Biomedical potential of Anabaena variabilis NCCU-441 based Selenium nanoparticles and their comparison with commercial nanoparticles

Selenium nanoparticles (SeNPs) are gaining importance in the field of medicines due to their high surface area and unique properties than their other forms of selenium. In this study, biogenic selenium nanoparticles (B-SeNPs) were synthesized using cyanobacteria and their bioactivities (antioxidant, antimicrobial, anticancer and biocompatibility) were determined for comparison with commercially available chemically synthesized selenium nanoparticles (C-SeNPs). Color change of reaction mixture from sky blue to orange-red indicated the synthesis of biogenic SeNPs (B-SeNPs). UV–Vis spectra of the reaction mixture exhibited peak at 266 nm. During optimization, 30 °C of temperature, 24 h of time and 1:2 concentration ratio of sodium selenite and cell extract represented the best condition for SeNPs synthesis. Various functional groups and biochemical compounds present in the aqueous extract of Anabaena variabilis NCCU-441, which may have possibly influenced the reduction process of SeNPs were identified by FT-IR spectrum and GC–MS. The synthesized cyanobacterial SeNPs were orange red in color, spherical in shape, 10.8 nm in size and amorphous in nature. The B-SeNPs showed better anti-oxidant (DPPH, FRAP, SOR and ABTS assays), anti-microbial (antibacterial and antifungal) and anti-cancer activitities along with its biocompatibility in comparison to C-SeNPs suggesting higher probability of their biomedical application.

. Optimization for B-SeNPs synthesis (a) for sodium selenite and cell extract concentration ratio (b) for temperature (c) reaction time. 441 showed resonance peak at 253 cm −1 (Fig. 4c). Scanning electron microscopy-Energy dispersive X-ray spectroscopy (SEM-EDX) analysis of B-SeNPs exhibited 12 nm size of nanoparticles ( Fig. 4d) with 94% weight of selenium. EDX spectrum gave a strong signal at 1.4 keV (Fig. 4e), which signified the presence of selenium. TEM analysis revealed that the synthesized cyanobacterial SeNPs were polymorphic, spherical and with an average  www.nature.com/scientificreports/ size of 10.8 nm (Fig. 4f). 19 nanoparticles have the size range between 9-10 nm, 18 nanoparticles exhibited the size range between 10-11 nm and the overall range was found between 6.4-15.8 nm (Fig. 4g).

Anti-oxidant activities of B-SeNPs and C-SeNPs.
Anti-oxidant activities were performed by DPPH, FRAP, SOR and ABTS assays. Ascorbic acid was taken as the positive control. In DPPH, assay, IC 50 Fig. 5a-d).  Table 4). The zone of inhibition (ZOI) of B-SeNPs, C-SeNPs and streptomycin against all the bacterial strains have been mentioned in Table 4. Both B-SeNPs and C-SeNPs were found to be more efficient against the gram positive strain (Bacillus subtilis, Staphylococcus aureus) than the gram negative strains (Klebsiella pneumonia, www.nature.com/scientificreports/ Escherichia coli). The B-SeNPs were effective in comparison to C-SeNPs against all the tested bacterial strains due to the larger ZOI (significant at P ≤ 0.05). Anti-fungal activity of B-SeNPs and C-SeNPs were determined against the three species of Candida sp., viz. Candida albicans, Candida glabrata and Candida krusei. B-SeNPs gave larger zone of inhibition than C-SeNPs against all the three strains at all the concentrations (20, 40, 60 and 80 µg/ml) against Candida krusei. No ZOI  Table 4. Anti-bacterial activity of B-SeNPs and C-SeNPs by disc diffusion method. Anti-cancer activity of B-SeNPs and C-SeNPs. MTT assay was performed to find out the cytotoxicity effect of