Green synthesis of selenium nanoparticles using Bacillus sp. strain STG-83: optimization, characterization, and prospects for cancer radiosensitization

Abstract

Selenium nanoparticles (SeNPs) have gained increasing attention due to their favorable biological properties and potential applications in cancer research. In this study, the ability of Bacillus sp. strain STG-83 to biosynthesize SeNPs was systematically investigated. This study comprehensively investigated how Bacillus sp. strain STG-83 can biosynthesize SeNPs. Response surface methodology (RSM) was utilized to optimize the bioreduction of selenite and to identify the key process parameters that play a significant role. The developed quadratic model showed a strong correlation with experimental data (R²=0.92). Statistical analysis demonstrated that time and selenium concentration significantly affected selenite reduction efficiency (P < 0.05), whereas bacterial inoculum percentage were not significant (P > 0.05). Increasing selenium concentration from 0.5 to 25 mM reduced the bioreduction efficiency from 100% to 29.37%, while extending time from 8 to 96 h increased efficiency from 42.03% to 61.51%. The biosynthesized SeNPs were characterized using UV–Vis’s spectroscopy, FTIR, EDX, SEM, TEM, and XRD analyses. The nanoparticles were predominantly spherical, with sizes ranging from 80 to 140 nm, and were coated with a bioorganic surface layer. Biological evaluation revealed that SeNPs induced dose-dependent cytotoxicity in U-87 line while exerting lower toxicity toward normal fibroblast cells. Flow cytometry analysis further demonstrated a significant increase in intracellular reactive oxygen species (ROS) levels following SeNP exposure, suggesting that oxidative stress plays a central role in the observed anticancer effects. The ROS generation triggered by SeNPs suggests they might serve as effective radiosensitizing agents. Future studies that combine radiation and in vivo approaches should confirm this potential.

Introduction

Nanotechnology is recognized as one of the most promising across various fields, including medicine, agriculture, and environmental sciences. In medicine, it enables precise targeting of tumor tissues through techniques such as targeted drug delivery, increased radiosensitivity, and molecular diagnosis of cancer. this approach helps minimize damage to healthy tissues while effectively treating cancer^1–3. Biological methods for synthesizing nanomaterials are important and emerging technologies. They aim to develop cost-effective and environmentally friendly procedures that adhere to the principles of green technology. Biological synthesis has emerged as a preferable alternative for medical applications due to issues with the chemical and physical production of nanoparticles, particularly the use of toxic reagents in various synthesis protocols^4–6. Biological synthesis utilizes plants, fungi, and microorganisms to produce nanoparticles, offering a more environmentally friendly alternative to chemical methods. Bacteria are excellent biocatalysts for synthesizing nanoparticles due to their easy cultivation and genetic modification. Selenium is utilized in solar cells, semiconductor devices, and photographic photometers. It is distinctive for its unique physical properties, including high anisotropy in thermal conductivity, optical conductivity, and X-ray response sensitivity. Also, selenium is an essential trace element found in humans, animals, and some microorganisms. It acts as a crucial cofactor for antioxidant enzymes such as thioredoxin reductase and glutathione peroxidase^7,8.

Among nanomaterials, SeNPs have gained significant attention as potential agents for cancer treatment and drug delivery carriers. Additionally, SeNPs possess unique properties as a form of selenium, exhibiting excellent antioxidant activity⁹. SeNPs can be generated through diverse physical and chemical methods, such as laser ablation, ultraviolet (UV) exposure, hydrothermal synthesis, catalytic reduction, and chemical decomposition. Despite the efficiency of these conventional techniques, the produced nanoparticles often necessitate additional stabilizing agents to maintain colloidal stability and prevent particle aggregation. In contrast, biologically mediated synthesis naturally coats SeNPs with organic molecules derived from the biological system, which function both as reducing and capping agents. This biologically inherent coating not only minimizes agglomeration but also improves colloidal stability, biocompatibility, and the functional performance of the nanoparticles in biological application^10,11. In a recent report by Long and colleagues, the novel strain Paenibacillus motobuensis LY5201 mediated the extracellular biosynthesis of SeNPs. The authors demonstrated that these biogenically produced nanoparticles exhibit considerable potential for development as a therapeutic strategy against liver cancer¹². Wang et al., investigated the synthesis of SeNPs using Bucillus licheniformis F1 to optimize the parameters¹³. Fahmi and his colleagues successfully synthesized silver and Se nanoparticles using Aspergillus carneus MAK 259 and characterized their antibacterial and antibiofilm properties. FT-IR analysis also confirmed the presence of proteins bound to nanoparticles. They concluded that further research is needed to optimize the synthesis protocol and explore additional medical applications to fully assess the effectiveness of these nanoparticles¹⁴. Also, Previous studies demonstrated that biological SeNPs synthesized by E. coli cultures exhibited a strong cytotoxic effect on A549 cell lines, while showing a relatively lower toxicity in healthy cells⁷. SeNPs synthesized by Bacillus sp. EKT1 also exhibit excellent antioxidant activity, making them valuable for various applications¹⁵.

