Characterization, in vitro cytotoxic and antibacterial exploitation of green synthesized freshwater cyanobacterial silver nanoparticles

Cyanobacteria-mediated silver nanoparticles synthesis approach has proven to be more efficient and eco-friendly in achieving biomedical applications compared to physical and chemical prototypes. In the present work, the silver nanoparticles were successfully synthesized by cell-free extract of freshwater cyanobacteria, i.e., Chroococcus turgidus and Characium typicum. The cyanobacterial silver nanoparticles (CSNPs) were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) analysis and were further tested for antibacterial and cytotoxic efficiency. The synthesis of CSNPs was confirmed through visible color change and shift of peaks at 430–445 nm by the UV-Vis spectroscopy. The size of CSNPs was between 22 and 34 nm and oval-shaped which were confirmed by SEM and TEM analyses. The FTIR spectra showed a new peak at the range of 3,400–3,460 cm−1 compared to control, confirming the reduction of silver nitrate. Furthermore, the antibacterial activity of CSNPs showed highest zone of inhibition with 6.9, 4.0, 2.0, and 3.0 mm against Salmonella paratyphi, Escherichia coli, Klebsiella pneumonia, and Staphylococus aureus, respectively, whereas in vitro cytotoxic activity of C. typicum and C. turgidus silver nanoparticles showed remarkable IC50 values with 43.3 and 40.9 ug/ml against MCF-7 breast cancer cell line and 20.8 and 55.7 ug/ml against HepG2 cancer cell, simultaneously.


INTRODUCTION
Nanoscience is an emerging field of nanobiotechnology, utilizing nanobased systems for various biomedical applications. The nanoparticles generally lie in the range of 10 −9 nm with dimensions of 1-100 nm (Sharma et al., 2016). Consequently, nanoparticles exhibit therapeutic properties because of their high surface volume, strong binding affinity, and their ability to easily diffuse into the cell (Sun et al., 2014). Among other nanoparticles, silver nanoparticles have prudent specific conductivity and stability with unique therapeutic, antibacterial, and catalytic properties which makes them important in nanooncology and in the diagnosis and treatment of cancer through nanodevices and therapeutic agents (Husain et al., 2015). The synthesis of nanoparticles through physical and chemical processes consumes extreme energy, pollutes the environment, produces toxic chemicals that stick to the surface of particles, and which have inimical effects in medical applications (Nadagouda and Varma, 2008). Green synthesis of nanoparticles is convenient over physical and chemical methods of nanoparticle synthesis, since biologically synthesized nanoparticles are more eco-friendly and energy efficient for the synthesis of inorganic nanoparticles (Jeffryes et al., 2015;Mittal et al., 2013) and economically feasible compared to physical and chemical synthesis.
Green synthesis of nanoparticles is generally carried out by the living forms of bacteria, fungi, viruses, plant extracts, plant parts, algae, yeast, and cyanobacteria (Keskin et al., 2016;Kharissova et al., 2013;Pirtarighat et al., 2019;SwaminathanáIyer, 2013). Among the biological systems, cyanobacteria are preferable since they can be easily cultivated with a high growth rate and they reduce silver ions expeditiously (Roychoudhury et al., 2016). Cyanobacteria are one of the prominent bio-factories that produce a wide range of bioactive compounds, i.e., secondary metabolites, like minerals, lipids, proteins, polysaccharides, carbohydrates, carotenoids, and vitamins, and number of other auxiliary metabolites with a wide scope of biological activities and applications (Kim, 2011). The bioactive compounds produced by cyanobacteria are chemically diverse sources of new pharmaceutical compounds that exhibit a wide range of biomedical applications. These metabolites are cytotoxic metabolites, antibacterial and antifungal metabolites, protein inhibitors, alkaloids, peptides, and terpenoids (Chlipala et al., 2011).
Freshwater cyanobacteria are able to synthesize nanoparticles, intracellularly as well as extracellularly. The functional groups like hydroxyl, amino, and carboxyl are generally responsible for capping and act as reducing agents. The size of the nanoparticle differs from intracellularly and extracellularly synthesized particles, i.e., 10 nm to 100-200 nm, respectively. The organic compounds like proteins attribute to the extracellular synthesis of silver nanoparticles (Sharma et al., 2016). The silver nanoparticles that are extracellularly synthesized are spherical in size and are between 100 and 200 nm (Ali et al., 2011). The size, number, and shape of the nanoparticle are directly proportional to the concentration and time period of exposure to silver ions (Tsibakhashvili et al., 2011) The silver nanoparticles synthesized from freshwater cyanobacteria evince the secretion of bioactive compounds, and when combined with AgNP's (silver nanoparticles) they show a wide variety of biomedical applications, including cytotoxic activity, which is exploited in cancer treatment, one of the deadliest diseases in the world. In our study, silver nanoparticles are synthesized extracellularly from freshwater cyanobacteria, such as Chroococcus turgidus and Characium typicum, and are characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. The cyanobacterial silver nanoparticles (CSNPs) were investigated for antibacterial and cytotoxic activities against human MCF-7 breast cancer cell lines and HepG2 cell line.

