Recently, the biosynthesis of nano-selenium structures has received high attention from researchers because they are considered necessary micronutrients for living organisms. Therefore, the synthesis of Se-NPs has increased in the last decades, especially by green approaches. Herein, Streptomyces vinaceusdrappus strain AMG31 was utilized to fabricate Se-NPs and to assess their biomedical applications. The formation of ruby color after interaction of Na₂O₃Se with the actinobacterial biomass filtrate confirmed the successful fabrication of Se-NPs. The intensity of this ruby color was monitored using UV-vis spectroscopy to measure the surface plasmon resonance (SPR). Figure 1A showed the maximum SPR for actinobacterial synthesized Se-NPs was 260 nm, which referred to the successful formation of small sizes and spherical shape [44]. Whereas the UV of biomass filtrate showing λmax at wavelength of 290 nm (Fig. S1, see supplementary data) which refers to the presence of aromatic compounds such as phenolic compounds (tannins and flavonoids), aromatic amino acids (tryptophane and tyrosine), and extracellular metabolites (such as enzymes, pigments, antibiotics) [3]. Recently, the maximum SPR of Se-NPs fabricated by different fungal strains appeared in the range of 260–280 nm [45]. The bacterial strain, Ralstonia eutropha, was utilized to fabricate Se-NPs with maximum SPR at 270 nm [46]. The Se-NPs reproducibility synthesis is mainly some parameter-dependents such as biomass filtrate production under optimum conditions for actinobacterial growing, pH, metal precursor concentration, contact times, and temperature. In our study, the S. vinaceusdrappus was grown under the same conditions at each batch (Czapek Dox broth at 30 ± 2 °C for 7 days with 150 rpm shaking) to confirm the production of same secondary metabolites that used as reducing agent. Moreover, the same concentration of Na₂O₃Se was mixing with biomass filtrate under pH value of 8 and stirring with 40 °C for 60 min followed by dark incubation for 24 h to ensure consistent batch-to-batch reproducibility. Under these optimum conditions, ensuring scalability and production of maximum yield at each batch.

FT-IR for actinomycetes biomass filtrate and biosynthesized Se-NPs exhibits different functional peaks at varied wavenumbers (Fig. 1B). As shown, the broad and strong peak at 3400 cm–1, shifted to 3380 cm–1 after nano-selenium fabrication, refers to stretching O-H of alcohol or N-H of amines [47]. The weak peaks at the wavenumbers in the 3100–2800 cm–1 range correspond to the stretching O-H group of phenol, alcohol, or water [48]. The strong peak at 1666 cm–1, deconvoluted into two peaks at 1685 and 1610 cm–1 upon Se formation, is related to the bending N-H of secondary amines, whereas the peaks at 1490 and 1400 cm–1 (shifted to1410 cm–1 in Se-NPs) correspond to the C = C or aromatic compounds [49]. The peaks in the ranges of 1365 to 1330 cm–1 in Se-NPs and biomass filtrate charts are related to the bending OH of phenol, whereas the peak at 1275 cm–1 signifies the C-N of aromatic amines, or S = O of sulfonates, or C-O of carboxylic acid, ethers, or ester [50]. The presence of different peaks in the ranges of 1000–1200 cm–1 indicates the presence of polysaccharides and C-O-C of sugars [51]. The peaks in the ranges of 400–600 cm–1 signify the alkaline halides (C-Se) [52]. The presence of these different functional groups indicates the efficacy of actinobacterial metabolites, such as proteins, polysaccharides, amines, and carbohydrates, in the reduction of Na₂O₃Se to form Se-NPs, followed by capping and improving their stability.