A significant challenge for oncologists and medical physicists is to enhance the effectiveness of radiation therapy while minimizing damage to surrounding healthy tissues. Despite the progress in radiation therapy techniques, patients show varying responses to treatment. These differences in response are attributed to the intrinsic resistance of tumor cells to radiation therapy. The microenvironment of cancer cells differs significantly from that of healthy cells, which contributes to increased tumor metastasis and recurrence. This difference often leads to tumors developing resistance to various treatment modalities, including radiation therapy. Typically, radiation therapy involves administering a dose of radiation aimed at overcoming the tumor’s resistance. However, this approach can also cause damage to the surrounding healthy tissues^16,17. Cancer treatment using nanoparticles to enhance radiotherapy effectiveness and address tumor radiation resistance has garnered significant research interest. Nanoparticles can enhance the sensitivity of cancer cells to radiation through direct interaction with ionizing radiation^18,19. SeNPs are known for their dual role in cellular mechanisms, namely as antioxidants in healthy cells and as prooxidants in cancer cells. Their interaction with the cell leads to the production of ROS and consequently DNA damage and triggering apoptotic pathways including p53, MAPK/Erk, and PI3K/Akt in cancerous cells, which is what has led to the therapeutic effects of SeNPs. Oxidative stress caused by ROS production can cause various forms of DNA damage, including DNA strand breaks and cross-linking, which is the cellular response to DNA damage, leading to apoptosis, cell cycle arrest, or necroptosis, depending on the degree of cellular damage^9,20,21. In the study conducted by Yang et al., the effectiveness of LNT-SeNPs in killing cells KYSE-150 was thoroughly investigated. The findings demonstrated that LNT-SeNPs have a radiosensitizing effect, significantly inhibiting the growth of KYSE-150 cells by blocking the S phase of the cell cycle, inducing apoptosis, and causing mitochondrial damage in these cells²². According to the study by Mahmood et al. bioconjugated selenium-chitosan nanoparticles (Se-chitosan NPs) caused apoptosis and mitochondrial damage in A549 lung cancer cells by enhancing ROS production²³. Mortazavi et al. assessed the response of A549 cells to a combined regimen of IMRT (intensity-modulated radiation therapy-4 Gy) and Se-Cur-PEG nanoparticles (50 µg/mL) over 48 h. The combined exposure resulted in a pronounced decrease in cellular viability relative to irradiation alone. Furthermore, their findings demonstrated that Se-Cur-PEG nanoparticles substantially increased intracellular ROS levels, suggesting a contributory role in enhancing radiation-mediated cytotoxic effects²⁴.

The biosynthesis of SeNPs using microorganisms is influenced by environmental conditions. Traditional methods of studying this process often control various factors at unspecified constant levels, which does not capture the combined effects of all the involved factors. These techniques are also time- consuming. The limitations of classical methods can be overcome by optimizing all influencing parameters simultaneously using statistical experimental design techniques, such as RSM. RSM encompasses various mathematical and statistical techniques used to define the relationships between the reaction and the independent variables. RSM analyzes the effects of independent variables both individually and in combination^25–27. According, this research aimed to investigate the capability of Bacillus sp. strain STG-83 to biosynthesize SeNPs and to optimize key process parameters using RSM. The study further sought to characterize the physicochemical properties and in vitro biological effects of the biosynthesized SeNPs as a sustainable nanobiotechnological platform for potential biomedical applications. Based on their selective cytotoxicity and ROS-inducing activity observed in this work, and supported by existing literature, these SeNPs may have potential implications for tumor radiosensitization.

Materials and methods

The source of the microorganism and growth condition

Bacillus sp. strain STG-83 had been isolated in 2006 from Neidasht Spring, Iran. The 16 S rDNA sequence of Bacillus sp. strain STG-83 had been deposited in the National Center for Biotechnology Information (NCBI) GenBank with the accession number EF051255. Bacillus sp. strain STG-83 is able to tolerate and reduce a significant concentration of toxic oxyanions of selenite, selenate and tellurite. Phylogenetic analysis based on 16 S rDNA sequencing had indicated that strain STG-83 exhibits high similarity to Bacillus pumilus. Its phylogenetic tree had been drawn which previously reported²⁸. For bioreduction studies, strain STG-83 was cultured in Tryptic Soy Broth (TSB) culture medium plus Na₂SeO₃ under an aerobic condition at 30 °C in a shaking incubator which is suitable for the growth of this strain²⁸. Under aerobic conditions, triplicate runs were performed for all experiments alongside negative controls.

Response surface methodology for modeling and optimization

In this research, RSM method was used to investigate the most effective factors for selenite bioreduction to SeNPs by Bacillus sp. strain STG-83 and their optimization. Three factors, percentage of bacterial inoculation, time, and the selenium concentration in five levels of -α, -1, 0, + 1, and + α were designated according to Table 1. Seventeen experiments were designed using RSM. The bioreduction efficiency R (%) of selenite was considered as a response (Table 1). All experiments were incubated under aerobic conditions at 30 °C on a shaker incubator at 150 rpm using TSB. Finally, after completion of the biosynthesis process, the reaction mixture was passed through a 0.22 μm syringe filter to remove bacterial cells and large debris. The filtrate containing SeNPs was then subjected to repeated centrifugation at 10,000 rpm for 15 min. The resulting pellet was collected and washed three times with distilled water to eliminate residual culture medium components and unbound biomolecules (Fig. 1). The purified SeNPs were subsequently air-dried and used for physicochemical characterization and in vitro biological evaluations²⁹. Also, the residual selenium content of the supernatant was investigated by ICP-Optical Emission Spectroscopy (Perkin Elmer Optima 7300DV).

Table 1.

Variables of the central composite design method and the coded levels.

Variables	Symbol	Surfaces
		-α	-1	0	+ 1	+α
Bacterial inoculation percentage	A	5	6.01	7.5	8.99	10
Time (hours)	B	8	25.84	52	78.16	96
Selenium concentration (mM)	C	0.5	5.47	12.75	20.03	25

Fig. 1.

An aqueous suspension of synthesized selenium nanoparticles by Bacillus sp. strain STG-83.

Characterization of biogenic SeNPs

The bioreduction of selenium ions into SeNPs was examined by the UV–VIS spectrophotometer (PG Instrument T80 + series UV-VIS spectrometer) in the range of wavelength 200 to 600 nm. For characterization of the SeNPs produced by Bacillus sp. strain STG-83, the samples were placed on the surface of carbon-coated TEM copper grids and analyzed with transmission electron microscopy (TEM) using Zeiss EM900 at a voltage of 15kv.