Isolation and cultivation of cyanobacterial cultures
Silver nanoparticles were synthesized from two cyanobacterial strains, C. turgidus and C. typicum, isolated from the freshwater habitats in Telangana. The cultures were maintained through a common subculturing method in the BG11 medium in laboratory conditions at 25°C under continuous cool white fluorescent light. The isolates were identified based on their morphological characters and molecular sequencing (AL-Sultan, 2017).

Preparation of cell-free extract for nanoparticle biosynthesis
The cyanobacterial cultures were grown in the BG11 medium and harvesting was conducted by centrifuging at 5,000 rpm for 10 minutes (REMI R-8C laboratory centrifugation) at 15°C and washed thoroughly with sterile distilled water to remove the trace elements of media. Wet biomass of 1.5 g of culture was then suspended in 30 ml of double distilled water and boiled at 80°C for 30 minutes in an Erlenmeyer flask. After boiling, the mixture was cooled and centrifuged at 5,000 rpm for 15 minutes and the supernatant was collected for biosynthesis (Jena et al., 2013).

Extracellular synthesis of silver nanoparticles
One hundred millimeter of AgNO 3 (Sigma Aldrich) solution (pH 7.0) was prepared. About 1 ml of cell-free extract was added to 19 ml of AgNO 3 solution and was incubated at 25°C ± 1°C under cool white fluorescent light (50 μmol photons m¯²s¯¹) and in the dark condition for 72 hours of incubation. A freshly prepared BG11 medium with AgNO 3 was used as control. During the incubation period, the change in color from pale yellowish green to brown indicated the synthesis of nanoparticles. The color change to a darker brownish color was time-dependent and it was quantified by absorbance spectra recording during the 72 hours of incubation period. One ml aliquot sample were taken every 12 hours, and the absorbance of the UV-Vis spectra at a resolution of 1 nm in the range of 300-800 nm were recorded by using a spectrophotometer (Shimadzu, Model: UV-2450).

Electron microscopic analysis
TEM images were obtained from an FEI TECHNAI G 2 TEM operating at an accelerating voltage of 200 KV. Samples were set up by putting a drop of sample solution on the carboncovered copper grid and dried under an infrared light preceding the assessment. Crystallinity of structures was affirmed by the selected area's electron diffraction pattern (Jena et al., 2013).

Scanning electron microscopy
SEM analysis (Hitachi, model: S-3400N) was used to observe the microalgal cell with entrapped silver nanoparticles. On a carbon coated copper grid, thin films of sample were prepared and dried under a mercury lamp for 5 minutes (Issa, 1999).

