with a Shimadzu 8400s Infrared

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Spectrophotometer (Kyoto, Japan) by potassium bromide (KBr) technique. Also, the crystalline nature of AgNPs was investigated using X-ray diffraction (XRD) (Rigaku D/MAX-

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2400 X-ray diffractometer with Ni-filtered Cu Kα radiation). Moreover, Energy-dispersive X-

equipped with an EDX attachment.

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ray (EDX) analysis of AgNPs was carried out on a Phenom prox (Netherland) using FE-SEM

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2.5 In vitro cytotoxicity of biosynthesized AgNPs

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The cytotoxic effects of different concentrations of biosynthesized AgNPs on A549 cells were measured by MTT assay. First, the cells were cultured and approximately 104 cells/well were

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seeded into the wells and incubated for 24 h prior to the experiments. Next, the cells were treated with different concentration of the biosynthesized AgNPs (0–100 µg/mL) and all

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cultures were kept in humidified incubator for 24 and 48 h at 37 °C. After 24 and 48 h of incubation, 50 µL of MTT solution was added to each well and incubated for 4 h. Finally, the purple color formazone crystals formed were dissolved in 100 µL of DMSO with gentle shaking at 37 °C, and absorbance was read at 570 nm using an EL× 800 microplate absorbance reader (BioTek Instruments, Winooski, VT) with a reference wavelength of 630 nm. All experiments were repeated 3 times.

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2.6 Morphological changes analysis A549 cells were cultured (105 cells/cover slip) and incubated with AgNPs at their IC50 values for 24 and 48 h. Then, the cells were fixed in 4% paraformaldehyde for 20 min. The cover slips were gently mounted on glass slides for the morphometric study. Morphological changes of A549 cells were studied by bright field inverted light microscopy (Olympus, Japan) at a

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magnification of 40X.

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2.7 DAPI for nuclear staining

After treating A549 cells with IC50 values of the AgNPs for 24 and 48 h, the cells were washed

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twice with PBS and fixed in 2.5% glutaraldehyde for 20 min. Subsequently, the cells were permeabilized in 0.1 % Triton X-100 for 15 min and stained with DAPI for 20 min in the dark.

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After being washed 3 times with PBS, the nuclear morphology of cells was observed by

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2.8 Cells cycle analysis

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fluorescence microscopy (Olympus, Japan).

Cell cycle progression was measured by staining the DNA with PI followed by FACS analysis.

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First, the 24-h seeded cells were treated with the concentrations of 63 and 42.5 μg/mL of the AgNPs. After 24 and 48 h, the cells were washed twice with PBS, trypsinized, harvested, and

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fixed with 70% ethanol at 4 °C for 72 h. After fixation, 10 µL ribonuclease A was added to tubes and incubated for 45 min in 37 °C and then, 10 µL propidium iodide (PI) was added to tubes. Finally, the cells were transferred to a sterile flow cytometer glass tube and analyzed the cell cycle status using flow cytometer (FACS Caliber flow cytometer; BD Biosciences, San Jose, CA, USA). The percentage of the cells in G1, S and G2 phases was calculated by Flowjo software for cell cycle distribution. Data were acquired in three separate sets of experiments.

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2.9 Determination of apoptotic cells by flow cytometry The apoptosis/necrosis in A549 cells treated with AgNPs was determined using Annexin V fluorescein isothiocyanate (FITC)/PI (eBioscience Annexin V Apoptosis Detection Kit). The externalization of phosphatidylserine, as a marker of early apoptosis, was assessed using an Annexin V FITC, while extensive membrane leakage due to late-stage apoptosis/necrosis was

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discovered by the binding of PI to nuclear DNA. In brief, A549 cells were seeded in 6-well

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plates at a concentration of 105 cells/well and treated with IC50 values of the AgNPs in 24 and

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48 h. The cells were then suspended in 500 µL PBS and incubated with FITC-Annexin V and PI in binding buffer for 15 min in the dark at room temperature prior to analysis. Data were

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analyzed by flow cytometer (FACS Caliber flow cytometer; BD Biosciences, San Jose, CA) and Flowjo software (Treestar, Inc., San Carlos, CA). Annexin V+, PI– cells were considered

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to be in the early stage of apoptosis, while Annexin V+, PI+ cells were considered to be in the

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2.10 Real-time PCR

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late stage of apoptosis.