B-SeNPs and C-SeNPs on the cell lines (MCF-7 and HepG2). The results of B-SeNPs and C-SeNPs showed the concentration-dependent cytotoxic effects in both the cell lines after 24 h exposure ( Fig. 8a- Table 6). These results showed that both types of SeNPs have anti-cancerous property but the B-SeNPs have better activity than C-SeNPs at lesser IC 50 values (significant at P ≤ 0.05). The microscopic observation of B-SeNPs and C-SeNPs treated cells at their IC 50 value showed considerable changes in the morphology of cells in comparison to untreated cells. The untreated cells appeared spindle shaped and closely adhered to each other, however the treated cells have fragmented and retracted morphology (Fig. 8d).  Table 5. Anti-fungal activity of B-SeNPs and C-SeNPs by disc diffusion method.  (Fig. 9). The difference between IC 50 value of normal cell and cancer cells of C-SeNPs were very close, that difference was much higher in case of B-SeNPs exposure than of C-SeNPs. So, the B-SeNPs were found to be nontoxic at their IC 50 value and C-SeNPs were toxic toward normal cells (significant at P ≤ 0.05) (Fig. 9).   www.nature.com/scientificreports/

Discussion
The biogenic SeNPs synthesis has got increased attention due to their wide applicability in biomedicine. The green synthesis method is preferred over physical and chemical methods as it is ecofriendly and does not depend on the use of harsh chemical and high energy 33 . For the very first time, cyanobacteria-Anabaena variabilis NCCU-441 has been used for the synthesis of SeNPs and comparison of their bioactivity with chemically synthesized commercial SeNPs. During the present investigation, B-SeNPs have been synthesized extra-cellularly using the crude cyanobacterial extract. Extra-cellular biogenic nanoparticles synthesis is much economical than intra-cellular synthesis as it saves chemicals, energy and time needed for cell disruption and purification. Use of cyanobacterial biomass have added advantage of their short life cycle. The synthesis of B-SeNPs was visually observed as the color change of reaction mixture from sky blue to orange-red. Control showed no color change that shows their inability to synthesized SeNPs. B-SeNPs having orange red color have been also obtained in other species e.g. Gliocladium roseum 15 , Allium sativum 22 , Emblica officinilis 26 , Pseudomonas putida KT2440 34 , Idiomarina sp. PR58-8 35 . UV-Vis spectrophotometric scanning of B-SeNPs exhibited peak at 266 nm. The absorption maxima of extract at 280 nm may be due to the presence of proteins in the extract and at 614 nm is due to the presence of phycobilin pigments. Sky blue color of extract and absorption maxima of extract at 614 nm confirms the presence of phycocyanin in the extract 36 . The peak at 614 nm from the extract completely disappeared after the synthesis of B-SeNPs which may be due to their involvement and utilization in the capping of synthesized nanoparticles. When nanoparticle size is smaller than its Bohr excitation radius, the band gap gets enlarged due to the quantum confinement effect and this also influences nanomaterial band gap due to capping biomolecules and the proteins present on the surface. Other organisms have showed slightly different λ max for SeNPs probably due to variation in their sizes e.g. at 330 nm in Zooglea ramera 14 , at 330 nm in Gliocladium roseum 15 , at 261 nm in Bacillus megaterium 17 , at 245 nm in Aspergillus terrus 19 , at 261 nm in Diospyros montana 21 , at 260 nm in Allium sativum 22 , at 381 nm in Psidium guajava 25 , at 271 nm in Emblica officinalis 26 and at 395 nm in Citrus limon 27 .
