Therapeutic potential of electron beam and gamma irradiation synthesized selenium nanoparticles for health care

Selenium nanoparticles (SeNPs) attract more and more interest due to good bioavailability and low toxicity, accompanied by various bioactivities consisting of antimicrobial, antioxidant, and anticancer activity. SeNPs could be generated by physical, chemical, or biological methods and their potential depends on the particle diameter, homogeneity, coating agents, etc Up to now, there has been no previous work reporting on the activities of SeNPs produced by electron beam yet. In our work, SeNPs created by electron beam (SeNP/EB) or gamma irradiation (SeNP/G), stabilized by gum arabic were evaluated for the anticancer capacity by MTT assay, the antioxidant activity by DPPH radical scavenging assay, and the antibacterial ability by agar well diffusion assay. The results showed that SeNP/EB and SeNP/G displayed growth inhibition on HeLa cervical cancer cells with IC50 values of 2.83 and 1.54 μg ml−1, while the values on MCF7 breast cancer cells were 27.70 and 38.80 μg ml−1 respectively. The SeNPs affected HeLa cancer cells more selectively than normal fibroblasts as evidenced by the high selectivity index of 7.98 and 26.25. Notably, the results demonstrated that SeNP/G is much safer than SeNP/EB when applying for cancer treatment in the future. Regarding the DPPH assay, SeNPs of both synthetic methods exhibited potential IC50 values (13.5 and 12 μg ml−1) compared with that of ascorbic acid (8.4 μg ml−1). In comparison to previous studies, our results sugessted that gamma and electron beam irradiation methods, accompanied by coating with gum arabic could be novel approaches in SeNP synthesis to enhance the antioxidant activity of the SeNPs. Besides, SeNPs also caused an inhibition towards Listeria monocytogenes and Escherichia coli, which was verified by the inhibition-zone diameter of approximately 8–12 mm, through inducing oxidative stress in bacterial cells. In conclusion, along with the advantages of physical methods such as time-saving, eco-friendly processes, SeNPs in our work could be a promising candidate for the research and development of healthcare products.