Also, the SeNPs were investigated by scanning electron microscope (SEM) (ZEISS EVO 18 Special Edition) -EDX analysis. For SEM, the samples were coated by gold using an Ion–Coater device (KIC-IA, COXEM) and Energy Dispersive X-ray analysis (EDAX) (0-20 keV) was used for chemical and elemental evaluation. XRD analysis (STOE, Germany) was carried out to investigate the crystalline phase of synthesized SeNPs at 40 keV/30 mA with Tube: Cu (K = 1.5406 A° at a range of 20°–80° at 2θ). To identify functional groups on the surface of SeNPs, the Bruker Tensor 27 FTIR spectrophotometer was used.

MTT assay

The human glioblastoma cell line U-87 MG (IBRC C10154) with cell number C531 was purchased from the human and animal cell bank of the Pasteur Institute of Iran. Cytotoxicity of the biological SeNPs was determined using a MTT assay. Briefly, the cells U-87 MG (1 × 10⁵ cells) were seeded into 96-well plates. Plates were then incubated in a CO₂ incubator for 24 h. Various concentrations of the SeNPs (1, 5, 10, 50, 100, 125, 250, 500 and 1000 µg/ml) were added to each well. Then the plate was kept incubated for 24 h. The cell culture medium was removed and 50 µl of the said MTT solution was added to each well. Then the cells were incubated for 4 h. The MTT solution was removed from each well and 50 µl DMSO was added to each cell culture. The cell culture plate was shaken for 20 min at 500 rpm for 10 min in the dark. The absorbance of all wells was measured by an ELISA reader at 570 nm. The cytotoxicity test was also performed on normal fibroblast cells.

Detection of reactive oxygen species

Intracellular ROS levels were assessed using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA, USA). Briefly, after treatment, cells were washed with phosphate-buffered saline (PBS), centrifuged at 1500 rpm for 5 min, and resuspended in PBS at a final volume of 1 mL. Cells were then incubated with DCFH-DA (2 µL) for 45 min at 37 °C in the dark to allow intracellular deacetylation and oxidation to the fluorescent compound DCF.

Following incubation, cells were washed with PBS to remove excess dye and resuspended in 500 µL PBS. Propidium iodide (PI, 3 µL) was added immediately before acquisition to exclude non-viable cells. Flow cytometric analysis was performed using a BD FACSCalibur system, and data were collected from PI-negative (viable) cells only. Intracellular ROS levels were quantified based on the mean fluorescence intensity (MFI) of DCF in the FL1 channel. Data were analyzed using appropriate gating strategies to eliminate debris and dead cells.

Results and discussion

Biosynthesis of nanoparticles, which uses biological materials such as plant extracts, bacteria, or fungi, is considered environmentally friendly and typically results in biocompatible products with low toxicity. However, these nanoparticles often show lower stability in harsh physical or chemical conditions due to organic capping agents. In terms of scalability, biosynthesis can be limited by slower reaction rates. In contrast, chemical synthesis enables highly scalable production with greater stability, but it commonly involves the use of toxic reagents and generates harmful by-products^30–32.

In this research, RSM was used to evaluate the biological production process of SeNPs by Bacillus sp. strain STG-83 and its optimization. Table 2 represents the experimental design of selenite bioreduction and calculated efficiency of selenite reduction. To establish the mathematical relationship between the independent variables and the response, polynomial regression was conducted, resulting in the optimal model for predicting selenite bioreduction as:

Table 2.

Values of variables and laboratory responses of the central composite design in the response surface methodology.

Experiments	Variable A (bacterial inoculation percentage)	Variable B (Time/h)	Variable C (selenium concentration/mM)	Response R (%)
1	7.5	8	12.75	44.44
2	6.01	25.84	20.03	18.61
3	8.99	25.84	5.47	83.5
4	10	52	12.75	49.73
5	7.5	52	12.75	57.67
6	7.5	52	25	45.23
7	5	52	12.75	49.73
8	8.99	78.16	5.47	90.08
9	7.5	52	12.75	50.33
10	8.99	25.84	20.03	18.61
11	6.01	78.16	5.47	90.34
12	6.01	25.84	5.47	85.12
13	6.01	78.16	20.03	36.51
14	7.5	52	12.75	52.30
15	7.5	96	12.75	65.04
16	7.5	52	0.5	99.21
17	8.99	78.16	20.03	33.33

The R² value of 0.92 shows that the proposed model is well able to predict the experimental values. The statistical importance of the effects of the CCD was evaluated by performing a ANOVA analysis, the outcomes of which are presented in Table 3. The F value of 15.59 and p value of 0.0002 indicate the accuracy of the proposed model. According to Table 3, effect of first-order terms (B: time, C: selenium concentration) and second order C² are statically significant (P-value < 0.05) while effect of A-factor (percentage of bacterial inoculation), AB, AC and BC are negligible (P-value > 0.05). Figures 2, 3 and 4 also show the response surface plot showing the effect of interactions between the three variables on selenite reduction by Bacillus sp. strain STG-83. The results indicated that the efficiency of selenite reduction decreased with the increase of selenium concentration. For example, the efficiency of selenite reduction decreased from 100% to 29.37% with the increase of selenium concentration from 0.5 mM to 25 mM in the central point (Figs. 3, 4 and 5). Our results were consistent with the previous observations, it is indicated that selenite is toxic to bacteria as well as many other organisms^33,34. Similar studies showed a decrease in biomass production with an increase in the initial concentration of selenite^35–38.

Table 3.

Analysis of variance (ANOVA).