The expression level of apoptotic signaling genes including Bax, Bcl-2, caspase-3 and caspase-

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7 were measured by real-time PCR method. At first, the total RNA of the treated A549 cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) based on manufacturers

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procedures. The purity and quantity of total RNA extracted was assayed by optical density measurement (A260/A280 ratio) with Nano Drop 1000 Spectrophotometer (Wilmington, DE, USA) and qualified by 1.5% agarose gel electrophoresis. cDNA was synthesized using Revert Aid First strand cDNA synthesis Kit (Fermentas, St Leon-Rot, Germany) based on manufacturer’s instructions. Gene expression studies were performed using the specific primers and SYBR Green-based PCR Master Mix. Furthermore, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. In this experiment, PCR was

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carried out for 40 cycles as follows: 95 ºC for 10 min, denaturation at 95 ºC for 10 min, annealing 60 ºC for 30s and extension step at 72 ºC for 30s. Finally, the real-time PCR data were analyzed using ∆∆CT method.

2.11 Statistical analysis

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The data are expressed as mean ± standard deviation (SD) values of triplicate measurements.

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P values were determined using Student’s t test. P<0.05 was used for the significance of

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differences between means.

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3. Results and Discussion 3.1 Green synthesis of AgNPs

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In the present work, we attempt to apply the aqueous leaf extract of M. chamomilla as a bioreductant for synthesis of AgNPs. The microtubes with samples of AgNO3, M. chamomilla leaf

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extract and AgNO3 with M. chamomilla leaf extract have been shown in Figure 1. According

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to these findings, the pallid yellow color of the aqueous leaf extract changed to a yellowishbrown color after reaction with Ag+. The emergence of a brownish color in the solution having

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M. chamomilla extract gave the clear indication of the formation of AgNPs in the reaction mixture and was due to the excitation of surface plasmon vibrations in the NPs (27). The color

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change shows that M. chamomilla extract can be used as a stabilizing and reducing agent in AgNPs synthesis, and further it acts as a part of the sign for a synthesis of AgNPs. Altogether, in this study, AgNPs were successfully synthesized by the aqueous extract of M. chamomilla, as a reducing agent, which was according to other reports of biological synthesis by different herbal extracts (28, 29).

3.2 Characterization of AgNPs

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3.2.1 UV-visible spectroscopy analysis of AgNPs The synthesized AgNPs were analyzed using UV-visible spectroscopy. UV-visible spectroscopy is a simple, cost-effective and precious technique to confirm the formation and stability of AgNPs (30). For the AgNPs prepared using an aqueous leaf extract of M. chamomilla, a strong surface plasma resonance (SPR) peak located at 430 nm was detected

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(Figure 2). The absorbance at 430 nm did not increase with the enhancing reaction time.

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In green synthesis of AgNPs using herbal extracts, several constituents may participate in the

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reduction process of silver ions. Hence, changing the chemical state (e.g., ionization) of these constituents can be affected on performance and rate of reduction procedure. For this reason,

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the effect of extract pH on the synthesis of AgNPs in various pHs (5, 7, 9 and 11) was studied using UV–visible spectrophotometer (Figure 3A). The results displayed that the rate of AgNPs

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synthesis enhanced with increasing pH up to 9. So, pH = 9 was selected as the optimum pH for further analysis.

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Synthesis of AgNPs was done at different temperatures in the range of 22–60 °C. The results

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presented in Figure 3B displayed that the efficiency of AgNPs synthesis was highest at 45 °C. Therefore, 45 °C was selected as the optimum temperature.

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To complete the reduction of silver ions to AgNPs, different concentrations of the extract were mixed with a constant volume of silver nitrate (1 mM). The findings showed that increasing

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the concentration of the extract causes synthesizing more AgNPs and eventually level off at concentration of 35 g/L (Figure 3C).