The detection of the sizes and shapes of the biogenic Se-NPs was achieved by electron microscopy analysis. The obtained images (Fig. 1C–E) revealed that the biosynthesized Se-NPs exhibited a spherical morphology with sizes ranging from 20 to 80 nm. Recently, Streptomyces parvulus was utilized to formation of semispherical Se-NPs with a size of 94 nm [53]. Also, Streptomyces minutiscleroticus was used to produce spherical Se-NPs with sizes of 100–250 nm, and the authors investigated antioxidant, antibiofilm, anticancer, wound healing, and anti-viral activities on it [54]. The activity of Se-NPs, especially in biomedical applications, mainly depends on their size, shape, agglomeration, stability, and surface charges. The activity was enhanced with smaller sizes compared to bigger ones. For instance, garlic-mediated biosynthesis of Se-NPs exhibited promising antimicrobial activity with sizes of 21–40 nm compared to their activity with sizes of 41–50 nm [55].

Characterization of actinobacterial-driven biogenic Se-NPs. (A) UV-vis spectroscopy shows the maximum SPR, (B) FTIR shows different functional biomolecules, and (C–E) TEM images at various magnification powers show a spherical shape

The crystallinity of actinobacterial-synthesized Se-NPs was assessed using XRD. Figure 2A showed that the sample containing Bragg’s diffraction peaks of (100), (101), (110), (102), (111), (201), (112), and (202) corresponding to 2θ° of 23.4°, 30.13°, 41.38°, 44.32°, 46.14°, 52.73°, 55.3°, and 65.7° respectively. The obtained diffraction confirmed that the biosynthesized Se-NPs were crystalline in nature according to the standard file of JCPDS No. 06–362 [56]. Recently, the biogenic Se-NPs using Penicillium crustosum exhibit diffraction peaks of (100), (101), (110), (102), (112), (202), and (210) at 2θ° 23.2°, 29.6°, 40.3°, 43.5°, 50.1, 55.5°, and 66.2°, respectively [57]. Singh and coauthors reported that the presence of peaks (100), (101), and (102) at 23°, 30°, and 43° values of 2θ confirmed the efficacy of microbial metabolites to reduce Na₂O₃Se and form crystallographic Se-NPs structure [58]. The presence of extra peaks in the XRD chart could be attributed to the scattering of actinobacterial capping agents [59].

The elemental compositions of actinobacterial synthesized Se-NPs were detected using EDX analysis (Fig. 2B). The EDX chart shows the presence of Se ions in addition to C, O, Na, and K ions with varied weight and atomic percentages. The presence of peaks at 1.4, 22.1, and 12.6 KeV bending energies confirmed the formation of Se-nanostructure [60]. The presence of other peaks (matched with the XRD chart) could originate from capping biomolecules such as proteins, enzymes, and carbohydrates [61]. The high weight and atomic% of O due to oxidation or the presence of oxygen-containing organic coatings [62]. The obtained findings were compatible with different published investigations that confirmed the presence of other peaks in EDX chart with Se and returned it to capping bioactive molecules [63].

The hydrodynamic sizes of biogenic Se-NPs in the liquid were determined by DLS (Fig. 2C). As shown, there are three peaks represented the sizes of 493.5 nm (for intensity 84.4%), 431 nm (for 13.2%) and 84.9 nm (for 2.3%) with an average hydrodynamic size of 460.1 nm. Similarly, the DLS of Se-NPs formed by exopolysaccharide secreted from Bacillus sp. showed hydrodynamic sizes in the ranges of 200–300 nm with an average of 209 nm [64]. Also, the sizes of Se-NPs formed by bacterial strain Zooglea ramigera ranged from 78 nm to 210 nm with an average of 152 nm as detected by DLS analysis [65]. Usually, the sizes obtained by DLS are higher than TEM sizes due to the DLS measuring the sizes in the hydrated state (hydrodynamic size), whereas TEM measures the particle at solid state [45]. Also, the DLS is affected by the capping agent in the liquid solution. Moreover, during sample preparation for TEM analysis involves drying and high-vacuum conditions, which leads to weak agglomerates. Whereas, DLS reflects the colloidal state in suspension, where leads to mild to moderate agglomeration due to electrostatic or van der Waals interactions, even if the particles are well-dispersed under TEM. The non-well distribution or aggregation of the NPs in the liquid affected DLS analysis and gave large sizes [66]. The homogeneity percentages of synthesized Se-NPs in the liquid solution can be detected by measuring the polydispersity index (PDI) during DLS analysis. PDI has a range from 0 to 1; the heterogenous increased when the PDI value was close or equal to 1, whereas the homogeneity increased at a PDI value less than 0.4 and decreased at a value greater than 0.4 to 1 [67]. Here, the PDI value of synthesized Se-NPs was 0.384, indicating the high homogeneity and stability of NPs in the colloidal solution. Similarly, the PDI of Se-NPs produced by Z. ramigera bacterial strain was 0.438 [65], which indicates (as the authors mentioned) high particle stability.

