Biomimetic synthesis of silver nanoparticles from Streptomyces atrovirens and their potential anticancer activity against human breast cancer cells

Abstract

Silver nanoparticles (AgNPs) have been undeniable for its antimicrobial activity while its antitumour potential is still limited. Therefore, the present study focused on determining cytotoxic effects of AgNPs on Michigan cancer foundation‐7 (MCF‐7) breast cancer cells and its corresponding mechanism of cell death. Herein, the authors developed a bio‐reduction method for AgNPs synthesis using actinomycetes isolated from marine soil sample. The isolated strain was identified by 16s ribotyping method and it was found to be Streptomyces atrovirens. Furthermore, the synthesised AgNPs were characterised by various bio‐analytical techniques such as ultraviolet–visible spectrophotometer, atomic force microscopy, transmission electron microscopy, Fourier transform infra‐red spectroscopy, and X‐ray diffraction. Moreover, the results of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay reveals 44.51 µg of AgNPs to have profound inhibition of cancer cell growth; furthermore, the inhibition of MCF‐7 breast cancer cell line was found to be dose dependent on treatment with AgNPs. Acridine orange and ethidium bromide double staining methods were performed for cell morphological analysis. The present results showed that biosynthesised AgNPs might be emerging alternative biomaterials for human breast cancer therapy.

1 Introduction

Nano‐biotechnology has emerged due to integration of biotechnology with nanoscience for developing biosynthetic and environmental‐friendly technology for synthesis of nanomaterials and with diverse applications [1, 2]. Silver nanoparticles (AgNPs) synthesised using physical, chemical and biological methods are well known [3, 4, 5, 6, 7]. However, in the recent times, the green pathways are in huge demand with a vision of minimising energy consumption and use of toxic chemicals benefitting environment. Therefore, there is a need to develop eco‐friendly, bio‐safe processes for the synthesis of NPs [8, 9, 10, 11]. Biological approaches have been establishing by using plants and microorganisms such as bacteria, actinobacteria, moulds, yeast and algae, which suggested as valuable alternatives for chemical and physical methods. Recent reports mentioned that the actinobacteria isolated from different ecosystems were recognised as potential synthesisers of gold and AgNPs. The biosynthesis of NPs has also been reported from actinomycetes such as Thermomonospora sp., Rhodococcus sp. and Streptomyces sp. [12]. Ag can exist in several oxidation states, though it is commonly encountered in its elemental AgO and monovalent Ag⁺¹ forms. Currently, AgO NPs are being used in antibacterial/antifungal agents in pharma and health sectors. It was widely used in biotechnology, textile engineering, water treatment and cosmetics. Nevertheless, there are reports on several bacterial strains, which are resistant to Ag, may accumulate it at their cell walls. AgNPs with various sizes and shapes could be synthesised by controlling its environment and the physicochemical parameters [13].

In recent years, nano‐scaled particle development had found to be the interested area for researchers in the field of biology and biomedicine due to its unique physical and chemical properties caused by quantum confinement and quantum size effects [14, 15]. Different fungal sources such as Fusarium oxysporum, Verticillium sp. and Aspergillus flavus were used for synthesising metallic NPs extracellularly. Several bacteria such as Escherichia coli, Lactobacillus, Pseudomonas etc. are involved in the NPs synthesis such as gold, Ag, titanium etc. [16, 17]. The biological important organism could secrete a great deal of enzymes involving enzymatic reduction of metallic ions. The biomass used for the synthesis of NPs is simple to handle in downstream process [18, 19]. The process of programmed cell death, or apoptosis, generally characterised by distinct morphological characteristics and energy‐dependent biochemical mechanisms. Apoptosis has emerged as an important mechanism for anticancer effects of many naturally occurring phenomenon [20]. Ag‐based antimicrobial agents show low toxicity to human cells due to the active Ag⁺ ions. It is also long‐lasting biocide with high thermal stability and low volatility [21]. Marine flora constitutes more than 90% of oceanic biomass that offers a great scope for discovery of new drugs in nanomedicine [22]. Nanomedicine concerns the use of precision‐engineered nanomaterials to develop novel therapeutic and diagnostic modalities for human use [23, 24]. This paper illustrates the efficacy of biologically synthesised AgNPs as an antitumour agent.