Introduction
Selenium (Se) is an essential trace element necessary for the biochemical metabolism processes of human body, taking part in the constitution of antioxidant enzymes including glutathione peroxidase (GPX), thioredoxin reductase (TXNRD), selenoprotein P (SELENOP), selenoprotein F (SELENOF) [1,2].The addition of selenium to daily diet helps to prevent damages caused by oxidation as well as lipid peroxidation [3].The compounds containing Se are divided into three main groups, consisting of organic, inorganic, and selenium nanoparticles (SeNPs) [4], which could be created by physical, chemical, or biological method [1].Along with the advantages of better bioavailability and lower toxicity when compared with the other Se-containing compounds, SeNPs possess diverse potential applications in food and pharmaceutical fields [5].To be more specific, SeNPs exhibit various bioactivities including antioxidant, antibacterial, anticancer abilities.According to previous reports, the antioxidant activity of SeNPs depends on the particle diameter, the homogeneity, the surface stabilizer, and could be variable owing to the purifying process [6,7].Notably, the increase in particle diameter could lead to a decline in antioxidant capacity in in-vitro assay [8].Therefore, the development of SeNPs aiming to more stability and smaller diameter is a target to take advantages of SeNPs in a variety of applications.
Additionally, the antibacterial activity of SeNPs has also been reported in former studies.Particularly, SeNPs could inhibit the growth of Vibrio parahaemolyticus, Staphylococcus areus, Enterococcus faecalis, Pseudomonas aeruginosa, etc [9][10][11][12].The increase in antibiotic resistance becomes a major challenge for clinical infection treatments, thereby posing a demand for novel antibiotic development.Thanks to the ability to cause oxidative stress and cell-membrane-disruption, SeNPs could inhibit varied bacterial species, in which smaller diameter SeNPs reach, the higher antibacterial ability of SeNPs was recorded [13,14].
Furthermore, selenium is also a candidate for investigating anticancer activity.More particularly, the organic and inorganic compounds containing selenium has been reported on the anticancer ability utilizing different experimental models [15].However, it is unfortunate that these constituents expressed non-targeted toxicity on both cancer and normal cells [16].Surprisingly, the production of selenium as nanoparticles was discovered to be less toxic when killing cancer cells more selectively than normal cells.This difference in response could be a result of the dissimilarity in cellular redox state as well as osmotic pressure between two cell types [17].In regard to properties of nanoparticle, diameter is considered as the most important parameter affecting the absorption of SeNPs [18], the distribution within the human body [18], as well as the anticancer activity [19].More particularly, small-size nanoparticles could evade the reticuloendothelial system and escape immunogenic responses [20,21].In addition, malignant tumors have the increased vascular permeability while the tight junctions of endothelial cells in normal tissue could prevent the invasion of particles greater than 2-4 nm [22].It was also reported that nanoparticles less than 100 nm could more easily penetrate tumor microenvironment together with tumor's core, helping to enhance the anticancer effect [23].In previous studies, the toxicity of SeNPs was investigated on several cancer cell lines including breast, lung, cervical, ovarian cancer cells, etc [4].However, most of these SeNPs were synthesized by chemical or biological methods with an average particle diameter greater than 30 nm, while there are few reports on the anticancer activity of SeNPs created by physical methods.
In our work, SeNPs supplied by Applied Research Institute of Natural Resources, Materials and Environment, Vietnam were created by two different physical methods, using electron beam or gamma irradiation combined with utilizing gum arabic as a stabilizer, which is a novel approach in SeNP synthesis [24].Electron beam irradiation acquire a lower penetration depth than gamma irradiation but this method creates higher energy due to constant emission intensity and high dose rate [25,26].Importantly, electron beam irradiation could be automatically processed thereby giving a uniform in product quality uniform when compared with gamma irradiation using a vicious radioactive source.Up to now, there are a few reports on the bioactivities of SeNPs generated by gamma irradiation [27,28], and there has been no previous work on the activities of SeNPs produced by electron beam yet.In addition, the stabilizing agents could affect the activities of SeNPs thanks to their ability in maintaining particle size and stability.Polysaccharides have preferences including low toxicity and high solubility as result of the presence of hydroxyl groups which could help to elevate the binding capacity of particles towards cell membrane [1].Among polysaccharides, gum arabic is commonly used in food and pharmaceutical industries.Thus, the use of gum arabic as a surface stabilizer for SeNPs could assist in maintaining particle diameter while ensuring non-toxic safety in applications [1].
In an effort to screen bioactivities of SeNP samples produced by electron beam irradiation or gamma irradiation in combination with gum arabic as a stabilizing agent, our report would evaluate the anticancer, antioxidant, and antibacterial activities of different SeNP samples through MTT cytotoxicity, DPPH, and agar well diffusion assays.

Selenium nanoparticles
Selenium nanoparticles (SeNP) supplied by Applied Research Institute of Natural Resources, Materials and Environment (Ho Chi Minh City, Vietnam) were created by electron beam irradiation (SeNP/EB) or gamma irradiation (SeNP/G), stabilized by gum arabic, then preserved as powder by freeze-drying method as reported in previous work [24,28].Thereupon, two experimental samples in our research include (1) SeNP/EB and (2) SeNP/G.
The morphology of the nanoparticles was observed using a transmission electron microscopy (TEM), whereas an x-ray diffraction spectrometer (XRD) with monochromatic Cu Kα radiation at a scanning rate of 2.25°/min was utilized to investigate the structure of the synthetic product, then the analysis was performed by Match!Software.
The samples were diluted in distilled water to gain tested concentrations.The samples were also filtered by sterile 0.22-μm membranes prior to cytotoxicity and agar well diffusion assays.

Bacteria strains
The bacteria strains utilized in this study consist of Escherichia coli (ATCC 25922) and Listeria monocytogenes (ATCC 15313).These species were cultured in Tryptone Soy Broth (TSB) medium (Himedia, India).