During optimization, it was observed that the best synthesis occurred when extract and salt where mixed in a ratio of 2:1 for 24 h at 30 °C. B-SeNPs derived from other organisms have shown maximum synthesis at 30 °C e.g. bacteria-Zooglea ramigera 14 , fungi-Gliocladium roseum 15  GC-MS analysis of extract was done to know the probable biomolecules present in the cyanobacterial extract that may have helped in the synthesis of B-SeNPs. Two major peaks of fatty acid esters were observed i.e. Peak 1 with area 38.46% (Hexadecanoic acid, methyl ester) and Peak 2 with area 49.64% (Methyl stearate). These compounds have been also recorded in the GC-MS profile of Streptomyces sp. 37 and Mentha spicata 38 respectively. They have also suggested their reducing nature. Two more fatty acid esters, viz. ϒ-Linolenic acid, methyl ester and 9, 12-Octadecadienoic acid, methyl ester were found in lesser quantity (1.09% and 1.41% respectively). These fatty acid esters with reducing potential have been also found in Allium saralicum 39 and Citrus wax 40 . Three esters (Heptadecanoic acid, methyl ester; 9-octadecenoic acid (Z)-, methyl ester; Oxalic acid, 6-ethyloct-3-YL heptyl ester) were also found in cyanobacterial extract. Heptadecanoic acid, methyl ester from Mentha spicata 31 , 9-octadecenoic acid (Z)-, methyl ester from Thesium humile Vahl 41 and Oxalic acid, 6-ethyloct-3-YL heptyl ester from Cakile maritima 42 were also reported for their good reducing potential. Apart from the esters, two carboxylic acids i.e. 1, 2-Benzenedicarboxylic acid and 2-(3-acetoxy-4, 4, 10, 13, 14-pentamethyl-) were also observed but in lesser amount. Some researchers have also reported the presence of these two carboxylic acids and reducing nature of cyanobacteria-Calothrix brevisima 43 and angiosperm-Punica species 44 .
Compounds of other classes like alkane (Heptane, 3,3-dimethyl), alkene (Neophytadiene), cycloalkene derivatives (1,3-cyclohexadiene, 5-(1,5-dimethyl-4-hexenyl and 3-methyl-5-(2,6,6-trimethyl-1-cyclohexen-1-YL)-), phenol (Phenol, 3,5-bis(1,1-dimethylethyl) and ether (1,3-Propanediol, decyl ethyl ether) have been also obtained in the present study using Anabaena variabilis NCCU-441 extract. Previous study have also reported some related compounds like Heptane, 2,5,5-trimethyl-, a derivative of Heptane, 3,3-dimethyl from Nostoc muscorum NCCU-442 45 , Neophytadiene from Plectranthus amboinicus 46  The presence of functional groups of alcohols, phenols, carboxylic acid, aromatic compounds, alkanes, alkenes and fatty acids in FTIR results perfectly correlates with the GC-MS results of extract where compounds having similar nature were observed. Ascorbic acid (a carboxylic acid) is a standard chemical reducing agent that has been used in many studies for the chemical and extracellular biogenic synthesis of SeNPs 29 www.nature.com/scientificreports/ structure of ascorbic acid has three functional groups (alcohol, ester and alkene), their presence in the FTIR of B-SeNPs and cell extract suggest that they have played an important role in the synthesis and capping of B-SeNPs in present study also. B-SeNPs were characterized by different techniques showed amorphous nature of SeNPs (XRD) having a size of 12 nm (SEM), 94% weight of selenium in EDX and having a prominent peak at 253 cm −1 in Raman spectrum with 10.8 nm size in TEM. Other biogenic SeNPs synthesized from Spirulina platensis 24 , Bacillus sp. MSh-1 1 and Pseudomonas aeruginosa ATCC 27853 51 as well as chemically synthesized also showed the amorphous XRD pattern 52 . Moreover, peak at 253 cm −1 is a characteristic absorption band for amorphous Se. Spirulina polysachcharides (SPS) based SeNPs have also shown a resonance peak at 253 cm −129 . SeNPs of different shapes and sizes have been also reported from different sources like from dried Vitis vinifera fruit (spherical, 3-18 nm) 24 , Arabic gum (spherical, 34 nm) 53 . Additionally, large sized spherical SeNPs have been also reported from Spirulina polysachcharides (90-550 nm) 29 and Capsicum annum leaf (80 nm) 54 . Aspergillus terreus mediated SeNPs have also shown an average size of 47 nm with signal at 1.37 KeV 19 . Gliocladium roseum formed spherical SeNPs of 20-80 nm size with some larger particles (100-130 nm), that showed EDX signal at 1.4 KeV 15 . SeNPs synthesized from Diospyros montana also showed 94.44% of weight % with signal at 1.5 keV) 21 .