2 Materials and methods

2.1 Ethics statement

The locations for the collection of marine soil samples (Marina Beach, Chennai) are very common marine soil area, so no specific permissions were required for these locations for collecting marine soil sample. It is also confirmed that these fields did not involve any endangered or protected species. We used sampling procedures that did not harm the marine plant or animal diversity of the locations.

2.2 Media, chemicals and cell line

The analytical grade Ag nitrate (AgNO₃) was obtained from Sigma‐Aldrich chemicals (St. Louis, USA). All the media components and analytical reagents were purchased from Hi‐Media Laboratories Pvt. Ltd. (Mumbai, India). Human breast cancer cell lines Michigan cancer foundation‐7 (MCF‐7) used in the present paper is procured from National Centre for Cell Science [National Centre for Cell Science (NCCS), Pune], India.

2.3 Isolation of actinomycetes

The marine soil sample was diluted by serial dilution method. The diluted samples (10⁻² and 10⁻³) were streaked on the starch casein NO₃ (CN) agar plates incorporated 50 µg/ml of cycloheximide and 35 µg/ml of nalidixic acid (Hi‐media, Mumbai). The plates were incubated at 37°C for 3 days. A typical whitish powdery growth was observed indicates the presence of actinomycetes.

2.4 Identification of actinomycetes

2.4.1 Biochemical test

Various standard biochemical tests were used for the identification of actinomycetes. Starch hydrolysis test were carried out for the confirmation of isolated actinomycetes. Starch is an insoluble polymer of glucose and it acts as a carbon source. Iodine solution added to one of the grown culture plate of actinomycetes. The blue–black colour appeared due to the formation of starch–iodine complex. Owing to the production of extracellular amylase enzyme from microbes, the starch was degraded to colourless area indicates the degradation of starch.

2.4.2 DNA isolation method

Mature culture of actinomycetes was preferred for isolation of DNA. The detailed protocol for DNA extraction is given in Supplementary Material.

2.4.3 Polymerase chain reaction (PCR) amplification, sequencing and restriction analysis

PCR amplification of the 16S rDNA of the Streptomyces sp. was performed using two primers: 9F (5’‐GAGTTTGATCCTGGCTCAG‐3’) and 1541R (5’‐AAGGAGGTGATCCAACC‐3’). The detailed procedure for amplification, sequencing is given in Supplementary Material. The restriction digestion of 16S rDNA was performed according to the method described previously [25].

2.4.4 Sequence alignment analysis

The deposited sequence (KF745859) in Genbank was compared with retrieving the similar sequence using National Centre for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (www.ncbi.nlm.nih.gov). The similar sequences were retrieved and the phylogenetic tree was constructed using the neighbour joining method in the molecular evolutionary genetics analysis software [26]. The outcome of the phylogenetic analysis was determined by the confidence values and estimated with the parameters of 1000 resampling using the bootstrap analysis [27]. The potential primer was identified using the primer blast (www.ncbi.nlm.nih.gov/tools/primer‐blast). This was employed to predict which region of the primer sequence is more potent to carry out the investigations.

2.5 Biosynthesis and characterisation of AgNPs

2.5.1 Biomass preparation of actinomycetes

The isolated actinomycetes were allowed to grow in 500 ml Erlenmeyer flask containing 100 ml sterile starch CN broth supplemented with cycloheximide and nalidixic acid in incubator shaker with 200 rpm at 37°C for 4 days. After the incubation period, the flasks were removed from the shaker and kept at steady condition at 5–10°C, so that mycelium biomass gets settled. Sterile distilled water was used for washing the cells and discards the supernatant. Again, the flasks kept at 5–10°C to settle the biomass for 30 min. By the centrifugation method (1500 rpm for 20 min), the mycelial mass was separated from the sterile distilled water. Moreover, pre‐weighted mycelial pellets were used for the synthesis of AgNPs.