Human cell lines
Two human cancer cell lines and one normal fibroblast cell line were investigated in this study consisting of MCF7 breast adenocarcinoma (ATCC HTB-22), HeLa cervical adenocarcinoma (ATCC CRM-CCL-2), and BJ-5ta fibroblast cell line (ATCC CRL-4001).

MTT cytotoxicity assay
Firstly, cells were detached from culture surface by 0.25% trypsin-0.53mM EDTA, centrifuged to obtain cell pellet, then resuspended in fresh medium, and finally identified cell density by Neubauer chamber (Hirschmann, Germany).The amount of 1 × 10 4 cells was transferred into each well of 96-well plate, incubated overnight for cell adhesion.After that, SeNP samples at experimental concentrations ranging from 0.31 to 160 μg ml −1 were supplemented, and the plate was incubated for 48 h before 5 μl of MTT (Sigma alrich, USA) (5 mg ml −1 ) was added into wells.Following a 4-hour incubation, 60 μl of lysis buffer (30% (w/v) SDS, 0.03N HCl) and 90 μl of 99.9% (v/v) DMSO were simultaneously introduced to each well, shaken at 800 rpm for 10 min, then the optical density (OD) was recorded at 550 nm wavelength by microplate reader.In parallel, non-treated negative control and blank samples were also established.The percentage growth inhibition of SeNPs concentrations was calculated by the formula: The experiment was performed in at least three replicates.The correlation between SeNP concentrations and inhibition percentages was established by Graphpad Prism, from which IC 50 (the concentration causing 50% growth inhibition) was interpolated.In addition, selectivity index (SI) of SeNP sample towards a cancer cell line was also calculated by the ratio of IC 50 on normal fibroblasts to IC 50 on cancer cell line.

DPPH radical scavenging assay
The antioxidant activity was evaluated by DPPH assay using 96-well plate.The DPPH powder (Sigma alrich, USA) was dissolved in ethanol 99.9% (v/v), while L-ascorbic acid (Sigma alrich, USA) and SeNP samples were dissolved in distilled water, then being preserved at 4 °C.The experimental SeNP concentrations were 1.25, 2.5, 5, 10, 15, 20, and 25 μg ml -1 .The final concentration of DPPH in each well of 96-well plate was 350 μM.Paralelly, negative control sample contained only DPPH and distilled water.Besides, the blank samples were also established.The reaction was conducted in light-preventive condition, incubated at 30 °C, and shaken at 800 rpm for 60 min before being measured at 492 nm wavelength.The inhibition percentage was calculated as follows: The whole assay was independently repeated in triplicate.After that, the IC 50 value, the concentration that 50% of DPPH• was reduced into DPPH-H, was identified by GraphPad Prism based on the linear-regression line exhibiting the correlation between I% and sample concentrations.

Agar well diffusion assay
The bacteria species were cultured in TSB medium overnight at 37 °C.The day after, the broth was subcultured into fresh medium (ratio 1:20 (v/v)) and incubated at 37 °C within 2-3 h until OD 600nm value gained 0.8-1.
Next, bacterial broth was diluted to the OD value of 0.1 in 0.5% (w/v) agar TSB, then being poured over a 2% (w/v) agar TSB layer in petri plates.Next, 7-mm wells were created on the upper agar layer, and 50 μl of experimental samples at concentrations of 50, 100, 200, 400, 800 μg ml −1 was transferred into wells.The positive control sample was ampicillin at the concentration of 500 μg ml −1 , while the negative control one was distilled water.The plates were incubated at 37 °C within 24 h, and then inhibition zone diameters were measured.