B-SeNPs and C-SeNPs were also tested and compared in the present study. B-SeNPs exhibited higher percentage of scavenging potential of free radicals by DPPH, SOR, APTS and FRAP. Higher percentage suggested the higher anti-oxidant potential. FRAP assay measures the anti-oxidant potential through the reduction of ferric ion (Fe 3+ ) to ferrous ion (Fe 2+) by anti-oxidants.
To the best of our knowledge, there is no report of such comparative analysis. However, few information is available regarding biogenic SeNPs. Diospyros montana mediated SeNPs showed IC 50 value of 0.225 μg/mL in DPPH scavenging assay 21 23 . Cyanobacterial SeNPs gave significantly better activity in all the assays, so they could be considered as the better anti-oxidant than commercially synthesized SeNPs (significant at P ≤ 0.05).
Antimicrobial (anti-bacterial and anti-fungal) study showed that 60 µg/ml B-SeNPs led to the formation of an inhibition zone of 10 mm and 9 mm against S. aureus and E. coli respectively. In case of C-SeNPs, lesser diameter inhibition zone (8.8 and 8.5 respectively) were observed at the same concentration. Angiosperm-Diospyros montana mediated SeNPs showed inhibition zone of 8 mm and 7 mm against S. aureus and E. coli respectively 21 . Psidium guajava (angiosperm) based SeNPs showed even bigger zone of inhibition of (17 mm and 20 mm) against E. coli and S. aureus respectively 25 . Similarly, B-SeNPs showed larger zone of inhibition during anti-fungal activities than C-SeNPs in Candida krucei. But, overall better anti-fungal activity was found in B-SeNPs (significant at P ≤ 0.05). Anti-fungal activity of Diospyros montana mediated SeNPs was studied against Aspergillus niger. The assay was performed using disc diffusion method and the zone of inhibition was recorded as 12 mm. Further, the anti-fungal activity of e-waste mediated SeNPs has also been observed against Candida albicans and Aspergillus niger 55 . They found the minimum inhibitory concentration (MIC) as 6.5 μg/ml and 12.5 μg/ml respectively. It has been also observed that SeNPs synthesized by Bacillus species Msh-1 was active against Aspergillus fumigatus and Candida albicans with MIC values being 100 μg/ml and 70 μg/ml respectively 56 . B-SeNPs were found to be more effective on MCF-7 (IC 50  Biocompatibility (anti-cancerous activity and low toxicity) can be considered as a remarkable property for any therapeutic agent directed towards the use of the human host. The results demonstrating the significant difference between the IC 50 values of cancer cell line and normal cell line (significant at P ≤ 0.05) clearly put forward the fact that biosynthesized SeNPs have selectivity towards the cancerous cells as compared to normal cells 25 .
More potent bioactivities and less toxic nature of B-SeNPs than the C-SeNPs is may be due to the presence of different biomolecules on the surface of biosynthesized nanoparticles which enhanced their biomedical potential. Also, the synthesized B-SeNPs was of amorphous in nature and amorphous nanoparticles have better bioactivities than the crystalline nanoparticles 59 . Thus, the present study suggests that SeNPs synthesized using Anabaena variabilis NCCU-441 can be used as a therapeutic agent which are eco-friendly, cheap and non-toxic.