Reference	Sum of squares	df	Mean square	F value	p-value
Model	9184.232	7	1312.033	15.59356	0.0002
A-Inoculation	1.870783	1	1.870783	0.022234	0.8848
B-Time	457.6663	1	457.6663	5.439379	0.0446
C-Concentration	8108.052	1	8108.052	96.36447	< 0.0001
AB	0.411598	1	0.411598	0.004892	0.9458
AC	0.2095	1	0.2095	0.00249	0.9613
BC	54.19066	1	54.19066	0.644058	0.4429
C2	561.8312	1	561.8312	6.677383	0.0295

Fig. 2.

Three-dimensional surfaces plot of the interaction of time and primary inoculation of bacteria in different concentrations of selenite salt by Bacillus sp. strain STG-83.

Fig. 3.

Three-dimensional surfaces plot of the interaction of selenium concentration and primary inoculation of bacteria in selenite reduction by Bacillus sp. strain STG-83.

Fig. 4.

Three-dimensional surfaces plot of the interaction of selenium concentration and time in selenite reduction by Bacillus sp. strain STG-83.

Fig. 5.

Diagrams showing how various factors influence the bioreduction of selenite to selenium nanoparticles by Bacillus sp. strain STG-83.

According to the results, time is also directly related to the efficiency of selenite bioreduction. The efficiency of selenite bioreduction increases over time. The efficiency increased from 42.03% to 61.51% with time passing from 8 to 96 h in the central point (Figs. 2, 4 and 5). The amount of selenite bioreduction by the Bacillus sp. strain STG-83 increased concurrently with the growth of this strain over time. In this study, the percentage of bacterial inoculation had little effect on the ability of Bacillus sp. strain STG-83 has a reduction efficiency of sodium selenite salt. Similarly, the results have been observed by others. Bioreduction is in the category of metabolism-dependent process in bacteria. Metabolism-dependent processes are performed by only live microbial cells^38–41.

According to Table 3; Figs. 2, 3 and 5, variable A (percentage of bacterial inoculation) statistically (p-value > 0.05) has the least effect on the reduction of selenite. The percentage of the initial bacterial inoculum frequently exhibits statistically insignificant effects on selenite bioreduction in controlled environments. This is mainly due to the fact that selenite reduction is generally limited by enzymes rather than the number of cells. Once there are enough cells to produce the required reductase enzymes, additional increases in cell concentration do not lead to a proportional rise in the reduction rate. Furthermore, in numerous experimental conditions, the lag phase for bacterial adaptation is either brief or minimal, particularly when utilizing pre-cultured or adapted strains, which lessens the influence of inoculum size on reduction kinetics. Over time, even minimal inoculum levels can result in complete or nearly complete reduction, thereby statistically diminishing the observed differences among treatments^28,42. These observations indicate that strain STG-83 may have an efficient and possibly constitutive metabolic system for selenium reduction that functions effectively, even with low biomass levels. The cells’ ability to adapt quickly and their high catalytic efficiency may allow them to start and maintain nanoparticle biosynthesis regardless of the initial cell density. Similar trends have been seen in other bacterial strains, where the production of nanoparticles is influenced more by environmental factors such as substrate availability and redox potential than by Initial bacterial inoculation. Additionally, research on mutants of Pseudomonas putida indicated that specific genetic modifications could enhance SeNP production without impacting overall biomass. This finding suggests that the metabolic processes related to selenium reduction can function independently of the initial cell density⁴³. These findings improve the feasibility of using strain STG-83 in large-scale biosynthesis processes, which may reduce costs and simplify production protocols.

The results indicated that Bacillus sp. strain STG-83 under optimal conditions, 5.18% bacterial inoculation and a specific concentration of 74.6 h, and 7.94 mM selenium can effectively reduce 74.97% selenite levels. These conditions were tested in triplicate, and the efficiency of selenite reduction was monitored at 77.88% ± 0.63. A comparison between the predicted bioreduction capacity and the experimental results demonstrates that the model possesses sufficient precision. The predicted values were compared with the experimental results, as shown in Fig. 6. The findings revealed that the actual results closely aligned with the predictions derived from the response surface analysis, confirming that RSM can be effectively utilized to optimize process parameters in studies employing statistical design of experiments.

Fig. 6.

Predicted versus actual for bioreduction of selenite to selenium nanoparticles by Bacillus sp. strain STG-83.

In the experiments, the formation of SeNPs over time due to the growth of bacteria and the reduction of selenite to selenium was visually identified by observing the color change of the reaction mixture from yellow to red and it was confirmed by using UV spectrum. This color transition has been widely reported as a qualitative marker of SeNPs formation^6,10. This indicates that the formation of SeNPs is through the enzymes produced by Bacillus sp. strain STG-83 (Fig. 7). The formation of SeNPs was confirmed using a UV spectrophotometer. The UV–visible spectroscopy analysis of the SeNPs revealed the two absorption peaks at 230 nm and 290 nm (Fig. 8). There are numerous researches associated with the formation of SeNPs that show various absorption peaks, indicating the presence of SeNPs. In these studies, the peaks have appeared to be at 290 nm^8,44 and the energetic absorption band has been located at 230 nm⁴⁵. It is indicated that the difference in the UV spectroscopy absorption spectra produced using bacterial reduction because of diversity in atomic structure²⁹. According to Fig. 8, the formation of SeNPs increased with increasing concentrations from 0.5 mM (Run C) to 12.7 mM (Run1) and above this concentration (Run D) it started to decrease¹⁵. The intensity of the peak decreased with respect to time (Run A and Run B), which probably represented the protein consumption during the reduction of selenite to SeNPs⁷.

Fig. 7.

Reduction of selenite to elemental selenium nanoparticles by Bacillus sp. strain STG-83 in TSB medium. TSB medium without selenite (A) control and TSB medium with selenite (B).

Fig. 8.

UV–vis spectra of selenium nanoparticles under different concentrations of selenium and at different times.