DCFH-DA assay
The ROS generation inside bacterial cells was assessed by the fluorescence probe, dichlorofluorescein diacetate (H2DCFDA).The assay was conducted following the protocol of Siva et al (2019) with modification [29].Firstly, 3 mL of inoculum at density of 1 ×10 6 CFU/mL was incubated with SeNP concentrations for 6 h at 37 °C.Subsequently, the cells were centrifuged at 6000 rpm for 5 min to gain cell pellet which was washed with PBS prior to being treated with H2DCFDA for 30 min in the dark.After being centrifuged, the biomass was resuspended in 50 μl PBS, and then 2 μl of 100-fold dilution was placed on a glass slide with a coverslip and visualized under a fluorescence microscope (Nikon Eclipse TiU).

Data analysis
Graphs and statistical analysis were performed by Graphpad Prism software.In this study, data were presented as mean ± SD.The mean differences were examined by unpaired Student's t-test (two-tailed), in which p-value 0.05 pointed out a statistically significant difference.

The properties of SeNPs synthesized by different physical methods
In the current study, selenium nanoparticles synthesized by electron beam irradiation (SeNP/EB) or gamma irradiation (SeNP/G) were subjected to the investigation.As shown in figure 1(a), SeNP samples consisting of powder and solution exhibit a characteristic orange-red colour of selenium nanoparticles (figure 1(a)).The observation of samples by TEM microscope revealed the spherical particle diameter was approximately 20-30 nm towards SeNPs created by electron beam irradiation, while that was relatively 60 nm for gamma irradiation samples (table 1, figure 1(b)).Besides, the crystal structure of the SeNPs was assessed by XRD analysis.The XRD patterns of two types of SeNPs were displayed in figure 1(c) showing the diffraction peaks corresponding to crystal planes of the trigonal phase of selenium.Particularly, there was the presence of (110) and (103) planes in the XRD pattern of SeNP/EB while (101) and (110) planes were observed in the pattern of SeNP/G [30].However, the broadness and low intensity of the peaks indicate that synthesized nano-selenium particles are poor in crystallinity [31][32][33].

Anticancer activity of SeNPs
The cytotoxicity of SeNPs against HeLa cervical and MCF7 breast cancer cell line was determined by MTT assay.
The inhibitory capacity of these samples expressed more impressively on HeLa than MCF7 cells.Particularly, for HeLa cells, the IC 50 values were 2.83 and 1.54 μg/ml towards SeNP/EB and SeNP/G samples, while the 50% growth inhibition on MCF7 cells only gained at concentrations of 27.70 and 38.80 μg/ml respectively (table 2).However, the cell inhibition between these two SeNP samples on a specific cell line showed no statistically significant difference (figure 2).
In comparison with previous reports on HeLa cell line (table 3), the anticancer capacity of SeNPs in this study expressed higher, equivalent to lower IC 50 values that might be due to the effect of the combination between small size and gum arabic as stabilizer towards our SeNPs.In relation with other reports, Zhenzhen et al (2022) generated SeNPs coated by dextran with diameter of 62.3 nm which is quite similar to that of our SeNP/G (~60 nm), but the IC 50 value of these SeNPs gained at higher level (279 μg ml −1 ), proving the stabilizers also play an important role in the anticancer activity of SeNPs on this cell line.In contrast, the tendency of IC 50 values of SeNPs on MCF7 cell line is irrelevant to particle diameters (table 4), while similar to the activity on HeLa cells,  the stabilizing agents might contribute to the cytotoxicity of SeNPs.More particularly, as summarized in table 4, folic acid seemed to be an efficient coater to enhance the SeNP's effect on breast cancer cells.In addition, as described in tables 3 and 4, it might be that the cytotoxic mechanism of SeNPs is different between cancer types because the activity of SeNPs is probably more dependent on particle diameter on HeLa than MCF7 cells with the size range from 20 to 200 nm.Interestingly, whilst the effect of SeNPs displayed no discrimination between MCF7 breast cancer and BJ-5ta normal cells owing to the fact that SI values fluctuated at approximately 1.0, the selective inhibition was obviously expressed on HeLa cells with SI values of 7.98 and 26.25 (table 2).Strikingly, although two SeNP samples with different diameters exhibited no difference on cancer cell lines, the SeNP/G with larger diameter displayed weaker activity (IC 50 40.42μg ml −1 ) than SeNP/EB (IC 50 22.57μg ml −1 ) on BJ-5ta normal fibroblasts (figure 2), which could be predicted that the SeNP/G is much safer than when applying for cancer treatment in future.
The analysis of HeLa and MCF-7 cells' morphology showed distinct alterations of SeNP-treated samples when compared with non-treated samples (figures 3, 4), which is in line with MTT results.More particularly, for HeLa cells, at the concentration 5 μg ml -1 of two SeNP samples, the cell density decreased and the cell appearance became abnormal with the shrunk shape and the accumulation of numerous intracellular vacuoles (figure 3).In relation to a former work, similar vacuolization phenomenon was also observed on HeLa cells treated with SeNPs, which was proved to be the cell endocytosis towards SeNPs [48].Besides, a decline in cell number was also observed on MCF7 cells at the SeNP concentration of 40 μg ml −1 and a loss of membrane integrity was recognized, implying necrotic cell deaths (figure 4).The difference in cell morphology in response to SeNP samples also suggested different inhibitory mechanism on distinctive cancer cell lines.