3.2.2 FTIR analysis of AgNPs FTIR spectroscopy analysis was carried out to anticipate the function of reducing and stabilizing capability of aqueous extract of air dried leaves of M. chamomilla. A FTIR spectra of control dried M. chamomilla leaf extract and biosynthesized AgNPs have been displayed in

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Figure 4. The FTIR signals of crude M. chamomilla leaf extract were observed at 3775 cm−1, 3421 cm−1, 2925 cm−1, 1616 cm−1,1072 cm−1 and 676 cm−1. OH stretching of alcohols and phenols appear at 3775 cm−1 and 3415 cm−1. The presence of OH (phenolic compound) functional group on the surface of AgNPs can be effective in Ag ion reduction. The band 2925 cm−1 was due to the presence of aldehydic C-H stretching in AgNPs. The absorption band at

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1626 cm−1 in M. chamomilla leaf extract corresponds to amide I vibrations and this band was

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shifted to 1616 cm−1 in AgNPs due to carbonyl group of amino acid residues in proteins that probably will bind to AgNPs through the amine groups. The sharp and strong band at 1072

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cm−1 is responsible for C-O-C stretching which could be ascribed to the reduction of Ag+ with

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extract. The extract of M. chamomilla was previously reported to contain phytochemicals such as sesquiterpenes, flavonoids, coumarins, and polyacetylenes. In addition, previous studies

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have shown that phenolic compounds can reduce Ag ion to AgNPs (31). Moreover, the results

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3.2.3 XRD analysis

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display that the phytochemical components give a high level of stability to the AgNPs.

XRD pattern confirmed the crystalline structure of the biosynthesized AgNPs. The XRD

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pattern of synthesized AgNPs using aqueous extract of M. chamomilla was shown in Figure 5. XRD results indicated three main characteristic peaks at 2θ range of 30-65° and these

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diffraction peaks were corresponding to the (1 1 1), (2 0 0), and (2 2 0). XRD spectra of the NPs derived from M. chamomilla extract propose that these NPs were crystalline in nature. The XRD patterns achieved were consistent to other studies that authenticate the cubic crystalline nature of synthesized AgNPs (32, 33).

3.2.4 EDX analysis

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The EDX analysis provides qualitative and quantitative status of the elements that may contribute to the formation of NPs. The elemental composition of synthesized NPs using the extract of M. chamomilla was determined by EDX spectroscopy. Figure 6 displays the energy dispersive spectrum of the synthesized AgNPs, which suggests the presence of Ag as the ingredient element. Metallic AgNPs generally show typical optical absorption peak

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approximately at 2.999 keV due to SPR (34). Previous studies revealed that biosynthesized

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3.2.5 Size and morphological characterization

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solution up to several months after biosynthesis (35, 36).

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NPs are surrounded by a thin layer of some organic capping material that can be stable in

Particle size and zeta potential are significant factors since they directly impact the stability,

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biodistribution and cellular uptake of NPs (37, 38). In this study, the average hydrodynamic size and size distribution of biosynthesized AgNPs were characterized by a zeta potential

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analyzer with DLS technique. DLS measurements revealed that the gained AgNPs had average

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diameter size around 45.12 nm with polydispersity index (PDI) of 0.339 and the zeta potential value of -34 mV (Figure 7).

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In fact, particle dispersion is stable if the zeta potential is more positive than +30 or more negative than -30 mV. However, the zeta potential of the particles powerfully changes with the

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electrolyte concentration and pH of the dispersion. The high negative potential value provides good colloidal nature, long term stability, and high dispersity of AgNPs owing to negative repulsion (39). To confirm the results acquired from DLS study, further characterization of the AgNPs was performed using FE-SEM and TEM (Figure 8). The FE-SEM and TEM images indicated mono-dispersed particles that were significantly uniform and spherical. A substantial proportion of the spherical and uniform AgNPs within average diameter size ranging 25-36 nm

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was identified in FE-SEM images. Also, TEM micrographs suggested the average size of the particles were around 34 nm. TEM studies confirm the role of the extract prepared from the M. chamomilla as the reducing, capping and stabilizing agent. The particle size acquired from DLS analysis is rather greater than the results attained from FE-SEM and TEM which likely due to the formation of surface hydration layer and pseudo-

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dehydrated for FE-SEM and TEM characterizations (40, 41).

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clusters on samples under particle size measurement using DLS, while they must be severely

3.3 In vitro cytotoxicity of AgNPs

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The cytotoxicity assays are one of the main factors to clarify the cellular responses to toxic agents and may reveal some information about metabolic activities, specially cell survival

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and death (42, 43). To study the influence of the AgNPs on mitochondrial function, cytotoxicity was determined after A549 cells treating with different concentration of the AgNPs (0-100

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µg/mL) (Figure 9). In consistent with others, our findings disclosed that the AgNPs show

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considerable cytotoxicity which meaningfully decrease the cell viability of the treated cells as a function of time and concentration (44, 45). The data showed that the concentrations between

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60 and 100 µg/mL of AgNPs (higher concentrations) had considerably more effect on the cell viability in relative to 0-50 µg/mL. The half maximal inhibitory concentration (IC50) values for

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the AgNPs against A549 cells were observed at 62.82 µg/mL and 42.44 µg/mL for 24 and 48 h, respectively.