Fourier transform infrared spectroscopy analysis
For FTIR spectroscopy, freeze-dried, cell-free extract silver nanoparticles were utilized. A limited quantity of dried biomass with potassium bromide (KBr) was crushed together. PerkinElmer FTIR system Spectrum GX model was used to record the sample spectrum with the measurements being carried out in the range of 400-4,000 cm −1 at a resolution of 4 cm −1 .

Antibacterial activity of CSNPs
CSNPs were tested for antibacterial activity, suggested by Srinivasulu et al. (2002). Gram-positive and Gram-negative bacteria like Staphylococus aureus (MTCC-3381), Escherichia coli (MTCC-1541), Klebsiella pneumonia (MTCC-3384), Salmonella paratyphi (MTCC-3220), and Micrococcus luteus (MTCC-1541), procured from the Microbial Type Culture Collection Center (Chandigarh), were used as test organisms. The test organisms were grown in fresh nutrient broth for 24 hours. The antibacterial activity was carried out by agar well diffusion method. Bacterial lawns were prepared by spreading the bacterial suspension of 100 μl (1×10 5 CFU/ml) on the surface of the agar plates. Agar wells (6 mm in size) were punched on the agar plates using a sterile stainless steel cork borer. The wells were loaded with 40, 60, and 80 μl of CSNPs with the same quantity of AgNO 3 as the negative control along with 30 μg/ml streptomycin as the positive control. The plates were incubated for 24 hours at 37°C and then examined for the presence of inhibition zones. The inhibition zone diameter was measured and the mean value for each organism was recorded and the relative percentage of inhibition with respect to positive control was calculated.

Cytotoxicity of silver nanoparticles
CSNPs were tested on MCF-7 breast cancer cell lines and human hepatocellular carcinoma (HepG2) cell lines using MTT-based cell cytotoxicity assay. The cells were procured from the National Centre for Cell Science, Pune, India. The harvested cells were cultivated in RPMI media with 10% fetal bovin serum, 1% of L-gultamin, and 1% of penicillin (100 U/ ml) along with 1% streptomycin (100 µg/ml). The cells in the media are diluted to the cell density from 1 × 10 6 to 1 × 10 3 i.e., 1,000-10,000 cells per well. The cells were seeded in a 96-well micro-titer plate by adding 100 µl per well in triplicate. The plates were incubated at 37°C in COD incubator maintain 5% CO 2 , allowing the cells to adhere overnight. After incubation, the media was discarded, and the wells were treated with CSNPs with concentrations of 100, 75, 50, 25, and 5 µg/ml and incubated at 5% CO 2 for 24, 48, and 72 hours, according to the study of test sample. The well with untreated cells was maintained as control.

RESULTS AND DISCUSSION
The synthesis of silver nanoparticles by C. turgidus and C. typicum was confirmed by color change from pale yellow to reddish brown. The intensity of color increased with the time of incubation till 72 hours. Synthesis of AgNPs by cyanobacterial cells was determined by studying the UV absorbance peak in the range of 300-800 nm (Shimazu, Model: UV-2450) because of the strong absorption of visible light due to excitation of the nanoparticles associated with surface plasmon resonance, which is a significant property of silver nanoparticles (Banerjee et al., 2014;Hartland et al., 2011;Krishnaraj et al., 2010;Kwon et al., 2012). The color change of AgNO 3 solution was observed only under light conditions by both the strains (Fig. 1). The UV absorbance of cell-free extract of C. typicum showed the wide peak with 0.663 at 435 nm (Fig. 2), whereas UV absorbance peak of 2.646 at 442 nm for cell-free extract of C. turgidus was observed (Fig. 3). Silver nanoparticle synthesis by cyanobacterial cells at 420 and 470 nm, corresponding to the plasmon absorbance suggested that cyanobacterial extract is efficient in nanoparticle synthesis. According to Pal et al. (2007), a narrow peak at 425 nm and a wide peak at 490 nm corresponded to 29 and 89 nm silver nanoparticles, respectively. Reduction of silver ions to silver nanoparticles may be due to capping of microalgal protein metabolites (Sudha et al., 2013). It is reported that the absorption spectrum of silver nanoparticle present maximum peak height between 420 and 450 nm with a blue or red shift with increment in particle size (Pal et al., 2007;Sönnichsen et al., 2002).