The structure and size of biological SeNPs were observed using a TEM and SEM (Figs. 9 and 10). The analysis showed the location of the biosynthesized nanoparticles. The nanoparticles were extracellular and their sizes were in the range of 80 to 140 nm which is acceptable at the nanoscale level. The particle size distribution histogram of the SeNPs showed in Fig. 10C. Previous studies have shown that the size of nanoparticles produced by different bacterial species can vary significantly. These variations are likely due to differences in bacterial species and the specific enzymatic and protein-mediated mechanisms each species uses for nanoparticle formation. Since different bacterial strains have unique biosynthetic pathways and biomolecular machinery, these biological differences can greatly influence the physicochemical properties of SeNPs^46,47. Overall particle size is a key factor influencing tumor penetration, cellular entry, and systemic clearance. SeNPs measuring between 10 and 200 nm can enhance the enhanced permeability and retention (EPR) effect associated with tumor blood vessels. This facilitates the exit and retention of nanoparticles within the tumor microenvironment, thereby improving the effectiveness of passive targeting. Smaller SeNPs, particularly those under 100 nm, offer a higher surface-to-volume ratio, which results in greater drug loading capacity and faster cellular uptake. However, nanoparticles that are excessively small may be rapidly excreted through the renal system^48–50. From a radiobiological perspective, selenium’s higher atomic number leads to increased local dose deposition due to the photoelectric and Auger effects when nanoparticles are positioned near DNA and other radiosensitive targets. While smaller nanoparticles may distribute more uniformly and penetrate deeper into tissues, larger nanoparticles have a greater cross-sectional area per particle for radiation interaction. Therefore, the overall impact on radiosensitivity results from a balance between size-dependent absorption and penetration, along with the increased radiation dose per particle^51,52. Therefore, the size optimization of SeNPs may enhance biological performance and represents an important direction for future work.

Fig. 9.

SEM image of SeNPs produced by Bacillus sp. strain STG-83.

Fig. 10.

Transmission electron microscopy of the cell bacteria (A), SeNP produced by Bacillus sp. strain STG-83 (B) and particle size distribution histogram of the SeNPs (C).

According to Figs. 9 and 10, these SeNPs have a spherical structure, which was consistent with the other studies^15,53. Furthermore, the non-aggregation of these nanoparticles indicates their stability, likely due to a coating that may consist of surface-associated biomacromolecules of microbial origin or components from the bacterial growth medium, which has been noted to stabilize SeNPs in other literature^15,54,55. These findings were supported by EDX analysis of the SeNPs which indicated the presence of selenium (Fig. 11). The EDX analysis of the biosynthesized SeNPs (Fig. 11) confirmed selenium as the elemental component, validating the successful bioreduction of selenite into elemental selenium. In addition to selenium, signals corresponding to Si, Ca, Mg, and Al were also detected. The presence of these elements can be reasonably attributed to residual components originating from the bacterial biomolecules, or the sample preparation process, rather than incorporation into the crystalline selenium core. Similar elemental profiles have been reported in previous studies on biogenic nanoparticles, where trace elements detected by EDX were associated with surface-bound proteins, polysaccharides, salts, or adsorption of medium-derived ions during biosynthesis and purification steps. Furthermore, the detection of silicon and aluminum may partially arise from the substrate, carbon-coated grids, or sample holders used during SEM–EDX analysis, which is a well-recognized limitation of surface-sensitive elemental techniques. Therefore, the EDX results collectively support the formation of elemental SeNPs, while the presence of minor metallic elements reflects the biogenic origin and surface complexity of the nanoparticles rather than contamination or compositional inconsistency⁵⁶.

Fig. 11.

Energy-dispersive X-ray spectroscopy of selenium nanoparticles (SeNPs) produced by Bacillus sp. strain STG-83.

The Fourier transform infrared spectroscopy of SeNPs showed an image of the active groups associated with these nanoparticles (Fig. 12). The FTIR spectrum of the SeNPs has several stretching vibrations at 3436, 2923.71, 2854.56, 1633.51, 1383.93,1262.14, 1028.17, 802.26 and 562.04 cm^− 1. The broad band at 3436 cm^− 1 is related to the hydroxyl groups⁵⁷. The two peaks at 2923 and 2854 cm^− 1 correspond to C-H⁵⁴. The peak at 1633.51 and 1383.93 cm^− 1 were assigned to the amide bond^15,58. The vibrational band can be attributed to elemental selenium (Se⁰) in the region near 563 cm^− 1 in the spectrum. The presence of functional groups such as hydroxyl, amine, and lipids on the surface of the SeNPs represents that these SeNPs have a polymer and/or protein coating on their surface which provides steric stability to them, as reported previously. This coating is also a natural capping agent to prevent their accumulation^15,54,58. The other researches indicate that proteins on the surface of biological SeNPs are responsible for controlling their size, facilitating the extracellular production of the nanoparticles, and assisting in their transport outside the cell^59–61. Also, this layer plays a critical role in governing both the physicochemical stability and biological behavior of the nanoparticles. Protein- and polymer-based surface coatings provide steric stabilization, preventing aggregation and enhancing colloidal stability under physiological conditions, which is a prerequisite for efficient cellular uptake. In addition, surface-associated proteins, enzymes, and lipids may facilitate nanoparticle internalization by promoting interactions with cellular membranes or enabling receptor-mediated endocytosis. Such biofunctional coatings have been reported to preferentially enhance uptake of biosynthesized nanoparticles by cancer cells. Beyond uptake, surface capping agents can significantly influence intracellular activity. Bio-coated SeNPs have been shown to exhibit enhanced redox activity, leading to increased intracellular ROS generation and mitochondrial dysfunction. This effect is particularly relevant in cancer cells, which already operate under elevated oxidative stress. Furthermore, the bioorganic surface layer improves biocompatibility and reduces nonspecific cytotoxicity, thereby contributing to therapeutic selectivity^62–64. In the context of radiotherapy, these surface coatings may further amplify radiation-induced biological damage by enhancing ROS accumulation, promoting mitochondrial stress, and potentially interfering with DNA damage response mechanisms. Collectively, the capping agents identified by FTIR analysis represent key determinants of cellular uptake, intracellular fate, and biological efficacy of biosynthesized SeNPs, rather than merely serving as stabilizing agents^63,65.

