Fe₃ O₄ /Ag nanocomposite biosynthesised using Spirulina platensis extract and its enhanced anticancer efficiency

Abstract

In this work, the authors investigated the apoptotic activities of Fe₃ O₄ /Ag nanocomposite biosynthesised by Spirulina platensis extract against MCF‐7 (human breast cancer cells). The physico‐chemical properties of prepared Fe₃ O₄ /Ag nanocomposite were studied by different spectroscopic methods. To evaluate the in vitro cytotoxic effect, MCF‐7 cells were treated with different concentrations of Fe₃ O₄ /Ag nanocomposite and examined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay. Moreover, apoptotic effects were also studied by Hoechst 33258 staining, caspase 3 activation assays, and annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide staining. Microscopic observations of Fe₃ O₄ /Ag nanocomposites indicated approximately spherical shape and small particles in the size range of about 30–50 nm. The MTT assay result revealed that the Fe₃ O₄ /Ag nanocomposite causes a dose‐dependent cell proliferation reduction in MCF‐7 cells (IC₅₀  = 135 μg/ml). Regarding to the Annexin V/PI staining result, the increase percentage of apoptotic cells (28.09%) was detected as compared to untreated cells. According to the caspase assay, Fe₃ O₄ /Ag nanocomposite enhances caspase 3 level. Furthermore, in vitro anti‐cancer activity of the nanocomposite was performed by Hoechst 33258 staining method. The proposed data suggest that Fe₃ O₄ /Ag nanocomposite may be an effective agent for the inhibition of breast cancer cells at in vitro level.

1 Introduction

Recently, nanomaterials with various compositions and shape have been increasingly discovered and overcome many restrictions of the traditional chemotherapeutic drugs [1]. Metal nanostructures such as Cu, Au, Ni, and Ag have attained more consideration in medicinal fields. Silver nanoparticles (AgNPs), among all nanomaterials, have been widely utilised in medicine with significant applications containing anti‐bacterial, cancer therapy, and drug delivery [2]. Various conventional physico‐chemical methods have been reported for the preparation of AgNPs. Among these, green approach seems to be an effective method to nanomaterial synthesis due to the cost‐effective, and non‐toxic, and environmentally benign method [3].

Owing to the rich phytochemical components, plants [4], microorganisms [5], and green algae [6] are favourable for fabrication of diverse inorganic nanoparticles, and they act as reducing agents and also as metallic nanoparticles stabilising during synthesis reaction.

Spirulina, which belongs to cyanobacteria division, has received enhanced attention because of their application in food supplements, and pharmaceutical industries. Spirulina platensis (SP), a filamentous cyanobacterium, has stood out for its minerals, essential fatty acids, great concentration of vitamins, besides amino acid composition [7, 8].‏ In these situations, an alternative magnetic nanoparticle constitutes especially Fe₃ O₄ have attracted much attention because of their unique advantageous properties in different fields such as nanomedicine, optofluidic sensor, electrocatalytic, and antibacterial properties [9]. Moreover, the utilisation of coated magnetite nanoparticles in clinical medicine has also amplified [10]. Recent reports are trying to design and fabricate biological synthesis method for different nanomaterials, which can be utilised very beneficially due to their environment‐friendly method for promising anti‐cancer and antimicrobial activity. For instance, Rejeeth et al. developed an anti‐cancer material using silver nanoscale particles using spirulina platensis extract [11]. Nakkala et al. [12] synthesised Ficus religiosa leaf extract mediated AgNPs and reported anti‐cancer mode of action in different cancer cell lines and antibacterial activity.

Recently, there is a need to develop the efficient biological activity of nanomaterials. The combination of magnetic properties of magnetite and surface properties of Ag nanoparticles cause efficient benefits in catalysis, targeting therapy, and biological targeting [13].