2.5.2 Biosynthesis of AgNPs

About 3 g of actinomycetes wet biomass was added to 50 ml of sterilised aqueous solution of metal ions at various concentrations (0.4, 0.8, 1.2, 1.6 and 2.4 mM) in 100 ml Erlenmeyer flasks and the flasks were kept on shaker with 200 rpm at 37°C for 7 days. The preliminary confirmation was detected by the colour change from white to brown. Ultraviolet (UV)–visible spectroscopy was carried out for the confirmation of AgNPs production in the culture. The wavelength scanning was performed every 5 day interval and the peak value was noted. The synthesised AgNPs were lyophilised and stored in screw capped vials for further characterisation studies.

2.5.3 Characterisation of AgNPs

Detailed characterisations of biosynthesised AgNPs performed by Fourier transform infra‐red spectroscopy (FTIR) spectroscopy analysis, atomic force microscopy (AFM) analysis, transmission electron microscopy (TEM) analysis and X‐ray diffraction (XRD) analysis are given in Supplementary Material.

2.6 Anticancerous activity of AgNPs

Human breast cancer cell line (MCF‐7) was obtained from, NCCS, Pune, India. It was grown as monolayer in Roswell park memorial institute 1640 medium with 10% foetal bovine serum and 0.2% antibiotics. Stock cultures were sub‐cultured every seventh day after harvesting the cells with trypsin‐ethylenediaminetetraacetic acid and then seeding them in tissue culture flask to maintain in exponential phase. The cell suspension was mixed gently and an aliquot was added to the 0.04% trypan blue solution‐I (100 ♣l cell suspension: 100 ♣l dye) and was then counted on haemocytometer.

2.6.1 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and cell morphological analysis

The cytotoxicity effects of different sizes of NPs on MCF‐7 were determined by MTT assay while acridine orange (AO) and ethidium bromide (EtBr) double staining was performed for cell morphological analysis [28, 29]. The detailed protocol is given in Supplementary Material.

3 Results and discussion

3.1 Isolation and identification of actinomycetes

The actinomycetes culture was isolated by using starch CN agar medium (SCNM) incorporated with 35 μg/ml of nalidixic acid and 50 μg/ml of cycloheximide chemicals are used to avoid the contamination during the isolation process. Furthermore, the biochemical test was pursued for amylase production, starch was degraded and zone formation occurs after the addition of iodine solution confirmed the positive test for starch hydrolysis (Figs. 1 a and b).

Fig. 1.

Biochemical test

(a) Pure culture of S. atrovirens in starch casein NO3 (CN) agar plates incorporated 50 µg/ml of cycloheximide and 35 µg/ml of nalidixic acid, (b) Its starch hydrolysing ability, (c) Synthesis of AgNPs from S. atrovirens

The 16s rRNA sequences obtained from the PCR amplification of the gene that encodes the ribosomal RNA using universal primers 27F and 1492R were aligned to database available in the NCBI for comparing with other sequences in the gene database/Genbank. The sequences were received from the Genbank with accession number as KF745859. Isolated MA7 was closely associated members of the diverse Streptomyces spectrum, with a sequence similarity of 99%. The similarity of MA7 with Streptomyces atrovirens was 99%. Apart from the understanding of biochemical characterisation and morphological studies of the strain, the molecular approach plays a crucial role to exhibit exact characteristics. Hence, the computational analysis was employed to predict the similarity analysis. The gene sequencing analysis of the isolate yield contains 1042 base pairs, and when performing the sequence alignment the seven sequence are shown with 99% of similarity to the other strains of Streptomyces. The NCBI includes AJ494865, EU876686, AJ002087, FN557016, EU741195 and NR043508. The phylogenetic relationships are carried out with the aforementioned sequences using neighbour joining method (Supplementary Material: SI.Fig. 1). The observed features from the phylogenetic approach were also confirmed by showing the similar characteristics from the mentioned strains. The primer result explores that the reverse primer of 3’–5’ has potential region containing the base pair regions AAGGAGG and GAGTT (Supplementary material: SI. Fig. 2). These results will be the benchmark of investigations in Streptomyces.