Antioxidant activity of SeNPs
The antioxidant activity of SeNP samples was investigated by DPPH radical scavenging assay.The activity rose following the increase in SeNP concentrations (figure 5) from 1.25 to 25 μg ml -1 .The SeNP/EB and SeNP/G samples exhibited potential IC 50 values of 13.5 and 12.0 μg ml -1 , which is 1.6 and 1.4 times higher than ascorbic acid (8.4 μg ml -1 ) (table 5).The SeNP/EB sample synthesized by electron beam accelerator showed no statistically significant difference when compared with SeNP/G sample created by gamma irradiation (figure 6).According to previous works, our SeNPs expressed the lowest IC 50 values; especially, IC 50 values of SeNPs in those reports were 2-to 150-fold higher than ascorbic acid (table 6).Therefore, independent of particle diameter, the antioxidant difference could be related to particle structure when synthesized by irradiation and coated by gum arabic which is a novel approach in SeNP synthesis.

Antibacterial capacity of SeNPs
The antibacterial capacity of SeNP samples was evaluated by agar well-diffusion assay utilizing E. coli and L. monocytogenes.As shown in figure 7, relating to E. coli species, SeNP/EB caused an inhibition zone from the concentration of 400 μg ml -1 while SeNP/G sample began to affect at the concentration of 200 μg ml -1 .On the other hand, regarding L. monocytogenes, the 200 μg ml -1 and above concentrations of both samples constrained the growth of this species.The diameter of inhibition zones also proved that the antibacterial activity of SeNP samples was in a dose-dependent manner (tables 7, 8).

Investigation of ROS level inside bacterial cells
To investigate the antibacterial mechanism of SeNPs, we conducted DCFH-DA assay to examine whether oxidative stress was triggered inside E. coli and L. monocytogenes contributing to bacterial growth inhibition caused by SeNPs.H2DCFDA when diffused into the cell would be deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ROS into highly fluorescent 2',7'-dichlorofluorescein (DCF).
As shown in figure 8, the results revealed that SeNP/EB-and SeNP/G-treated samples exposed increased signals of ROS which were similar to the positive sample treated with H 2 O 2 , an oxidative stress inducer [53].However, while ROS levels increased in almost all bacterial cells treated with other SeNP samples, for E. coli treated with SeNP/G, the positive signals only appeared in a small proportion of the population, thus it is possible that there is another mechanism that SeNPs created by gamma irradiation affected the bacteria apart from causing cellular oxidative stress.Interestingly, the morphological change was observed in some E. coli cells with elongated features along with brighter signals, proving an intense stress level in these cells.In conclusion, the above results suggested that the antibacterial property of SeNPs was contributed by inducing the increase in ROS levels in both E. coli gram-negative and L. monocytogenes gram-positive species.