The zeta-sizer of synthesized Se-nanostructure showed two peaks with a zeta potential value of −37.9 mV for the first peak (high peak) and −16.9 mV for the second peak (low peak) (Fig. 2D). The presence of one charge (-ve) on the Se-NPs surface indicates the electrostatic repulsion between particles and each other’s, leading to aggregation stopping, and hence high stability [68]. Some authors reported that the capping agents, such as flavonoids, terpenoids, polysaccharides, and alkaloids, secreted by biological entities and used for reducing the metal and production of the nanoscale structure have a role in improving stability via adding the -ve charge to NPs surfaces and hence enhanced electrostatic repulsion between particles [69].

XRD analysis showing the crystalline nature of biogenic Se-NPs (A), elemental compositions of biogenic Se-NPs by EDX (B), size distribution in the colloidal by DLS (C), and stability detection using zeta-potential analysis (D)

All four assays, including DPPH, ABTS+, TAC, and FRAP, showed that Se-NPs possessed concentration-dependent antioxidant activity with promising antioxidant potential. In the measurements done with DPPH and ABTS, Se-NPs increased their radical scavenging ability with increasing concentration, which also meant that a greater degree of inhibition was achieved. In particular, at the highest concentration range, 1000 µg mL–1 of Se-NPs, the results were DPPH of 86.7% scavenging and ABTS antioxidant activity of 84.6%. At the lowest tested concentration of 1.95 µg mL–1, Se-NPs exhibited a 23.9% DPPH scavenging activity. As concentration increased, the scavenging percentage also increased. At 7.8 µg mL–1, the scavenging percentage was 36.7%, while at 15.6 µg mL–1, it reached 45.5%. A notable increase was observed at 31.25 µg mL–1, with a 52.9% scavenging activity. The scavenging percentage continued to rise, reaching 59.4% at 62.5 µg mL–1, 66.4% at 125 µg mL–1, and 72.9% at 250 µg mL–1. Its IC50 value was 26.06 µg mL–1 compared to ascorbic acid (2.54 µg mL–1).

At a concentration of 500 µg mL–1, Se-NPs exhibited a 79.4% DPPH scavenging ability, which further increased to 86.7% at the maximum tested concentration of 1000 µg mL–1 (Fig. 3A).

Antioxidant activity of actinobacterial-mediated biosynthesis of Se-NPs. (A) DPPH scavenging activity was compared to a positive control (ascorbic acid), and (B) ABTS scavenging activity of Se-NPs was compared to gallic acid as a positive control. Different letters (a and b) on the bars at the same concentration indicate the results are significantly different (Mean ± SD, n = 3, P ≤ 0.05)

A similar pattern was demonstrated by Se-NPs in ABTS·+ assay, as the scavenging activity increased with an elevation in the concentration. Where at its lowest concentration of 1.9 µg mL–1, the ABTS scavenging percentage was 25.0%. This percentage increased to 30.7% at 3.9 µg mL–1, 40.2% at 7.8 µg mL–1, and 45.2% at 15.6 µg mL–1. A noticeable increase was observed at 31.2 µg mL–1, with a 53.5% scavenging activity. The IC50 value for Se-NPs was determined to be 25.17 µg mL–1 compared to 2.54 µg mL–1 of gallic acid. The scavenging percentage continued to rise, reaching 59.0% at 62.5 µg mL–1, 65.4% at 125 µg mL–1, and 71.6% at 250 µg mL–1. At a concentration of 500 µg mL–1, Se-NPs exhibited a 79.0% ABTS scavenging ability, which further increased to 84.6% at the maximum tested concentration of 1000 µg mL–1 (Fig. 3B).