Fig. 2.

UV–vis absorption spectra of AgNPs synthesised from S. atrovirens, the curves A, B correspond to biomass and supernatant, respectively

3.2 Synthesis, optimisation and characterisation of AgNPs

S. atrovirens biomass was added to 1.6 mM concentration (50 ml) of AgNO₃ solution and incubated it for 7 days. The colour changes were observed from pale water to yellowish brown in samples, whereas no colour change was observed in the control (without AgNO₃) which was incubated in the same conditions as mentioned above (Fig. 1 c). The appearance of colour change from pale water to yellowish brown indicated the presence of AgNPs, and also due to the excitation of surface plasmon vibrations in the AgNPs [30, 31, 32, 33, 34]. Thus, it was evident that the cultures produce enzymes that reduce AgNO₃ to Ag atoms. This reduction method clearly indicated that the reduction of ions occurred through extracellular method.

Furthermore, synthesis of AgNPs was confirmed by using UV–visible spectroscopy. Many research groups followed this basic technique and proved to be successful for the synthesis of AgNPs. The aqueous AgNO₃ was reduced during exposure to bacterial biomass which showed the colour change and was further analysed using UV–visible spectrophotometer for AgNPs confirmation. It is observed from the spectra that the AgNPs surface plasmon resonance (SPR) peak occurs at 418 nm with high absorbance, which is very specific for AgNPs [35, 36]. A UV–visible (UV–vis) spectrum is one of the important techniques to ascertain the formation of metal NPs, provided SPR exhibits from the metal. A sharp clear peak, assigned to a surface plasmon, was developed for various metal NPs with different sizes, has already been reported [37]. The UV–vis spectra recorded for the present paper from S. atrovirens biomass and supernatant were represented graphically in Fig. 2.

3.2.1 Optimisation of AgNPs production

Various concentrations of AgNO₃ (0.4, 0.8, 1.2, 1.6 and 2.4 mM) were added to the S. atrovirens biomass. The colour change was observed at 1.6 mM concentration of AgNO₃ that was added to the pellet collected, but no colour change observed in the supernatant of the same. This showed that 1.6 mM is the optimum concentration required for AgNPs production and also showed that the pellet has the ability to convert AgNO₃ to AgNPs. This was initially confirmed by taking reading in UV‐spectrophotometer at 418 nm in 1.6 mM concentration (Fig. 2). This is very specific for AgNPs which corroborates with previous studies [21, 28, 29].

3.2.2 AFM studies

AFM studies were conducted to reveal the detailed size, shape and morphology of AgNPs and agglomeration of Ag atoms. The earlier report suggested that the AFM approach is eminent to develop the three‐dimensional image of the Ag nanoisland formation, which can be clearly visible [32, 38]. The supernatants with the maximum time duration after the synthesis of AgNPs were used for this experiment. AFM images were taken with silicon cantilevers with force constants 0.02–0.77 N/m, tip height 10–15 nm and contact mode. It is noted that the AgNPs agglomerated and formed distinct nanostructures as shown in Fig. 3. The topographical image of irregular shaped AgNPs was noted along with the formation of nanoisland. Apart from that there was an agglomeration of Ag, which confirms the presence of AgNPs.

Fig. 3.