Fig. 12.

FTIR spectrum of SeNPs produced by Bacillus sp. STG-83.

The crystal structure of the synthesized SeNPs was analyzed by X-ray diffraction analysis. The results (Fig. 13) indicated that the recorded X-ray diffraction peaks at 2θ correspond to the (100), (101), (102), (111), (201), (112) and (202) crystallographic planes of the spherical shape crystals with a hexagonal structure of Se crystals. The diffraction peaks were identified by comparison with the Powder Diffraction File (PDF) database, and were found to be in good agreement with PDF card No. 00-027-060⁶⁶. This result agrees well with the previous literature data^8,29 which was in accordance with the TEM analysis. Thus, these results suggest that the synthesized SeNPs by Bacillus sp. strain STG-83 were well-crystallized. The crystallinity of nanoparticles can significantly affect properties such as dissolution rate, surface reactivity, and interaction with biological systems. Crystallized SeNPs exhibit significant biological activities, including antioxidant, antimicrobial, and anticancer properties. Crystallized SeNPs can enhance antioxidant defense mechanisms, and SeNPs also exhibit significant antimicrobial properties, including the production of ROS, which lead to membrane disruption and leakage of cellular contents. SeNPs can enhance the efficacy of some chemotherapeutic agents by improving drug delivery and targeting. Overall, crystallized SeNPs with diverse biological activities have become promising candidates for therapeutic applications^67,68.

Fig. 13.

XRD pattern of the synthesized selenium nanoparticles by Bacillus sp. strain STG-83.

The strong anticancer effects of SeNPs can arise from their ability to induce apoptosis, or programmed cell death, through multiple pathways. SeNPs achieve this by activating intrinsic pro-apoptotic signals while simultaneously dismantling important pro-survival mechanisms. Specifically, they initiate apoptosis via the p53-mediated and mitochondrial pathways, while also inhibiting key survival pathways such as the PI3K/AKT and the EGFR-mediated Ras/Raf/MEK/ERK pathways. This dual action—encouraging cell death while blocking survival signals—creates a significant stress that ultimately leads to the activation of the executioner caspase-3 pathway, resulting in irreversible commitment to apoptosis. This coordinated, multi-faceted approach highlights their potential to overcome common resistance mechanisms encountered in cancer therapy^69,70. In this study, cytotoxicity of the synthesized SeNPs in normal cell lines fibroblast and U-87 MG was investigated by MTT assay. As shown in Fig. 14, the results indicated that the viability of U87-MG cells decreased in a dose-dependent manner with increasing nanoparticle concentration, with an IC₅₀ value of 591.3 (µg/mL). At the IC₅₀ concentration of 591.3 (µg/mL) determined for U87-MG cells, the nanoparticle formulation did not induce significant cytotoxic effects in normal fibroblast cells, which exhibited a markedly higher estimated IC₅₀ of 4444 (µg/mL), indicating selective cytotoxicity toward tumor cells. Moreover, MTT assay results revealed a statistically significant difference in cell viability between U87-MG tumor cells and normal fibroblasts at concentrations of 250 (p = 0.043), 500 (p = 0.003), and 1000 (p < 0.001) (µg/mL). The synthesized SeNPs demonstrated a strong cytotoxic effect on U-87 MG cells but caused relatively less toxicity in normal cells. This finding aligns with research conducted by Jiang et al., which showed that GLP-SeNPs exhibited greater cytotoxicity toward tumor cells compared to normal human cells. Specifically, GLP-SeNPs showed the highest sensitivity to U87 cells, with an IC₅₀ value of 9.1 ± 1.53 µM. In contrast, GLP-SeNPs were not toxic to normal cells, with IC₅₀ values of 159.9 ± 9.73 µM for brain glial cells, 79.5 ± 4.26 µM for Hk-2 cells, and 95.6 ± 7.68 µM for L02 cells⁷¹. Also, the study’s results⁷ indicated that the viability of A549 cells was approximately 70%, 45%, and 25% when treated with the biosynthesized SeNPs at concentrations of 20 µg/mL, 60 µg/mL, and 100 µg/mL, respectively. According to Zou et al., LET-SeNPs exhibited a significant inhibitory effect on the proliferation of PC3 cells, with the effect increasing as the drug concentration rose. The study reported an IC₅₀ value of 81.22 µM⁷². By using the MTT method was demonstrated that (Polyporus umbellatus polysaccharide) PUP -SeNPs nanoparticles inhibited the growth of the MDA-MB-231, HepG2, HeLa, and HT29 cancer cell lines and MDA-MB-231 cells proved to be the most sensitive cell line, displaying an IC₅₀ of 6.27 µM. Importantly, no toxicity was observed in normal cells, including LO2, 293T, and NIH3T3 cells⁷³. Based on the research by Hashem et al. the cytotoxicity of SeNPs on the Vero CCL-81 cell line was determined to have an IC₅₀ value of 173.2 µg/mL, confirming their non-toxicity. Additionally, anticancer activity against HepG2 cells was validated, showing an IC₅₀ value of 102.8 µg/mL⁷⁴. Collectively, compared with previously reported biosynthesized SeNPs exhibiting anticancer activity across diverse tumor models, the SeNPs produced in this study demonstrate comparable tumor-selective cytotoxicity with a notably higher safety margin toward normal cells. Although the absolute IC₅₀ values vary due to differences in nanoparticle size, surface chemistry, and experimental models, the consistent pattern of preferential toxicity toward cancer cells reinforces the therapeutic relevance of biosynthesized SeNPs and supports their further evaluation as selective anticancer agents. According, broader validation across multiple cancer types and normal cell lines is essential to generalize the findings of this study. therefore, future studies will extend these experiments to additional cancer models relevant to radiotherapy, as well as corresponding normal tissues, to comprehensively evaluate selectivity and efficacy.