Characterization of CSNPsScanning electron microscopy
The morphology and characterization of biosynthesized silver nanoparticles by the bio-reduction of cell-free extract of C. turgidus and C. typicum were revealed through SEM studies. The light microscopy showed that the bio-reduced silver nanoparticles seemed to be trapped on the cellular structures either in aggregates or dispersed. Further study showed that the size of the particle synthesized by cell-free extract ranged between 22 and 34 nm, and nanoparticles synthesized by biomass are mostly oval in shape (Figs. 4 and 5).

Transmission electron microscopy
TEM analysis provided information regarding the morphology and shape of the synthesized nanoparticle. The results were interpreted through images of TEM and revealed that the size and shape of the synthesized particles varied significantly among species. The dispersion of nanoparticles was evenly distributed in the cell-free extract with shape of the particles varying from spherical to oval and irregular shapes. TEM analysis showed the synthesis of polydisperse AgNPs in the range of 20 nm, with an average size of 18.10 nm in C. turgidus (Fig. 6), with the smallest particle size being 9.87 nm, an average of 20.67 nm in C. typicum (Fig. 7), and the smallest particle size was 19.78 nm.

Fourier transform infrared analysis
FTIR spectroscopy is an epic tool that uses electromagnetic radiation frequencies for verification of organic compound and structure determination between 4,000 and 400 cm −1 of frequency. Chemical bonds of different compounds absorb different intensities at different frequencies. Therefore, IR spectroscopy works by collecting absorption information, analyzes it, and gives the output in the form of spectrum. The peak and signal frequencies of absorption are directly relate to the bonds within the compound (Smith, 2018;Yang et al., 2015). FTIR analysis was carried out on C. turgidus and C. typicum silver nanoparticles synthesized from cell-free extract. The FTIR spectral peaks were compared with control, i.e., cell extract without AgNO 3 .
The IR spectra of bio-reduced silver nanoparticles by C. turgidus cell-free extract showed a major vibration band at 3,440.87 cm −1 , which is generally assigned to N-H 1 0 amine or amide with O-H bend of alcohols. The similar stretching bands were observed ranging at 1,382-1,386 cm −1 , which is assigned to the protein bending of methyl, ʋ s (C-O) of carboxylic acid, and C-F stretch of the alkyl halide functional group. Another band was at     the range of 1,023-1027 cm −1 , which was assigned to carbohydrate (C-O-C) polysaccharide (Fig. 8). Inasmuch as C. typicum cellfree extract silver nanoparticles IR spectral data revealed a band at 3,450.44 cm −1 , which is assigned to 2 0 amide in the N-H stretch, 1,382.42 cm −1 is assigned to protein δ as (CH 2 ) and δ as (CH 3 ) bending of methyl, carboxylic acid ʋ s (C-O), and C-F stretch of alkyl halide (Fig. 9). The frequency bands were compared with control FTIR spectra by noting the appearance of new bands in the FTIR spectra of cell-free extract of cyanobacterial strains at N-H peaks of 1 0 and 2 0 amide (Fig. 10).