AFM image showed formation of AgNPs by S. atrovirens

3.2.3 FTIR analysis

FTIR spectrometer analysis revealed the presence of functional groups, which may be present between amino acid residues and protein during the AgNPs synthesis. It is also carried out to identify the biomolecules responsible for the reduction of Ag⁺ ions and capping of the bio‐reduced Ag bio‐NPs synthesised by S. atrovirens [32]. The synthesised AgNPs showed strong bands at 3697.44 and 2923.77 cm⁻¹, 1648.20 cm⁻¹ corresponds to alcohol stretch free and acid stretch in OH bands, respectively. Here, 3432.05 cm⁻¹ represents the amine stretching frequency of the amide band present in the AgNPs. The peaks present at 2102.87 cm⁻¹ confirm the alkyne stretch (–C≡C–). The value 1254.39 cm⁻¹ shows the presence of –CN stretching of amine and –C=O of acid in the AgNPs. The peaks at 812.95 and 834.24 cm⁻¹ represent the alkene bending frequencies in AgNPs. Besides, the ester stretching frequencies might correspond to 1048.53 and 1206.72 cm⁻¹ estimated to be present in AgNPs (Supplementary Material: SI.Fig. 3). This evidence suggests that the biomolecules could possibly perform the dual function for the formation and stabilisation of the AgNPs in aqueous medium [39, 40, 41].

3.2.4 TEM and XRD analyses of AgNPs

The size and morphology of the synthesised AgNPs were also characterised by TEM (Fig. 4 a). The average size of the NPs was found to be in the range of 58 ± 2 nm with smooth surface and well dispersed. Morphology of the NPs was found to be spherical in shape, which is in agreement with the observations from AFM images. It is observed that the synthesised particles are in much physical contact; this may be either due to the biogenic nature of NPs. Similar results have been observed by Christensen et al. [42] in the biosynthesis of AgNPs using Murrayakoenigii leaf extract. The result correlate well with the value obtained from XRD. The XRD method was used to investigate the crystalline nature of the synthesised NPs. Fig. 5 shows the XRD pattern of the AgNPs. The two main characteristic peaks of metallic Ag were recorded at 2θ value of 38.4° and 44.5°, corresponding to the Ag crystal plane (111) and (200) of the face‐centred cubic structure (JCPDS 00‐004‐0783). From the Scherrer equation, the average crystallite size of AgNPs was found to be 55 ± 5 nm. The dynamic light scattering (DLS) data showed the average particle size around 58.5 nm and poly dispersity index of 0.250 (Fig. 4 b).

Fig. 4.

Size and morphology of the synthesised AgNPs

(a) TEM micrograph image of AgNPs synthesised using S. atrovirens showed the spherical shaped particle the average size around 58 nm, (b) Supporting data for the NPs size distribution with DLS showed the average particle size around 58.5 nm

Fig. 5.

X‐ray diffraction analysis of synthesised AgNPs from S. atrovirens

3.3 Evaluation of in‐vitro anticancerous activity of AgNPs

3.3.1 Effect of AgNPs on cell cytotoxicity

Mitochondrial function‐based MTT conversion to formazan crystals is often used as cell viability test in cell culture system. MTT was reduced to purple formazan by mitochondrial succinate dehydrogenase of living cells. In this paper, AgNPs exerted cytotoxic effects on MCF‐7 human breast cancer cell line at the concentration of 2–50 µg for a period of 24, 48 and 72 h. NPs inhibited the growth of the MCF‐7 human breast cancer cell line significantly in a dose and time‐dependent manner. The cytotoxic activity of this cell line was determined based on the concentration of the compound required to reduce the survival of cells by 50% (IC₅₀). NPs were highly cytotoxic to the MCF‐7 cell lines at very low concentrations and IC₅₀ dosage differ with time and dose‐dependent manner. The IC₅₀ values for NPs are shown in Fig. 6, for MCF‐7 cell line. Since NPs at a dose 44.51 µg were found to be effective for 24 h treatment which was fixed for further analysis [12, 43].

Fig. 6.