Discussion
SeNPs are usually syntheszied by biological and chemical methods, while there have been only a few reports on physical methods.Biological methods create SeNPs by using organisms consisting of bacteria, yeast, fungi, and plants, while chemical methods utilize reducing agents to reduce selenite to selenium nanoparticles [54].Compared with those methods, physical method-irradiation has advantages including the purity of obtained SeNP samples and the capability of producing large volumes at a competitive price level.Concerning physical methods, it is reported that the high energy ball milling technique could create SeNPs with sizes of 37-85 nm [55], while the pulsed laser ablation synthesized particles with diameter of 60-100 nm [56,57].Therefore, the synthesis of SeNPs by electron beam irradiation could be an efficient approach to lower the particle size, aiming to enhance the strength of bioactivities as previously reported [13,14,19].Besides, the diameter of SeNPs in our research is also comparable to the diameter range of SeNPs (10-300 nm) synthesized by chemical or biological methods [54].
For anticancer effect, SeNP/EB and SeNP/G samples inhibited HeLa cells with IC 50 values of 2.83 and 1.54 μg/ml, while these values were 27.70 and 38.80 μg/ml respectively for MCF7 cells.According to Indrayanto et al (2021), IC 50 less than 100 μM, equivalent to 7.9 μg/ml of SeNPs, implies strong anticancer potential of a pure compound [58]; thus, our results indicated high cytotoxicity of SeNPs synthesized by physical methods (electron beam and gamma irradiation) against HeLa cervical and MCF7 breast cancer cells.In addition, selective growth inhibition of SeNP/EB and SeNP/G on HeLa cells when compared with normal fibroblasts was also recorded as evidenced by SI values of 7.98 and 26.25.A promising anticancer agent should express a high toxicity on cancer cells without causing harm to normal cells in initial screening tests, due to the fact that the current chemotherapeutic drugs sometimes cause serious side effects on patients [59,60].Thus, SI values greater than 2 in cytotoxicity assay indicates a specific inhibitory capacity towards cancer cells [61].In terms of the selective toxicity of SeNPs on maglinant cells, the difference in response between cancer and normal cells is due to the redox imbalance in malignant cells as demonstrated by a high ROS level recorded [17].When being internalized into cancerous cells, SeNPs play pro-oxidant role [62], triggering the formation of free radicals, causing the cellular oxidative stress [40,63,64], the disruption of mitochondrial membranes, and the induction of mitochondrial-dependent apoptosis [17,[65][66][67].In comparison with former studies, SeNPs created by biological or chemical method inhibited HeLa cells at higher IC 50 (4-30 μg ml −1 ) despite similar particle diameters [34][35][36].The cytotoxic difference between our particles and other SeNPs might be related to the particle size in a combination with the effect of stabilizing agent.Actually, stabilizers could also contribute to the anticancer activity of SeNPs, helping to lower the IC 50 value when compared with non-coated SeNPs (IC 50 11.1 versus 29.4 μg ml -1 on HeLa cells respectively) [68].However, on MCF7 breast cancer cell line, independent of particle sizes, the growth inhibition might be dependent on synthetic method and particle design with IC 50 values spanning in a broad range (4-160 μg ml −1 ) (table 4); thus, our SeNPs with diameter of 20-60 nm gaining IC 50 values of 27.7 and 38.8 μg ml −1 are comparable to other SeNPs.To sum up, in the current study, SeNP samples created by electron beam irradiation and gamma irradiation, coated by gum arabic expressed cytotoxicity towards both MCF7 breast and HeLa cervical cancer cell lines; however, the particles showed more impressively selective killing-effect on HeLa cervical cells.
Regarding the antioxidant activity, our results demonstrated that SeNPs created by electron beam irradiation expressed the ability similar to those generated by gamma irradiation.Interestingly, the IC 50 values of SeNP samples in our study (12-13.5 μg ml −1 ) were much lower than SeNPs in preceding studies (IC 50 22.5-600 μg ml -1 ) synthesized by other methods with particle sizes ranging from 4 to 150 nm (table 6).Therefore, it seems to be that independent of the particle size, the antioxidant ability of SeNP samples might be related to the synthetic method.For example, Wane Chen et al (2015) reported that SeNPs synthesized by chemical method and coated by chitosan as a stabilizing agent showed the IC 50 values of 23-31 μg ml −1 in DPPH assay [6], implying stabilizing agent could also contribute to antioxidant activity of SeNP samples.Therefore, in the current study, the synthetic method using electron beam or gamma irradiation accompanied by the use of gum arabic could be a promising approach to improve antioxidant activity of SeNPs.