Results from numerous works described that both the AgNPs and Ag+ released by AgNPs are contributed in the cytotoxicity mechanism in various pathways: indeed, AgNPs maybe present a perfect surface outside the mitochondria for the univalent reduction of oxygen to superoxide from electron via the respiratory chain. Additionally, Ag ions binds to DNA and proteins, restricting their roles. Furthermore, ROS generation and oxidative stress take place as an early

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consequence resulting in NP-induced cytotoxicity. It should be noticed that these mechanisms cytotoxicity can be assisted or hampered by changing a diversity of parameters, ranging from nanoparticle the shape, size and surface properties of AgNPs to environmental stability and bioavailability to the duration of cell exposure, cell type and target cell (46, 47). Surface charge plays a pivotal role in the cytotoxicity of AgNPs. Frey (2017) found that

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positively-charged AgNPs had superior cytotoxicity against human cancer cells than

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negatively-charged AgNPs (47). In fact, AgNPs with positively-charged capping agents

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interact with negatively charged phospholipid membrane of mammalian cells and kill the cells more so than those with negatively-charged capping agents. Authors suggested that

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investigators exploring AgNPs for targeted cancer therapies should apply positively-charged AgNPs over negatively-charged AgNPs on the premise of improved bioavailability.

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Although the biosynthesized AgNPs in this study had negative surface potential charge but we found a cytotoxic effect against the cancer cells, suggest that other parameters such as size and

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shape of the biosynthesized AgNPs may be involved in the detected deleterious effects.

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Our results are in accordance with previous studies. Lee et al. showed that the AgNPs inhibited the viability of A549 lung cancer cells in a dose-dependent manner (51). Elshawy and

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coworkers revealed that the biologically synthesized AgNPs induced apoptosis dosedependently in MCF-7 and MCT cancer cell lines through activation of caspase-3 (49). In

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addition, it has been shown that biosynthesized AgNPs had cytotoxic activity against numerous human cancer cell lines, such as HepG-2 and MCF-7 cell lines (52, 53).

3.4 Induction of apoptosis by biosynthesized AgNPs 3.4.1 Morphological analysis After treatment with AgNPs, the morphological changes of A549 cells were observed using bright field microscopy. As shown in Figure 10, after exposure to AgNPs for 24 and 48 h, many

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of the cells showed cytoplasmic condensation as separated from each other and or suspended in the medium. In comparison to 24 h, it was found a considerable expansion in the number of rounded cells with progressive nuclear shrinkage after exposure to the AgNPs for 48 h. The cytoplasmic vacuolation and shrinkage, and cellular fragmentation were the most identifiable morphological alterations in the biosynthesized AgNPs treated cells detected in this work

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(Figure 10A- C).

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3.4.2 DAPI for nuclear staining

DAPI is a fluorescent stain that binds to minor groove of double-stranded DNA and is used to

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highlight the nuclear changes during apoptosis and also to assess the percentage of apoptotic cells with condensed and fragmented chromatin. To observe nuclear morphology changes

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related to apoptosis, the cells treated with the biosynthesized AgNPs were stained by DAPI after 24 and 48 h incubation. As shown in Figure 10D-F , the untreated cells displayed normal

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nuclei (smooth nuclear), normal organelle and intact cell membrane while in the treated cells

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with the AgNPs, typical characteristics of apoptosis, such as condensation of chromatin in the nucleus, shrinkage of nuclei and appearance of nuclear apoptotic bodies were observed.

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cancer cells (54).