In the current study, Fe₃ O₄ /Ag nanocomposite using Spirulina platensis cyanobacteria extract has been reported for the first time for their anti‐cancer performance against breast cancer cell line. The produced nanocomposite has been determined using Fourier‐transform infrared spectroscopy (FTIR) spectroscopy, powder X‐ray diffraction (PXRD), and transmission electron microscopy (TEM) analysis. The in vitro cytotoxic properties of Fe₃ O₄ /Ag nanocomposite were evaluated in human breast cancer (MCF‐7) cells. Also, the ability of nanocomposite to induce apoptotic cell death was evaluated by caspase assay, Annexin V/PI staining, and Hoechst 33258 staining.

2 Materials and methods

All materials were obtained from commercial sources and used as received without further purification. Fe₃ O₄ magnetic nanoparticles were prepared via chemical co‐precipitation method as reported, previously [14]. FTIR spectra of samples in the form of KBr pellets were recorded using an Alpha‐Bruker FTIR spectrophotometer. PXRD data were collected with a Philips pw 1830 diffractometer (Co‐Kα X‐radiation, λ  = 1.79 Å). TEM analysis was carried out using a Zeiss‐EM10C microscope operating at 80 kV.

2.1 Preparation of pure cyanobacterium extract

The experimental cyanobacterium Spirulina platensis in this study obtained from Persian Microalgae Corporation, Iran. For water extracts preparation, 1.5 g of S. platensis powder was added to 25 ml of deionised sterile water and maintained in a 55°C water bath for 20 min. Then, the solution was centrifuged at 6000 rpm (Eppendorf, Germany) for 10 min and the supernatant filtered as the water extract through Whatman No. 1 filter paper. The filtrate concentration was increased using a rotary evaporator vacuum pump (Rotavapor® R‐100 Rotary Evaporator, Buchi, Switzerland) and after determining the concentration, stored at 4°C for the synthesis of Fe₃ O₄ /Ag nanocomposite particles.

2.2 Biosynthesis of Fe₃ O₄ /Ag nanocomposite by Spirulina platensis

About 250 mg of Fe₃ O₄ nanoparticles was sonicated in 500 ml deionised water for 30 min to achieve a uniform dispersion. About 100 mg of AgNO₃ was added and sonicated for 45 min. Then, 50 ml of aqueous extract of Spirulina platensis was added to the mixture and stirred at room temperature for 24 h. The solid material was washed with water and ethanol several times and dried in an oven at 70°C for 8 h.

2.3 Cell culture

MCF‐7: Human breast cancer cell line was procured from the cell bank of Pasteur Institute of Iran. The cells were cultured for 24 h in the Dulbecco's modified eagle medium containing 10% (v/v) foetal bovine serum, 100 µg/ml streptomycin and 100 U/ml penicillin in 5% CO₂ plus 95% air at 37°C.

2.4 MTT viability assay

The cytotoxicity of Fe₃ O₄ /Ag nanocomposite was evaluated by a commercial cell viability assay using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay. In order to assess the Fe₃ O₄ /Ag nanocomposite cytotoxic properties, the cells were plated at a cell density of 1 × 10⁴ cells/well in 96‐well plate and grown at 37°C under 5% CO₂. After overnight plating, cells were allowed to treat for 24 h with 15.625 to 500 µg/ml of the nanocomposite. In order to perform the assay, the Fe₃ O₄ /Ag nanocomposite‐treated and untreated cells were incubated by 20 μl of MTT dye solution (5 mg/ml) for 4 h at 37°C. To solubilise the formazan crystals in the reaction, the supernatants were discarded and added with 100 µl of dimethyl sulfoxide. The absorbance was calculated at 570 nm using plate reader.

2.5 Annexin V/PI assay

Identification of apoptotic and necrotic cells was evaluated, according to manufacturer's protocol of the Annexin VFITC/PI detection kit (Hoffman‐La Roch Ltd., Basel, Switzerland). MCF‐7 cells (1 × 10⁶ per well) were grown in 6‐well plates, and treated with IC₅₀ concentration of Fe₃ O₄ /Ag nanocomposite. Then, cells were washed twice with PBS and re‐suspended in binding buffer. The fluorescent dye containing Annexin‐V‐FLUOS and propidium iodide (PI) was added to each well and incubated in dark for 15 min. Finally, the contents were analysed with a flow cytometry (Partec, Germany).