Relating to the antibacterial capacity of SeNPs, there was no significant difference in antibacterial activity of nanoparticles created by the two physical methods.As previously depicted, phytosynthesized SeNPs (diameter of 23.2 nm) coated by chitosan (20 μg ml −1 ) could inhibit L. monocytogenes with zone diameter of 17.2 mm in diffusion test [69], while bio-SeNPs (particle diameter of 50-200 nm) synthesized by Moringa oleifera extract at  the concentration of 700 μg ml −1 displayed an inhibitory diameter of 14.7 mm [70].However, for E. coli species, while some works reported on E. coli inhibition at SeNP concentrations of 100 μg ml −1 and 950 μg ml −1 with diameter of 9 mm and 19 mm respectively [71,72], the others described the resistance of E. coli treated with these nanoparticles [9].In brief, in agreement with previous studies, the antibacterial activity of our SeNPs was shown against E. coli and L. monocytogenes in agar well-diffusion assay; however when compared with those reports, this activity was expressed lower (d < 12 mm).
The difference between gram-positive and gram-negative species is the structure of cell wall and outer membrane which would affect the surface charge of bacteria.Accordingly, the antibacterial properties of SeNPs could depend on the electrical charge at the surface of SeNPs (zeta potential).In regard to gram-negative bacteria, the outer membrane is covered with phospholipids and lipopolysaccharides creating a strongly negative charge on bacterial surface, thus a negative zeta potential of nanoparticles could be a barrier for them to attach the membranes of these bacteria.Geoffrion et al (2020) produced SeNPs with positive zeta potential (+66 ± 3 mV) exhibiting the strong inhibition on MRSA (methicillin-resistant Staphylococcus aureus), MDR-Escherichia coli, and Pseudomonas aeruginosa which are gram-negative species [65].In this study, SeNPs exhibited quite similar inhibition zones on both gram-positive Listeria monocytogenes and gram-negative Escherichia coli since our SeNPs possess a slightly negative zeta potential (approximately −15 to −20 mV) making the particles approach easily not only positive-but also negative-gram bacteria.However, to examine this hypothesis, more gram-negative and -positive species should be utilized to investigate in next studies.The antibacterial mechanisms of selenium nanoparticles follow various pathways.Particularly, arabinogalactane-capped SeNPs could disrupt the integrity of the cell membrane and cause a cellular respiratory reduction due to the inhibition of dehydrogenase enzyme in phytopathogen Clavibacter michiganensis sepedonicus (Cms) [73].In another report, chemically-synthesized SeNPs stabilized by polyvinyl alcohol could inhibit methicillin-sensitive and methicillin-resistant Staphylococcus aureus through triggering a significant decrease in adenosine triphosphate (ATP) level, promoting ROS production inside bacterial cells, and causing a rupture of cell membranes [74].Similarly, antibacterial mechanism of SeNPs synthesized by Providencia sp.DCX was inducing the increase in ROS intensity and the change in membrane permeability towards various gram-positive and gram-negative bacteria investigated [75].In addition, interestingly, SeNPs biosynthesized by Staphylococcus aureus exposed photocatalytic activity, which ROS generated at SeNPs interface could produce a leakage of cell membrane leading to the death of Escherichia coli [76].Although there has been no report on how SeNPs penetrate the cells, it is likely that bacterial cell wall breaks or membrane ruptures by the effect of SeNPs are the entry gates for the movement of the particles into cells [77], or that SeNPs only interacted with the outer surface of bacteria but inducing intracellular stress.In the present study, the data of DCFH-DA assay indicated that the growth-inhibitory ability of our SeNPs was due to the induction of oxidative stress inside the cells of both gram-positive L.mocytogenes and gram-negative E. coli species which is in line with the antibacterial mechanism of SeNPs in previous reports.Additionally, in terms of SeNPs created by gamm irradiation, besides causing oxdiative stress, it is suggested that other antibacterial mechanisms might contribute to the activity on E. coli due to the fact that increased ROS signals only appeared in a small population.The response difference of SeNP/G on E. coli might be resulted from the particle size and the strutures of outer membrane or cell wall of gram-negative species.In brief, SeNPs created by gamma and electron beam irradiation own antibacterial activity which is related to the induction of oxidative stress inside bacterial cells.