Fig. 14.

Effect of SeNPs on the viability of U87-MG glioblastoma cells and normal fibroblasts. Cell viability was assessed using the MTT assay after 24 h of treatment with varying concentrations of SeNPs (1–1000 µg/mL). U87-MG cells exhibited a dose-dependent decrease in viability, while normal fibroblast cells maintained relatively high viability across the same concentration range. Statistically significant reductions in viability were observed in U87-MG cells compared to normal fibroblasts at 250, 500, and 1000 µg/mL (*p < 0.05), suggesting selective cytotoxicity of SeNPs toward tumor cells.

Intracellular levels of ROS can significantly affect cell growth. Excessive production of ROS leads to oxidative stress within cells, resulting in DNA damage. This damage can alter the distribution of the cell cycle, ultimately impacting cellular lifespan. Additionally, ROS can play a crucial role in the destruction of tumor cells, particularly when using SeNPs, as has been widely suggested⁷⁵. For this reason, we detected levels of ROS in U-87 MG cells with synthesized SeNPs by Bacillus sp. strain STG-83. As shown in Fig. 15, flow cytometry analysis using the DCFH-DA probe revealed a concentration-dependent increase in intracellular ROS generation within the IC₅₀ range of the nanoparticle (250–1000 µg/mL). Notably, a significant negative correlation was found between ROS levels and cell viability at these concentrations, suggesting that ROS-mediated oxidative stress may contribute to the observed cytotoxicity in U87-MG cells (R² = -0.85, P = 0.004). Several studies report that internalization of SeNPs leads to excessive production of ROS causing DNA damage, resulting in disruption of mitochondrial membrane potential and activation of the caspase-9 and caspase-3 apoptotic cascades, indicating an intrinsic mitochondrial apoptosis pathway that is often observed in cancer cells treated with SeNPs, including glioma models^76,77. A significant factor in this context is the documented role of certain substances as redox modulators that generate ROS. These ROS can induce oxidative stress, cause mitochondrial dysfunction, and activate both intrinsic and extrinsic apoptotic pathways. Since the nanoparticles used in our study were synthesized by bacteria, the bioactive coating layer—comprised of proteins and lipids—may further affect cellular uptake and specific interactions⁷⁸.Consequently, it is highly likely that bio-based SeNPs exert cytotoxic effects on U-87 glioblastoma cells through a convergent mechanism involving ROS-induced stress. This mechanism may lead to the modulation of important pathways, including p53, PI3K/AKT, and MAPK/ERK⁶⁹. We recognize that additional research is needed to validate this hypothesis, and we consider this an important area for future studies.

Fig. 15.

Analysis of intracellular ROS generation in U87-MG cells following treatment with selenium nanoparticles (SeNPs) by Bacillus sp. strain STG-83. (A) Flow cytometry histograms of DCFH-DA staining show the percentage of ROS-positive (DCFH⁺) cells after 24-hour treatment with SeNPs at concentrations of 250, 500, and 1000 µg/mL. A progressive increase in the DCFH⁺ population was observed with increasing nanoparticle concentration, indicating dose-dependent ROS generation. (B) Quantitative analysis of mean fluorescence intensity (MFI) confirms a significant, concentration-dependent elevation in intracellular ROS levels, correlating with the increase in SeNP concentration.

The radiosensitizing potential of biosynthesized SeNPs is likely governed by a combination of physical, chemical, and biological mechanisms. selenium (atomic number Z = 34) does not possess as high an atomic number as gold or platinum, it still exceeds that of biological tissue. This property allows selenium to interact more effectively with ionizing radiation, particularly in the kilovoltage to low megavoltage range^79,80. The primary mechanism for radiosensitization by SeNPs is considered to be the enhancement of ROS levels. SeNPs participate in the redox cycle, owing to selenium’s multiple oxidation states. It is important to note that cancer cells already exist in a state of high oxidative stress. SeNPs can further elevate ROS levels beyond the cytotoxic threshold^77,81,82. In 2018, MCF-7 cells were treated with SeNPs along with radiation. A significant decrease in survival cells was observed. In addition, a significant increase in apoptosis, autophagy and cell cycle arrest were observed in G2. Analyzes also showed increased ROS production and cell death in cells treated with selenium⁸³. Also, in another study, it was shown that selenium has a radiosensitizing effect on A549 cancer cells. The results showed that selenium increased the tumor response to radiation from 10% to 60%⁸⁴. The combination of nano-Se and radiotherapy was used in NSCLC cancer cells in 2020, and the effect and mechanism of this combination therapy were discussed. First, through the CCK-8 test, this study confirmed that the effect of SeNPs combined with radiation therapy on the proliferation of NSCLC cells was either greater than treatment with SeNPs alone or radiation alone. It is shown that SeNPs and radiation therapy had the same effect. They are synergistic in inhibiting the activity of cell proliferation or promoting each other⁸⁵. A study investigated the use of conjugated SeNPs with folic acid (FA@SeNPs) as a cancer-targeting agent. These nanoparticles have the potential to enhance the anticancer effects of ¹²⁵I radioactive beads and help prevent metastasis. Notably, the ¹²⁵I beads exhibited a strong synergistic effect when combined with FA@SeNPs, leading to excessive production of ROS. This increase in ROS resulted in apoptosis and ultimately disrupted the cell cycle in human cancer cells⁸⁶. A study in 2024 confirmed that combining LET-SeNPs (Lentinan-functionalized SeNPs) induced a time-dependent increase in intracellular ROS. LET-SeNPs in combination with X-ray radiotherapy effectively inhibited prostate tumor growth and ultimately, it enhances the effectiveness of radiation therapy by inhibiting the mechanisms that repair DNA. Consequently, LET-SeNPs emerged as a powerful radiosensitizer used in conjunction with radiotherapy, enhancing the effects against prostate cancer⁷². Also, LNT-SeNPs were shown to enhance the efficacy of radiotherapy for treating esophageal squamous cell carcinoma (ESCC) by increasing the production of ROS in 2025²². LNT-SeNPs amplify radiotherapy-induced ROS production, resulting in apoptotic cell death. Moreover, these nanoparticles attenuate GPX2 antioxidant activity and activate apoptosis via the p53/IGFBP3 pathway²². Other research has indicated that nano-heterojunction Se@PB acts as a radiosensitizer by increasing ROS production in tumor cells. This process leads to mitochondrial dysfunction and DNA damage, ultimately enhancing apoptosis in cervical cancer cells⁸⁰.