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Interestingly, it has been shown that AgNPs can also cause DNA damage and apoptosis in

3.4.3 Cell cycle analysis In order to explore the mechanism involved in AgNPs mediated inhibition of cell growth, cell cycle distribution was quantified using flowcytometric analysis. The cell cycle profile was evaluated in A549 cells after exposure to 63 and 42.5 µM of the AgNPs for 24 and 48 h. The results displayed that treating cells with the AgNPs caused a considerable inhibition of cell cycle progression in A549 cells at mentioned intervals (Figure 11), resulting in a considerable

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accumulation of the cells in S phase, which is consistent with the reported findings by Chairuangkitti and et al (55). Moreover, no considerable apoptosis was detected as showed by the absence of cell population in sub G1 (Figure 11). These results revealed that AgNPs could hamper the cell proliferation through induction of an S phase cycle arrest, proposing that obstruction of cell cycle progression may be one of the mechanisms underlying the antitumor

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effect of the AgNPs. DNA damage was suggested to be the principal reason of cell cycle arrest

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(56). It has been reported that oxidative stress in AgNPs treated cells implies to the probability

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of DNA damage and chromosomal abnormalities which was considered the main factors resulting in cell cycle arrest (57). The cells with reversibly damaged DNA will accumulate in

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G1, DNA synthesis, or in G2/M phase, but cells that carry irreversibly damaged DNA will undergo apoptosis (58). AshaRani et al. showed that AgNPs induced cell cycle arrest at

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S/G2/M phase of cell cycle (59). Also, Xue and coworkers revealed that AgNPs caused cell

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cycle arrest at G2/M phase (60).

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3.4.4 Determination of apoptotic cells by flow cytometry To study the effects of the AgNPs on the induction of apoptosis in A549 cells, flowcytometric

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analysis by annexin V assay was applied to measure the proportions of apoptosis in the total cell population. Results of the Annexin V assay for determining the percentages of early and

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late apoptotic cells have been provided in Figures 12. After 24- and 48-h treatment, the proportion of early and late apoptotic cells increased significantly in relative to the control cells (P<0.05). There were no changes registered in the percentage of necrotic cell death. This finding revealed apoptotic activities of the AgNPs on A549 cells. The process of programmed cell death, or apoptosis is a well-documented phenomenon in multicellular organisms which has been recognized as a major anticancer therapeutic response (61). The loss of cell membrane phospholipid asymmetry that leads in the externalization of phosphatidylinositol serine bonds

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to the outer membrane is an early marker of early-stage apoptosis. Fluorochrome-labeled Annexin-V dye binds very specifically to externalized phosphatidylinositol serine ligands on the surface of cells resulting in the detection of early apoptotic cells (62). Consequently, it can be concluded that the biosynthesized AgNPs conducted the cells towards apoptosis which is

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crucial in cancer therapy.

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3.4.5 Expression of apoptotic genes

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To further explore the mechanisms involved in AgNPs -mediated inhibition in A549 lung cancer cells, real-time PCR was used to analyze the expression of apoptotic genes including

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Bcl-2, Bax, Caspase-3 and Caspase-7 in the cells treated with 10 and 40 μg/ml of AgNPs after 48 h incubation. As shown in Figure 13, the mRNA expression of caspase-3, caspase-7 and

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Bax were significantly up-regulated and the expression of anti-apoptotic gene, Bcl-2, was significantly down-regulated in both concentrations in relative to the untreated cells (P< 0.001).

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As our outcomes displayed, the expression of Bax and Bcl-2 genes could be regulated inversely

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by the AgNPs, proposing a balance in the expression levels of the genes which may be responsible for regulation of the apoptosis process. The significant regulatory mechanisms of

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apoptosis comprise receptors of death, caspase activation, mitochondrial responses, and the regulation of Bcl-2 and Bax expression levels (31). Bcl-2 is the founding member of the Bcl-2

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family of regulator proteins that regulate cell death. Members of the Bcl-2 family (i.e. Bcl-2 and bax ) can have both pro-apoptotic and anti-apoptotic activities (63). BCL2 family members form hetero- or homodimers and the relative levels of the available dimerization partners shift the balance of cell fate in favor of either cell death or viability (31). Caspase-3 and Caspase-7, members of the cysteine protease family are well-known to be the major regulators of the apoptotic machinery and play a central role in the apoptotic pathway (64). The effect of AgNPs in decreasing and increasing expression levels of Bcl2 and Bax has been detected in human

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colon and breast cancer cell lines (17, 31). In this study, we demonstrated that the biosynthesized AgNPs exert cytotoxic effects on A549 lung cancer cells via down-regulation of an anti-apoptotic gene (Bcl-2) and up-regulation the pro-apoptotic members (Bax, caspase3 and caspase-7). However, further investigations are needed to provide insight into the

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mechanisms involved in the elicited anti-cancer effects of biosynthesized AgNPs.