2.6 Caspase 3 assay

In vitro quantitative evaluation of Caspase‐3 enzyme activity was assessed, according to the protocol of the ApoTarget ™ Caspase Colorimetric Protease Assay Kit (Invitrogen Corporation). MCF‐7 cells were seeded at a density of 3–5 × 10⁶ per well in 6‐well plate followed by treatment of 135 µg/ml Fe₃ O₄ /Ag nanocomposite for 24 h. Lysations of the cells were carried out using 50 μl of cell lysis buffer followed by incubation on ice for 10 min. The cells were centrifuged at 10,000g for 1 min. The DEVD‐pNA (caspase 3) substrate was then incubated with supernatant and the absorbance of samples was calculated using a microplate reader at 405 nm wavelength.

2.7 Cell morphology analysis

The cells were seeded in plate (6‐well and 4 × 10⁵ cells per well) and incubated with the IC₅₀ concentration of Fe₃ O₄ /Ag nanocomposite at 37°C for 24 h. The cells were then stained with Hoechst 33258 dye at 37°C in the dark. The morphology of stained nuclei was viewed under fluorescence microscope (Incell Analyser 2000, USA).

2.8 Statistical analysis

The one‐way analysis of variance and post‐hoc test were evaluated by means of SPSS v.16.0 software. In all cases, p ‐value <0.05 were considered significant.

3 Results and discussion

3.1 Nanoparticle characterisation

Recently, the researchers have attempted to find innovative alternatives for cancer chemotherapy [15]. Among these, the uses of nanomaterials are attractive in different fields, especially medicine. Herein, we describe Spirulina platensis cyanobacteria extract as reductive agent for Fe₃ O₄ /Ag nanocomposite preparation. Moreover, the anti‐cancer activities of biofabricated Fe₃ O₄ /Ag nanocomposite were evaluated against a human breast cancer MCF‐7 cell line. The use of different chemical reducing agents involving dimethyl hydrogen sulfide, sodium borohydride, and hydrous hydrazine, is the most usual protocol for fabrication of metallic nanoparticles [16]. However, most of these chemical compounds are toxic and harmful to environment and organisms [17]. In recent years, there is a growing need to investigate biological methods, which do not utilise harmful agents during synthesis methods. Moreover, biological protocols are one of the promising alternatives to conventional strategies such as physical and chemical methods.

For fabrication of metal nanoparticles, cyanobacteria attract consideration as bionanofactories due to their advantages such as inexpensive growth media eco‐friendly, and produce a large amount of bioreductive compounds for production of nanoparticles [18, 19].

The FTIR spectra of Fe₃ O₄ NPs and Fe₃ O₄ /Ag nanocomposite are shown in Fig. 1. Absorption bands at around 1700 and 1630 cm⁻¹ can (Fig. 1 b) be due to the stretching vibrations of C = O moieties and aromatic C = C bond or intramolecular hydrogen bonds, respectively, mainly resulting from algae extract molecules [20]. The peaks observed at about 11,150–1350 cm⁻¹ (Fig. 1 b) can be attributed to the deformation of C–O–H, C–H stretching of epoxy groups, and C–O alkoxy groups [21]. Two sharp bands at about 595 and 435 cm⁻¹ (Figs. 1 a and 1 b) were assigned to stretching frequency of Fe–O [22]. The broadband at about 3400 and 1627 cm⁻¹ are due to the bending vibration of absorbed H₂ O and the stretching vibration of O–H bonds [23].

Fig. 1.