The TAC and FRAP values provided an overall assessment of the antioxidant capacity of Se-NPs, expressed in terms of ascorbic acid equivalents (AAE). For Se-NPs, the TAC value was reported as 965.9 ± 4.9 µg/mg. This value represents the concentration of ascorbic acid that would exhibit an antioxidant capacity equivalent to 1 mg of Se-NPs. Meanwhile, the Ferric FRAP assay evaluates the ability of an antioxidant to reduce Fe3+ to Fe2+. For Se-NPs, the FRAP value was reported as 727.7 ± 7.6 µg/mg. This value indicates the concentration of ascorbic acid that would exhibit a ferric-reducing ability equivalent to 1 mg of Se-NPs (Table 1).

Following our findings, a phytosynsized Se-NPs with an average size of 80 nm from Nyctanthes arbortristis L exhibited potent antioxidant activity in DPPH and H2O2 assays [70]. Another spherical Se-NPs sized 80 nm derived from Diospyros montana exhibited robust antioxidant activity, with IC50 of 24.7 ± 0.6 µg mL–1 and EC50 of 46.3 ± 0.2 µg mL–1 in DPPH and reducing power assays [71]. Nocardia concave was utilized to fabricate Se-NPs that was spherical and showed antioxidant capacity in DPPH and ABTS assays with IC50s = 31.4 µg mL–1 and 35.7 µg mL–1, respectively [72]. Similarly, cyanobacterium Anabaena was employed to biofabricate Se-NPs that was spherical and sized 50 nm that demonstrated high antioxidant activity at 50 µg mL–1 in the DPPH assay [73].

ROS and reactive nitrogen species (RNS), referred to as free radicals, are produced during normal metabolic functions and have an essential role in the physiological processes of a cell. Conversely, when present in excessive amounts, these free radicals harm essential cellular components such as proteins, nucleic acids, and membranes [74]. It is worth mentioning that the Se-NPs can exhibit some antioxidant activity by lowering these oxidant species by donating electrons and converting them into non-reactive species. Furthermore, Se-NPs enhance and stimulate the production of essential antioxidant enzymes, including superoxide dismutase (SOD), and catalase (CAT) which scavenges H2O2 together with lipid and phospholipid hydroperoxides and converts them to water and alcohol [15]. Also, it has been reported that Se-NPs functioning as Glutathione peroxidase (GPx) mimetics that decompose peroxides via glutathione-mediated reactions, while concurrently stimulating Nrf2 transcription factors to boost production of vital protective enzymes as SOD, CAT, and heme oxygenase-1 [75].

The scratch wound healing assay results indicated that Se-NPs at 209.87 µg mL–1 promoted enhanced wound healing compared to the control cells. Notably, the Se-NPs-treated cells exhibited a higher migration rate (10.4 μm h–1) than the control cells (10.4 μm h–1), suggesting an accelerated wound closure process. Furthermore, the percentage of wound closure was markedly higher in the Se-NPs group (73.6%) compared to the control group (62.6%), corroborating the wound-healing effect of Se-NPs. Moreover, the area difference percentage, which quantifies the change in the wound area in µm², was also considerably higher for the Se-NPs-treated cells (445876.7 μm²) than the control cells (379461.5 μm²), corresponding to area difference percentages of 73.6% and 62.6%, respectively further reinforcing the efficacy of Se-NPs in promoting wound healing (Table 2)(Fig. S2, see supplementary data).

Se-NPs offer dual protection in wound microenvironments through their powerful anti-inflammatory and antioxidant effects. They reduce inflammation by shifting macrophages from pro-inflammatory to anti-inflammatory states, speeding healing [76]. Also, their strong antioxidant properties combat oxidative stress in wounds, supporting essential cellular healing functions [77]. Furthermore, Coating Se-NPs with red blood cell membranes improves their stability and helps them evade immune detection, making them more effective against infected wounds [78]. This enhanced efficacy is evident in a study where fungal-derived Se-NPs significantly reduced Staphylococcus aureus infections, resulting in smaller wound areas and faster healing [79].