Antibacterial activity of biosynthesized silver nanoparticles
The antibacterial activity of biosynthesized silver nanoparticles of two cyanobacterial strains were studied against S. aureus (MTCC-3381), E. coli (MTCC-1541), K. pneumonia (MTCC-3384), S. paratyphi (MTCC-3220), and M. luteus (MTCC-1541), through agar-well diffusion method. The agar well was loaded with 40, 60, and 80 µl with silver nanoparticles along with streptomycin as the negative control and silver nitrate as the positive control. 80 µl silver nanoparticle solution of C. turgidus and C. typicum showed high biocidal activity against S. paratyphi with 4.1 and 6.9 mm zone of inhibition, 3.3 and 4 mm zone of inhibition against E. coli, respectively, whereas 60 µl silver nanoparticle solution of C. typicum showed 4.2 mm zone of inhibition against S. paratyphi as seen in Table 1; the antibacterial activity plates clearly showed the zone of inhibition (Fig. 11).  Cytotoxic assayThe MTT cell assay works on the principle for determination of cell viability, cell proliferation, and effect of cytotoxic chemical entities. The assay is the quantitative measurement of insoluble formazan crystals formed through the reduction of the yellow colored water soluble Tetrazolium dye, i.e., MTT extracellularly by metabolically active cells that are conciliated by lactate dehydrogenase of mitochondria. The color intensity of formazan crystals is directly proportional to the number of viable cells which are assessed spectrophotometrically at 570 nm (Van Meerloo et al., 2011;Tolosa et al., 2015). The assay procedure includes reconstitution of the preestimated MTT reagent in the assay buffer, trailed by its addition to the culture system. Subsequent to dissolving the formazan crystals in the solubilization solution, results can be directly perused.
In vitro cytotoxicity studies carried out by silver nanoparticles of three cyanobacterial strains dissolved in DMSO were evaluated against MCF-7 breast cancer and HepG2 cell lines with different concentration of 5, 10, 25, 50, 75, and 100 ug/ml for each sample in comparison with the standard tamoxafin drug. All three cyanobacteria-mediated silver nanoparticles cytotoxicity were dose-dependent and compared with untreated cells. The cytotoxic activity of C. typicum against MCF-7 breast cancer cells  of 5 ug/ml concentration showed the cell viability of 64.7% then decreased to 33.4% by 100 ug/ml concentration; this explains the death of cancer cells increased by increasing concentration of silver nanoparticles ( Table 2). The overall cytotoxicity of C. typicum against MCF-7 breast cancer cells gave the IC 50 value of 43.375 ug/ml. IC 50 is the half maximal inhibitory concentration which is the measure of the potency of a drug in inhibiting biological cell growth. The cytotoxic activity of C. turgidus against MCF-7 breast cancer cells resulted in decrease of cell viability from 64.1% to 32.7% and from 5 to 100 ug/ml concentration, respectively, giving out the IC 50 value of 40.917 ug/ml (Table 3).
The cytotoxic activity of cell-free extract silver nanoparticles against HepG2 cancer cell line by C. typicum showed the cell viability of 59.3% with 5 ug/ml concentration, where 100 ug/ml showed 27.03% viability with IC 50 value of 20.8 ug/ml (Table 4). The cytotoxicity of C. turgidus killed the cell with 40.9% cell viability with 100 ug/ml concentration, whereas 5 ug/ml concentration showed more cell viability of 63.3%, and the IC 50 value of MBKG 35 silver nanoparticles against HepG2 cancer cell line was 55.7 ug/ml (Table 5). The cell viability was observed under a compound microscope in the T 25 cell culture flask (Fig. 12).

CONCLUSION
Our present study indicates that the green synthesis of silver nanoparticles by biological protocol using isolated purified C. typicum and C. turgidus cell-free extract acted as both equally reducing and stabilizing agents for the successful bio-reduction of nanoparticles. The characterization study using UV-Vis, FTIR, SEM, and TEM detailed that the synthesized nanoparticles were 19-34 nm in size and the proteins and polysaccharides were important factors in the biosynthesis of silver nanoparticles. Because of their small size, the silver nanoparticles were able to exhibit some synergetic bactericidal potentiality against human pathogenic bacteria, such as S. paratyphi and E. coli. Silver nanoparticles of C. typicum and C. turgidus exerted significant cytotoxicity against MCF-7 and HepG2 cancer cell lines. Our study emphasized that this approach was simple, eco-friendly, and efficient, which resulted in the production of silver nanoparticles without the use of any toxic reducing or dispersing agents at an ambient temperature.