FTIR spectrum of

(a) Fe₃ O₄ NPs,

(b) Fe₃ O₄ /Ag nanocomposite

The X‐ray diffraction (XRD) patterns of Fe₃ O₄ NPs and Fe₃ O₄ /Ag nanocomposite are shown in Fig. 2. The reflections along (111), (220), (311), (222), (400), (422), (511), and (440) (Figs. 2 a and b) are consistent with the face‐centred cubic phase of Fe₃ O₄. In addition, the characteristic peaks of the Ag at 2θ  = 41.4°, 51.1°, and 76.6° correspond to (111), (200), and (220) planes (Fig. 2 b) and are clearly evident in Fe₃ O₄ /Ag nanocomposite pattern which is in well agreement with the standard pattern (JCPDS card no. 04‐0783).

Fig. 2.

XRD patterns of

(a) Synthesised Fe₃ O₄ NPs,

(b) Fe₃ O₄ /Ag nanocomposite

Fig. 3 shows the TEM image of Fe₃ O₄ /Ag nanocomposite. The TEM image of synthesised Fe₃ O₄ /Ag nanocomposite showed that majority of the nanoparticles were in quasi‐spherical shape. The TEM image also shows aggregated nanoparticles in which lighter Ag domains were nucleated on the dark magnetic Fe₃ O₄ particles. The estimated sizes of the adsorbed Ag species and Fe₃ O₄ are about 10–20 and 60–70 nm, respectively. The TEM data serve as an important piece of evidence for the formation of Fe₃ O₄ /Ag nanocomposite.

Fig. 3.

TEM image of Fe₃ O₄ /Ag nanocomposite. The TEM image shows aggregated nanoparticles in which lighter Ag domains were nucleated on the dark magnetic Fe₃ O₄ particles

3.2 In vitro cytotoxicity against MCF‐7 cells

In order to investigate the effect of Fe₃ O₄ /Ag nanocomposite on the viability of MCF‐7 breast cancer cells, MTT assay was performed. MCF‐7 cell proliferation was decreased in dose‐dependent manner for 24 h with an IC₅₀ of 135 μg/ml, when compared to untreated cell.

As illustrated in Fig. 4, no significant cell cytotoxic activity were calculated for Fe₃ O₄ /Ag nanocomposite at the concentration of 15.625 µg/ml, although a significant decrease in MCF‐7 cell viability was investigated at the concentration above 31.25 µg/ml. This is the first study to evaluate the cytotoxicity of Fe₃ O₄ /Ag nanocomposite using the extract of Spirulina platensis cyanobacteria against human breast cancer MCF‐7 cell line.

Fig. 4.

Cell viability of Fe₃ O₄ /Ag nanocomposite in MCF‐7 cells was performed by MTT assay; asterisks (*) exhibit a significant difference with the untreated control group (*p < 0.05, **p < 0.01, *** p < 0.001)

Based on Sharaf et al. report, no in vitro cytotoxic effect was detected for spirugenic iron oxide nanoparticles for tested concentrations on the human epithelial cell line [24].

Herein, FITC‐conjugated Annexin V and PI staining was utilised to assess the mode of death of cells treated with IC₅₀ concentrations of Fe₃ O₄ /Ag nanocomposite (derived from respective MTT assay) for 24 h (Fig. 5).

Fig. 5.

Flow cytometric analyses by Annexin V‐FITC/PI double staining of MCF‐7 cells treated with Fe₃ O₄ /Ag nanocomposite at 24 h. Dot plots of Annexin V/PI staining are exhibited in

(a) Untreated MCF‐7 cells,

(b) MCF‐7 cells treated with 135 μg/ml Fe₃ O₄ /Ag nanocomposite showed 13.2% early stage apoptosis and 15.7% late stage apoptosis

3.3 Effects of Fe₃ O₄ /Ag nanocomposite on apoptosis

Apoptosis, programmed cell death, plays a crucial function in the promotion of tumorigenesis and is contributed in deregulation of cancer. Nowadays, induction of apoptosis death pathway has been indicated as a rational strategy to killing tumour cells [25].

The result indicates an enhancement in the number of both Annexin V⁺, PI⁻ stained cells (early apoptotic) and Annexin V⁺, PI⁺ stained (late apoptotic) cells in a Fe₃ O₄ /Ag nanocomposite‐treated cell. The percentage of nanocomposite‐treated MCF‐7 cells entering early and late stage apoptosis reached about 13.2 and 15.7%, respectively. Our data revealed that Fe₃ O₄ /Ag nanocomposite has the potential to trigger apoptosis in MCF‐7 breast cancer cells.