Materials and methods
Chemicals used. All cyanobacterial media chemicals (highly analytical grade) were procured from Merck except sodium selenite (Na 2 SeO 3 ), Luria broth, Luria agar and yeast extract peptone dextrose (YPD) which was purchased from Himedia. Milli Q water was used for media preparation. Commercially synthesized SeNPs (average size of 50 nm) with ultra-high purity (99.9%) were procured from Nano Research Elements (CAS No 7782-49-2). www.nature.com/scientificreports/ Extract preparation and its characterization. Lyophilized biomass powder was used to prepare 1 L aqueous extract by keeping it in water bath for 10 min at 60 °C. It was followed by centrifugation at 6000 rpm for 15 min at 4 °C and filtration by Whatman filter No.42. The chemical profile of cyanobacterial extract was analysed using GC-MS to find out the probable compounds that have reducing potential and aided the synthesis of nanoparticle. Samples for GC-MS analysis were prepared by dissolving the dried extract in methanol. The GC-MS analysis was done by Shimadzu GC-MS QP 2010 Plus equipment in electron ionization (EI) mode fitted with a RTX-5 capillary column (60 m × 0.25 mm × 0.25 μm). Helium was used as carrier gas with 0.7 ml min -1 of flow rate. The temperature of the injector was fixed at 260 °C. The initial and final temperature of the column was 80 °C and to 280 °C at the rate of 10 °C min -1 and 15 °C min -1 respectively. A 3.5 min solvent delay was used. Mass spectra were recorded under scan mode in the range of 40-650 m/z. Compounds were identified by comparing with NIST11/ WILEY library.

Culture collection and maintenance. Anabaena variabilis
Biogenic SeNPs synthesis and optimization. Color change of the reaction mixture was observed for the synthesis. Their UV-Vis spectra was scanned in between 200 to 700 nm. Sodium selenite and cell extract concentration ratio, reaction time and temperature were optimized for SeNPs synthesis. Tested ratio of sodium selenite (1 mM) and cell free extract (4 g/L) were 1:1, 1:2, 1:3 and 1:4. The best ratio of sodium selenite and cell free extract (1:2) of the reaction mixtures was then kept constant for determining effects of temperature (30 °C, 32 °C, 35 °C, 40 °C). After temperature optimization, reaction time was optimized. For this, the reaction was allowed for 6, 12, 18 and 24 h at optimized temperature (30 °C) and ratio (1:2). In all above conditions, reaction was allowed in the presence of fluorescent light with 2000 ± 200 lx intensity.
Purification of B-SeNPs. Synthesized B-SeNPs were purified by centrifugation at 12,000 rpm for 20 min and repeated washing by double distilled water. The supernatant containing unreduced salt and unused capping agents were discarded while the pellet containing B-SeNPs was re-suspended in double distilled water and washed thrice. Finally, pellet was lyophilized at − 40 °C for physico-chemical and biological characterization.

Physico-chemical characterization of B-SeNPs.
FTIR was performed to determine the functional groups present on the surface of SeNPs. Powdered SeNPs were used and spectra were scanned between the range 600-3500 cm −1 by using TENSOR 37 FTIR Spectrometer. XRD was carried out to observe the diffraction pattern of synthesized SeNPs. For that, Lyophilized SeNPs were placed on glass slide and the diffraction pattern was studied by using Ultima IV X-ray diffractometer (Rigaku). The Raman spectroscopy was done at room temperature via Raman microscope (LabRAMHR800, HR800, JY). Freeze dried SeNPs were placed on a thin film and spectra were recorded in between the spectral range of 100-500 cm −1 with an interval of 10 s. SEM-EDX was performed to determine the shape, size, elemental composition and purity of synthesized nanoparticles. For that, SeNPs were dispersed in water and sonicated in Ultrasonicator (Thermotech PID-41 S). A drop of the sample was placed on carbon coated copper grid followed by coating of gold. The micrographs were captured on SEM (Nova NanoSEM 450), at accelerating voltage of 5 keV. The purity and elemental composition of synthesized SeNPs were obtained by EDX operated at 20 keV. The average size and range of synthesized nanoparticles was determined by performing TEM. SeNPs dispersed in water was placed on carbon coated grid and analyzed by using HRTEM-FEI 300 VHRTEM TECHNAI G2 30S TWIN. The size was calculated by using Image J software.
Biological characterization. All the biological activities (anti-oxidant, anti-microbial, anti-cancer and biocompatibility) of B-SeNPs and C-SeNPs were determined for the comparative study.