MTT assay – inhibition of cell viability at different concentrations of AgNPs in various time intervals

3.3.2 Effect of AgNPs on cell morphology

The proliferation activity of cell populations under different treatment conditions were determined by the MTT assay based on the detection of mitochondrial dehydrogenase activity in living cells. The first and most readily notable effect following exposure to any toxic compound is the alteration in cell shape or morphology. The effects of NPs at a fixed IC₅₀ dose on cultured MCF‐7 human breast cancer cell line was evaluated by exposing the cells for 24 and 48h (Fig. 7). Fluorescent microscopic analysis with untreated control and treated cells revealed that IC₅₀ dose of NPs induced changes in cellular shape. Most of the cellular contents were found as small fraction, so the cells became more granular in appearance. Owing to the presence of swelling in their cytoplasm, colony morphology was less well defined. There were dissembled gaps between neighbouring cells and remaining adherent cells had become more round shaped, which are characteristics of apoptosis. This confirms the cell death through apoptosis. The cell death also takes place in a time‐dependent manner. This was represented in Fig. 7; the fluorescent green colour on the surface of the cells represents the presence of AgNPs absorbed by the cell line [44, 45].

Fig. 7.

Determination of anticancer activity of synthesised AgNPs against MCF‐7 human breast cancer cell line

[I] (A, B) Control – without treatment of AgNPs, (C, D) After 24 h AgNPs cancer cells showing the dissemble gaps, granular in appearance and death cells. [II] (A, B) Control – without treatment of AgNPs, (C, D) After 48 h AgNPs treated cancer cells showing the dissemble gaps, granular in appearance and many death cells

3.3.3 AO and EtBr double staining observation of MCF‐7 cell line

The effects of NPs on apoptotic‐induced morphological changes in MCF‐7 cell line were also noted. MCF‐7 cells were first treated with the respective IC₅₀ doses for 24 and 48 h; it was stained with AO and EtBr and was observed under fluorescent microscope. AO and EtBr double staining was used for cell morphological and mode of cell death analysis. After MCF‐7 cell lines were exposed to various concentrations for 24 and 48 h, the cells were classified into four types according to the fluorescence emission and the morphological feature of chromatin the stained nuclei (Fig. 8): (i) viable cells had uniformly green fluorescing nuclei with a highly organised structure. (ii) Early apoptotic cells had green fluorescing nuclei, but the peri‐nuclear chromatin condensation was visible as bright green patches or fragments. (iii) Late apoptotic cells had orange–red fluorescing nuclei with condensed or fragmented chromatin. (iv) Necrotic cells have uniformly orange–red fluorescing nuclei with no indication of chromatin fragmentation, and the cells were swollen to large size. This treatment caused more cell death in 48 h than 24 h in MCF‐7 human breast cancer cell lines. This paper revealed that synthesised AgNPs have potential for tumour reduction. The cytotoxic efficacies of AgNPs are the result of physicochemical interaction of Ag atom with functional group of intercellular protein and DNA, the details of mechanism of which has been explained in our previously published report [39, 46, 47, 48]. The present paper also revealed that AgNPs are having the capacity to inhibit the cancer cells. It could be concluded that this approach can be undergone for further trails and it will be a milestone against cancer through nanomedicine approach.

Fig. 8.

Effect of AgNPs on MCF‐7 human breast cancer cell line stained with AO and EtBr

(a) Control – without AgNPs treatment, (b) AgNPs treated breast cancer cell: (A, B) After 24 h AgNPs treated cells and (C, D) After 48 h AgNPs treated cells

4 Conclusion

Overall, the present paper confirmed that S. atrovirens are capable for producing AgNPs by extracellular reduction. This eco‐friendly approach was found to be easier as compared with the conventional physicochemical synthesis. The UV–vis analysis had confirmed the biosynthesis of AgNPs by showing the absorbance at 418 nm. Besides, AFM, TEM and XRD confirmed the AgNPs formation with 55–78 nm in size having spherical, irregular morphology and are crystalline in nature. The intrinsic properties of AgNPs are responsible for outstanding cytotoxicity effect against MCF‐7 human breast cancer cell. The data of our paper revealed that the low concentrations are highly toxic which are found to be time, dose dependent. This approach contributes to novel materials as an alternative potential therapeutic treatment (nanomedicine) for human breast cancer. Therefore, further studies are needed to fully identify the mechanisms of anticancer activity and toxicity of the AgNPs.