Conclusion
Taking advantages of physical methods consisting of time-saving, the purity of obtained SeNPs, and importantly the large production at competitive price, SeNPs created in our work were evaluated for antioxidant, antibacterial, and anticancer activities.The IC 50 values in the DPPH assay of SeNPs produced by electron beam and gamma irradiation reached 13.5 and 12.0 μg ml -1 respectively, while that of ascorbic acid was 8.4 μg ml-l.In addition, SeNP samples could inhibit the growth of Listeria monocytogenes and Escherichia coli; however, this activity was not impressive since the diameter of inhibition zone was less than 12 mm at the highest concentration of 800 μg ml −1 .Interestingly, our SeNPs generated by electron beam and gamma irradiation caused highly selective toxicity towards HeLa cervical cancer cells with low IC 50 values of 2.83 and 1.54 μg ml −1 , and high SI values of 7.98 and 26.25 respectively.In conclusion, SeNPs could be a candidate for the development of antioxidant healthcare products; additionally, anticancer capacity could also be investigated on other cancer cell lines accompanied by the exploration of underlying mechanisms.

Figure 1 .
Figure 1.The properties of SeNP samples.(a) The colour of SeNPs in form of freeze-drying powder or solution (400 μg ml −1 ).(b) The images of SeNPs captured by transmission electron microscopy (TEM).(c) The XRD patterns of SeNP samples.

Figure 2 .
Figure 2. The comparison of IC 50 values between SeNP samples on HeLa and MCF-7 cells.Data represent as mean ± SD of at least three replicates.The statistical difference was analyzed by two-tailed Student's t-test, ** p 0.01, ns, p > 0.05.

Figure 3 .
Figure 3.The effect of SeNP samples on HeLa cell morphology at different concentrations after 48-hour incubation.Negative control (Neg.Ctrl) sample was treated with distilled water without SeNPs.The images were captured at the magnification of 400X.

Figure 4 .
Figure 4.The effect of SeNP samples on MCF7 cell morphology at different concentrations after 48-hour incubation.Negative control (Neg.Ctrl) sample was treated with distilled water without SeNPs.The images were captured at the magnification of 400X.

Figure 5 .
Figure 5.The antioxidant activity of SeNP samples by DPPH radical scavenging assay.Data represent mean ± SD of triplicate.

Table 1 .
The particle diameter of different SeNP samples.

Table 2 .
The IC 50 values and selectivity index (SI) of SeNP samples.

Table 3 .
The comparison with previous reports on the activity of SeNPs against HeLa cervial cancer cell line.

Table 4 .
The comparison with previous reports on the activity of SeNPs against MCF7 breast cancer cell line.

Table 5 .
The IC 50 values of SeNP samples in DPPH radical scavenging assay.

Table 6 .
The comparison with previous reports on the activity of SeNPs in DPPH assay.

Table 7 .
The diameter of inhibition zone (mm) on E. coli caused by SeNP samples in agar well diffusion assay.

Table 8 .
The diameter of inhibition zone (mm) on L. monocytogenes caused by SeNP samples in agar well diffusion assay.