Se-NPs promote tissue regeneration by stimulating fibroblast proliferation and collagen synthesis, key elements in wound healing [80] Their effectiveness increases when combined with other treatments, as seen when paired with platelet-rich plasma (PRP), where they work synergistically with PRP’s growth factors to speed healing significantly [81]. Similarly, when integrated into nitric oxide-generating gels, Se-NPs enhance both collagen deposition and epithelialization, where wounds are healing [82].

The hemolytic activity of Se-NPs was assessed against a positive control (complete hemolysis by deionized H2O, representing 100% hemolysis). Negative control (isotonic solution), where concentrations from 1000 to 25 µg mL–1 exhibited minimal hemolytic effects: 1000 µg mL–1 showed 1.8% hemolysis, 800 µg mL–1 causing 0.5% hemolysis, 600, 400, and 200 µg mL–1 causing 0.8%, 0.4%, and 0.2% hemolysis respectively.

On the other hand, the concentrations from 100 to 25 µg mL–1 maintained a consistent 0.1% hemolysis (Table 3). When compared to the complete hemolysis control (absorbance 1.211), these values demonstrated negligible membrane disruption (Fig. S3, see supplementary data), confirming the safety profile of Se-NPs towards red blood cells even at high concentrations.

Hemolytic assays are crucial for assessing how Se-NPs interact with red blood cells, with research showing that specific biologically produced Se-NPs demonstrate minimal hemolytic activity, indicating favourable blood compatibility [83]. As evidence of this pattern, chitosan-stabilized Se-NPs caused only 7.2% hemolysis, substantially below toxic control levels, suggesting their suitability for safe medical applications [84].

Assessing blood compatibility for biogenic Se-NPs necessitates thoroughly examining multiple crucial factors to confirm their safety profile and effectiveness in medical safety and function. Manufacturing techniques, dimensional measurements, structural formations, and RBCs reactivity are central factors when determining compatibility between selenium-based nanomaterials and blood components. Phytogenic Se-NPs and microbial-synthesized Se-NPs exhibited distinct biological functionality and toxicity signatures compared to chemically manufactured versions [85].

Notably, environmentally sustainable fabrication using Hybanthus enneaspermus extracts [86] or Bacillus halotolerans cultures [87] has yielded remarkably compatible selenium nanostructures with minimal harmful effects. Dimensional and structural characteristics of Se-NPs play essential roles in their functionality; particularly, minute small particles measuring 5–50 nm typically demonstrate superior cell-level engagement and may significantly alter blood compatibility outcomes [88]. Also, Round-formed Se-NPs consistently show improved tissue integration and lower rates of blood cell rupture [89].

The evaluation of biocompatibility of Se-NPs against normal WI-38 cells showed minimal or no cytotoxicity for all the concentrations studied with the exception of concentrations 1000 and 500 µg mL–1, where the percentage of cell viability dropped to 11.1 and 33.3%, respectively (Fig. 4). It is observed that as the concentration decreased, the cytotoxicity was reduced, while the cell viability was significantly high, remaining at 93.8% for 250 µg mL–1 and above 98% for concentrations ranging from 125 µg mL–1 to 31.25 µg mL–1, likely with minor or no morphological changes as in the case of control cells (Fig. S4, See supplementary data). Interestingly, the IC50 value was found to be noteworthy, 419.7 ± 5.2 µg mL–1.