The caspase family proteins play a significant role in two apoptosis pathway: extrinsic and intrinsic. Among them, Caspase‐3 is mediated in the induction of both extrinsic and intrinsic pathways. Herein, the role of executioner caspase‐3, a key mediator of apoptosis, in the Fe₃ O₄ /Ag nanocomposite‐induced apoptosis was determined. As shown in Fig. 6, MCF‐7 cells exposed for 24 h with Fe₃ O₄ /Ag nanocomposite caused 1.2‐fold increase in caspase 3 activity compared to that of untreated cells.

Fig. 6.

Activity of caspase 3 was increased after Fe₃ O₄ /Ag nanocomposite‐exposed cell line, which demonstrated apoptosis pathway

Our result agrees with recent investigate, indicating that graphene oxide–Ag nanoparticle nanocomposites using Tilia amurensis extracts induce the cell cytotoxicity in ovarian cancer cells (A2780) and trigger apoptosis via caspase‐3 activity [26].

Shokoofeh et al. [27] reported the efficacy of a Fe₃ O₄ /Ag nanocomposite in ciprofloxacin‐resistant Staphylococcus aureus. Zhang et al. indicate that the Fe₃ O₄ @Ag nanoparticles have the strong ability to increase the radiosensitivity of Glioblastoma (U251) cell line compared with Ag nanoparticles or Fe₃ O₄ ‐OA alone.

Their findings showed the significant application of Fe₃ O₄ @Ag nanoparticles as a mostly promising nano‐radiosensitiser for the treatment of glioblastoma cells [28]. Recently, no study has yet investigated the use of Fe₃ O₄ /Ag nanocomposite on the level of caspase protein in breast cancer MCF‐7 cells, so, here, caspase 3 assay used to determine the caspase 3 level after treatment by IC₅₀ concentrations of Fe₃ O₄ /Ag nanocomposite. Up‐regulation of the caspase 3 indicates their toxicity to the cancer cells.

Apoptosis was further conducted using Hoechst 33258 staining for 24 h. Nuclei of the cells were stain by Hoechst stain [29], allowing one to determine whether or not nanocomposite led to morphological alterations in nuclei, which was the sign of the apoptosis pathway. Fig. 7 showed DNA fragmentation and cell shrinkage in Fe₃ O₄ /Ag nanocomposite‐treated cells when compared with the untreated cells. Overall, the results suggest that the Fe₃ O₄ /Ag nanocomposite is an effective agent to trigger the apoptosis in breast cancer cells.

Fig. 7.

Fluorescence images of MCF‐7 cells treated with Fe₃ O₄ /Ag nanocomposite at 135 μg/ml for 24 h

(a) Control,

(b) Fe₃ O₄ /Ag nanocomposite

4 Conclusions

This is the first study to develop a simple, cost‐effective, and environmentally friendly method for synthesis of Fe₃ O₄ /Ag nanocomposite using A. Spirulina platensis extract. Interestingly, the determination of synthesised Fe₃ O₄ /Ag nanocomposite was characterised using FTIR spectroscopy, PXRD, and TEM; these methods proved the presence of nanocomposite with an average size of 60–70 nm. The Fe₃ O₄ /Ag nanocomposite possessed significant cytotoxic effect in human breast cancer MCF‐7 cells. The Annexin V/PI staining results showed that the increase percentage of apoptotic cells (28.09%) was detected as compared to untreated cells. Furthermore, the caspase 3 level and Hoechst 33258 staining method revealed that Fe₃ O₄ /Ag nanocomposite has the potential to trigger apoptosis in MCF‐7 breast cancer cells. Therefore, it has been suggested that Fe₃ O₄ /Ag nanocomposite may be an effective agent for the inhibition of breast cancer cells at in vitro level.