Determination of anti-oxidant activities of SeNPs by DPPH assay. 2,2-diphenyl-1-picrylhydrazyl scavenging activity of B-SeNPs and C-SeNPs was determined by modified method of Brand-Williams 60 . 80% ethanol was used to prepare 0.1 mM DPPH. 3 ml DPPH and 1 ml SeNPs (B-SeNPs or C-SeNPs) having different concentrations (25 µg to 1000 µg) were mixed together and incubated in the dark for 30 min. Then, the absorbance was taken at 517 nm. Mixture of 3 ml DPPH and 1 ml of water was taken as a control and same procedure was followed for the control. % scavenging of SeNPs for DPPH was calculated by using the following formula: where Ac = Absorbance of control, At = Absorbance of test. Free radical scavenging activity of SeNPs was calculated from the percentage scavenging. IC 50 values were calculated and compared with the standard-Ascorbic acid.
Determination of anti-oxidant activities of SeNPs by SOR assay. According to Zhishen et al. (1999), for performing superoxide radical scavenging activity, 1 × 10 −2 M methionine, 3 × 10 −6 M Riboflavin, and 1 × 10 −4 M nitroblue tetrazolium (NBT) (50 μM) were mixed together in 0.05 M of phosphate buffer (pH 7.8) 61 . 300 µl from the different concentrations of SeNPs (B-SeNPs or C-SeNPs) and 3 ml of the above mixture were mixed together and incubated in light for 30 min. One more similar set was kept in dark for the same time as blank. Absorbance was taken at 560 nm. % scavenging was calculated by adopting Eq. (1).

Determination of anti-oxidant activities of SeNPs by FRAP assay.
Ferric reducing anti-oxidant power assay was performed as described by Benzie and strain (1999) 62  www.nature.com/scientificreports/ tate buffer (300 Mm, pH-3.6) in 1:1:10 ratio. In 200 µl of SeNPs (B-SeNPs or C-SeNPs) 1.5 ml of FRAP reagent was added. The mixture was then incubated in dark at room temperature for 30 min. Determination of anti-cancer activity of SeNPs by MTT assay. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to check cytotoxicity of SeNPs (B-SeNPs or C-SeNPs) using the method of Mossman (1983) 66 . 1 × 10 4 mL −1 cell lines (MCF-7 and HepG2) in their exponential growth phase were grown in a 96-well plate, then incubated in a CO 2 incubator at 37 °C for 24 h. Different concentrations of B-SeNPs/C-SeNPs (0, 6.5, 12.5, 25, 50,100, 200, 400 and 800 μg/ml) was then added to the plate in triplicates. After 24 h of incubation, 20 μL MTT reagent was added in each well and incubated, formazan crystals were formed after 4 h of incubation. After adding 150 μL of detergent in each well, plates were read in a microplate reader at 570 nm (BIO-RAD microplate reader-550). Untreated and 24 h treated cell lines by 0, 6.5, 12.5, 25, 50,100, 200, 400 and 800 μg/ml of SeNPs for 24 h were used for cell viability test. Images were captured before and after the treatment. Doxorubicin was used as the standard drug. Percentage viability was calculated using equation: where Ac = Absorbance of control, At = Absorbance of test.
Bio-compatibility assay of SeNPs on HEK-293 cell line. MTT assay was also performed to check the compatibility of B-SeNPs and C-SeNPs towards HEK 293 cells (normal kidney embryonic cell line) 25 .
Statistical analysis. Data were presented as mean ± SD. Statistical analysis was performed by one way ANOVA followed by least significant difference at the level of 95% significance using Graph Pad software 8.1, San Diego, California.

Concluding remarks
The present study highlights the Anabaena variabilis NCCU-441 cell extract mediated synthesis of B-SeNPs, its optimization and its comparative bioactivities with the commercial SeNPs. Orange-red colored, small (10.8 nm), spherical shaped SeNPs were successfully synthesized which were of high purity (94%). B-SeNPs showed better anti-oxidant, anti-microbial and anti-cancer activity than C-SeNPs. Thus, cyanobacteria mediated synthesis can be considered as safe and non-toxic way to synthesize SeNPs, that can be used as a probable drug candidate against cancer, microbial disease etc. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.