For the anticancer activity against the Caco-2 cell line, Se-NPs also demonstrated concentration dependence, where increased concentrations were more cytotoxic and accompanied by visible morphological changes. Particularly, the most profound cytotoxicity occurred at the highest tested concentration of 1000 µg mL–1, where the cell viability decreased to only 3.8% (Fig. 4), signifying cytotoxic effects, evidenced by noticeable cytomorphological changes. At this concentration, cells rounded up and completely lost attachment to the surface, which are signs of cell death or apoptosis caused by the potent cytotoxic impact of Se-NPs during treatment (Fig. S5, See supplementary data). A similar concentration-dependent trend was observed at the concentration of 500 µg mL–1, where cell viability deteriorated to as low as 4.2% (Fig. 4), suggesting considerable cytotoxicity was present along with striking morphological distortions, possibly resembling the cellular deformations observed at the highest concentration (Fig. S5, See supplementary data). As the concentration dropped to 250 µg mL–1, the cell viability increased to 10.5% (Fig. 4). However, this still reflected severe cytotoxicity, and these are likely to be accompanied by changes in the morphological features like cell shrinkage or atypical cellular shapes, pointing to the fact that Se-NPs still interfered with the cell structure. Even at 33.2% viability with a 125 µg mL–1 concentration, the morphological alteration was rather expected but less severe than that at higher concentrations. Remarkably, it is interesting to note that at lower concentrations of 62.5 and 31.25 µg µg mL–1, the observed cell viability increased significantly to 80.7% and 99.9%, respectively (Fig. 4), evidencing very low levels of cytotoxicity and slight morphological changes as compared to the untreated control cells (Fig. S5, See supplementary data). Furthermore, the determined IC50 of 102.5 ± 2.1 µg mL–1 complemented this concentration-dependent cytotoxic effect, with cell death and disruption of cell morphology being significant at concentrations above this value and less morphometric abnormalities with some cytotoxicity at concentrations below.

Cell viability of Wi38, Caco-2, and PANC-1 cells treated with biogenic Se-NPs (31.2–1000 µg mL–1). Different letters (a, b, and c) on the bars at the same concentration indicate the results are significantly different (n = 3, P ≤ 0.05)

Our quantitative analysis of the PANC-1 pancreatic cancer cell line showed a concentration-cytotoxic inhibitory effect of Se-NPs with an IC50 value of 100.4 ± 1.9 µg mL–1. Cells treated with Se-NPs at 250, 500, and 1000 µg mL–1 showed a severe cytotoxic effect as the viability percent ranged between 3.5% and 4.5%. In contrast, lower 125 and 62.5 µg mL–1 concentration had a viability of 69.2% and 16.8%, respectively (Fig. 4).

The notable increase in cell viability at lower Se-NPs concentration also points toward the potential of minimal cytotoxicity and changes in cell morphologies since the untreated PANC-1 cells displayed seemingly epithelial-like morphology, which is characterized by a more or less fair cobblestone appearance with well-defined cell borders (Fig. S6, see supplementary data).

Microbial and photosynthesized Se-NPs, with a size range of 79 to 500 nm, have been reported to be promising candidates for malignancies treatment [90]. For example, spherical Se-NPs sized 22 nm bioformed from Portulaca oleracea have been reported to have anticancer activity against HepG2 with IC50 = 70 µg mL–1 [69]. Another mycofabricated spherical Se-NPs from Penicillium verhagenii was documented to have anticancer activity against MCF7 and PC3 cell lines [45]. Similarly, spherical Se-NPs derived from P. crustosum inhibited the cancerous cells of T47D and HepG2 cells lines in vitro [57]. Also, spherical Se-NPs with an average size of 48.9 nm bioformed from P. corylophilum were documented to have an IC50 = 104.3 ppm against Caco-2 cell lines, while retaining biocompatibility against normal WI38 with IC50 = 171.8 ppm [91].

Se-NPs have been proven to be effective against various cancers, including breast, prostate, and other cancers. In breast tumours, they demonstrated anticancer action by decreasing levels of certain pro-inflammatory cytokines such as IL-17, IL-2, IL-12, IFN-γ, and TNF-α, and improved delayed-type hypersensitivity and natural killer cells activity, which resulted in reduced tumor size and prolonged the life span of a mouse model of breast cancer [75, 92]. In prostate cancer PC-3 cells, the ameliorative effect of Se-NPs was demonstrated by the upregulation of necroptosis-related factors IRF1 and TNF, decreased levels of prostate-specific antigen (PSA) and androgen receptor (AR), and an enhanced ROS-dependent necroptosis in PC-3 cells [