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5. Conclusion

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In this study, M. chamomilla leaf extract was used as a reducing and capping agent to synthesize AgNPs without production of any harmful chemicals. This strategy possesses some benefits

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which support the combinatorial application of two anti-cancer agents (M. chamomilla leaf extract and AgNPs) in a short time and with a low cost. Our findings revealed that the

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biosynthesized AgNPs with uniformity, monodispersity and good stability exhibited remarkable anti-cancer effects against A549 lung cancer cells. According to the results, it can

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be suggested that the AgNPs synthesized by the facile and eco-friendly method might be used

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Acknowledgments

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as an anti-cancer agent for lung cancer therapy.

The authors thank the Student Research Committee, Tabriz University of Medical Sciences,

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Tabriz, Iran, for all support provided.

Conflict of Interest The authors declare that they have no competing interests regarding the publication of this paper.

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Figure Legends Figure 1. Synthesis of AgNPs using M. chamomilla leaf extract. (A) AgNO3, (B) M. chamomilla leaf extract and (C) AgNO3 with M. chamomilla leaf extract. Figure 2. Ultraviolet–visible spectra of M. chamomilla extract and AgNPs synthesized with M. chamomilla leaf extract. The absorption spectra of the biosynthesized AgNPs showed a

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strong broad peak at 430 nm which was ascribed to surface plasmon resonance (SPR) of the

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NPs.

concentration of plant extract and (C) temperature.

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Figure 3. UV-vis spectra of the biosynthesized AgNPs recorded as a function of (A) pH, (B)

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Figure 4. FT-IR spectra of (A) M. chamomilla leaf extract and (B) biosynthesized AgNPs from M. chamomilla leaf extract.

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Figure 5. XRD pattern of the AgNPs synthesized using M. chamomilla leaf extract exhibiting the facets of crystalline.

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Figure 6. EDX of biosynthesized AgNPs from M. chamomilla leaf extract. EDX spectrum

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displayed higher percentage of silver signals. Figure 7. DLS analysis to determine size and zeta potential of Biosynthesized AgNPs by M.

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chamomilla leaf extract. Synthesis conditions: AgNO3 concentration (1 mM), temperature (45 ∘C), extract pH (pH= 9) and extract concentration (35 g/L)

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Figure 8. FE-SEM (A) and TEM (B) images of biosynthesized AgNPs. Synthesis conditions: AgNO3 concentration (1 mM), temperature ( 45 ∘C), extract pH, (pH= 9), extract concentration (35 g/L) Figure 9. Cytotoxic effects of AgNPs on A549 lung cancer cells studied using MTT assay. Values are presented as mean ± S.D. of three parallel measurements.

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Figure 10. Morphological changes in control cells (A and D) and the cells exposed to the AgNPs at 24 h (B and E) and 48 h (C and F) detected using bright filed microscopy (A-C) and fluorescencent microscopy (D-F). Figure 11. Cell cycle analysis of A549 cells exposed with IC50 concentration of the biosynthesized AgNPs for 24 and 48 h. The results of this analyze revealed a significant

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accumulation of cells in the S phase. All experiments were carried out in triplicate.

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Figure 12. Biosynthesized AgNPs-induced apoptosis in A549 cells were treated with IC50

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concentrations of AgNPs for 24 and 48 h and analyzed using flow cytometry after staining with Annexin V and PI. The amount of apoptosis was assessed as the percentage of Annexin V+/PI-

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and Annexin V+/PI+ cells.

Figure 13. The expression levels of Bcl-2, Bax, Caspase-3 and Caspase-7 genes in relative to

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reference gene (GAPDH) in A549 cancer cell line treated with the biosynthesized AgNPs. *P

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< 0.05, **P< 0.01 and ***P< 0.001 vs. control was considered significant.

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Highlights 

Matricaria chamomilla leaf extract was used as a reducing and capping agent to synthesize AgNPs without production of any harmful chemicals.

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the biosynthesized AgNPs with uniformity, monodispersity and good stability exhibited

Using M. chamomilla in combination with AgNPs via green synthesis approach may

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be an efficient strategy for effective treatment of lung cancer.

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remarkable anti-cancer effects against A549 lung cancer cells.

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