Although SeNPs demonstrated pronounced cytotoxicity and ROS generation in U-87 glioblastoma cells, it should be emphasized that the present study did not directly evaluate their radiosensitizing efficacy under combined nanoparticle and ionizing radiation exposure. Radiosensitization is a context-dependent phenomenon that requires experimental confirmation through clonogenic survival assays, DNA damage analysis, or apoptosis measurements following irradiation. The observed increase in intracellular ROS induced by SeNPs, however, represents biological features that are highly relevant to radiation response. Given that ionizing radiation exerts its cytotoxic effects primarily through oxidative stress and DNA damage, these findings suggest that SeNPs may possess potential radiosensitizing properties. Nevertheless, this hypothesis remains to be experimentally validated, and future studies incorporating combined radiation and nanoparticle treatments are essential to substantiate this effect. Extracellular biosynthesis of SeNPs by Bacillus sp. strain STG-83 is a promising method for large-scale synthesis of these nanomaterials with potential bioactivity. RSM optimization studies highlighted that both selenium concentration and time play a crucial role in optimizing the yield of these nanoparticles. These SeNPs have considerable potential to be integrated into cutting-edge technologies due to their optimal physicochemical and biological properties. Large-scale synthesis is also feasible due to the extracellular synthesis and natural capping mechanisms. These studies and future research will enhance the translation of these SeNPs from bench-scale experiments to industry and clinics, providing a sustainable route for the exploitation of the therapeutic potential of selenium.

Conclusion

Biosynthesized SeNPs have gained increasing attention owing to their desirable biological properties, such as antioxidant, antimicrobial, and anticancer properties, as well as the theoretical potential to affect therapeutic responses related to oxidative stress. In the current study, Bacillus sp. strain STG-83 was found to be a potent biological system for the extracellular synthesis of SeNPs. RSM was found to be an effective and accurate statistical method for the modeling and optimization of the biosynthesis process, where the parameters that had the most significant effect on the efficiency of selenite bioreduction were found to be selenium concentration and incubation time. Spherical SeNPs with diameters ranging from 80 to 140 nm were successfully produced under optimized conditions. FTIR spectroscopy was used to confirm the presence of surface-bound functional groups, possibly of biological origin such as proteins and lipids, which are important for the stability of nanoparticles. Although qualitative evidence suggests acceptable colloidal stability, comprehensive quantitative stability assessments of the SeNPs, including their behavior over time, are required, which will be the focus of future work.

The results of the biological evaluation revealed that the cytotoxic effect of biosynthesized SeNPs was significant against the U-87 MG cancer cell line, while the cytotoxic effect of the SeNPs was relatively low against normal fibroblast cells. In addition, flow cytometry analysis revealed that the intracellular level of ROS in cancer cells significantly increased after exposure to SeNPs. This study suggests that the anticancer effect of SeNPs may be due to the induction of oxidative stress in cancer cells.Importantly, despite the lack of any experimental studies on the effect of radiation, the cytotoxic effect mediated by ROS offers a mechanistic rationale for the potential use of these SeNPs as a radiosensitizer, similar to previous studies on SeNPs and ionizing radiation. However, such radiosensitization remains a hypothesis in the context of the present work and requires direct experimental validation through combined radiation–nanoparticle studies. Although the synthesized SeNPs are larger than many previously reported bio-SeNPs (< 100 nm), the results indicate that nanoparticles within the 80–140 nm size range can still achieve effective cellular interaction and anticancer activity, potentially due to extracellular biosynthesis, biomolecular surface capping, and sufficient intracellular ROS induction. Overall, this study demonstrates the potential of Bacillus sp. strain STG-83 as a biological source in the synthesis of SeNPs with functional potential and their anticancer activity in vitro. Future investigations incorporating multiple cancer models, in vivo validation, detailed stability analyses, and direct radiation-combination assays will be essential to fully define their therapeutic applicability and translational relevance.

Author contributions

Authors Contributions: The manuscript was a part of the PhD. thesis by S. A-K. All the authors contributed to the conception and design of the study. S. A-K.: conducting experiments, writing (original draft preparation). Dr. S-M-R. A.: writing (review and editing), supervision. Dr. S. S-A.: writing (review and editing), supervision. Dr. P. T-M-G.: writing (review and editing), supervision, software. Dr. A. B-S.: supervision. Dr. S-M. M.: writing (review and editing), supervision. M-R. D.: adviser. All authors read and approved the final manuscript.

Data availability

All data generated or analyzed during this study are included in this article. The 16 S rDNA sequence of Bacillus sp. strain STG-83 had been deposited in the National Center for Biotechnology Information (NCBI) GenBank with the accession number EF051255 (https://www.ncbi.nlm.nih.gov/nuccore/EF051255).

Declarations

Competing interests

The authors declare no competing interests.