Biogenic synthesis of bioactive zinc nanoparticles from cyanobacterial Nostoc sp. MK-7 for biomedical applications.

Introduction
Nanotechnology has revolutionized the synthesis of metal oxide nanoparticles (NPs), attracting significant attention across various research fields, particularly in biogenic nanomedicine [1]. Biogenic NPs, due to their small size, exhibit unique biological properties that are not seen in bulk materials [2]. Among these, zinc oxide (ZnO), titanium dioxide (TiO2), and silver oxide (Ag2O) NPs have gained particular focus in biomedical applications because of their versatile functionality in mammalian systems [3]. ZnO NPs, in particular, are recognized as safe by the Food and Drug Administration (FDA) and are known for their high stability under air, ultraviolet (UV) and visible light, and chemical exposure [4]. Their applications span antimicrobial treatments and therapeutic interventions [5]. Despite the advantages of conventional physiochemical methods for synthesizing ZnO NPs, these approaches involve expensive, hazardous, and non-biodegradable chemicals, posing environmental and health concerns [6]. In contrast, green approaches offer environmentally friendly alternatives. These methods utilize natural resources to mediate NP synthesis, providing biocompatible, non-toxic, and sustainable materials [7]. Importantly, biologically synthesized NPs often demonstrate superior activity compared to those produced through conventional techniques [8]. Phytochemicals, including flavonoids, terpenoids, and phenols, act as natural reducing and stabilizing agents, facilitating NP formation while enhancing biological functionality [9]. In recent years, microbial sources have emerged as promising agents for NP biosynthesis due to their rich bioactive metabolite profiles [10]. Cyanobacteria are gaining recognition for their potential in NP biosynthesis, owing to their ability to produce novel compounds with significant biotechnological value [11]. They are efficient biomass sources, requiring minimal time and resources for growth. Nostoc sp. biomass, rich in compounds like amino, hydroxyl, and carboxyl groups, serves as a reducing agent for NP synthesis [12]. As a filamentous cyanobacterium, Nostoc sp. has attracted attention for its role in green nanotechnology. Known for producing bioactive compounds such as phycocyanin, polysaccharides, amino acids, fatty acids, terpenoids, alkaloids, and bioactive peptides, Nostoc sp. exhibits antioxidant, anti-inflammatory, antimicrobial, and anticancer activities [13]. These metabolites make Nostoc sp. an ideal candidate for the eco-friendly synthesis of metal oxide NPs. The bioactive compounds present in Nostoc sp. MK-7 strain reduce zinc ions (Zn2+) to form MK-7–ZnO NPs, while simultaneously enhancing their biological activity, stability, and therapeutic potential [11]. Green synthesis of MK-7–ZnO NPs with bioactive compounds from Nostoc sp. MK-7 has the potential to synergistically enhance anti-inflammatory and anticancer activity [14]. The MK-7 cyanobacterial metabolites not only facilitate green synthesis but also confer additional biological benefits, such as cytotoxicity against cancer cells, reduction of oxidative stress, and modulation of inflammation. Triple-negative breast cancer (TNBC) is a particularly aggressive form that is negative for estrogen, progesterone, and HER2 receptors, making it challenging to treat with conventional therapies [15]. MDA-MB-231 breast cancer cells, a widely used model for TNBC, are recognized for their invasive and metastatic characteristics [16]. The integration of green nanotechnology with cyanobacterial bioactive compounds represents a sustainable, eco-friendly, and cost-effective approach to advanced nanomedicine [17]. This study aims to synthesize and characterize MK-7–ZnO NPs using Nostoc sp. MK-7 strain, evaluate their biological activities, bridging the fields of nanotechnology, phytomedicine, and cancer therapy. Characterization techniques i.e., UV–vis, FTIR, XRD, Raman, FESEM, TEM, and EDS confirm their structural, morphological, and chemical properties [18].

Materials and methods

Isolation of cyanobacterial strain
A water sample was obtained from Gujar Khan, in the Rawalpindi District, and stored at the Department of Plant Sciences, Quaid-i-Azam University (QAU), Islamabad, Pakistan. The Nostoc sp. MK-7 strain was isolated, cultured, and purified on a Blue-Green Algae 11 (BG11) medium, then incubated in a growth chamber at 25 °C under a light/dark photoperiod of 8/16 h with cool white light at 3000 Lux [19]. After colony formation, purity was confirmed, and the Nostoc sp. MK-7 strain was preserved for further analysis. Morphological characteristics of Nostoc sp. MK-7 were observed using a light microscope. Taxonomic identification was performed and confirmed with a botanical expert at QAU following standard methods [20]. For biomass production, axenic cultures of Nostoc sp. MK-7 were cultivated in 100-, 250-, and 500-mL Erlenmeyer flasks containing 70, 200, and 350 mL of BG-11 medium, respectively. Cells were harvested at the optimal growth phase by centrifugation at 4000 rpm for 15 min. The collected biomass was washed with double-distilled water to remove residual culture medium and then air-dried [21]. The dried biomass was thoroughly ground and passed through a 100 μm sieve to obtain a uniform fine powder, which was subsequently stored in an airtight container to prevent moisture absorption and rehydration prior to further biological applications.

Biosynthesis of MK-7 ZnO NPs
The biosynthesis of MK-7 ZnO NPs using Nostoc sp. MK-7 aqueous extract follows a green synthesis approach [22]. The process begins by preparing the Nostoc sp. MK-7 biomass. A 15 g sample of dried Nostoc sp. MK-7 powder is dissolved in 250 mL of distilled (DI) water to prepare a cyanobacterial MK-7 extract [13]. The solution is then homogenized for 10 min with constant stirring using a magnetic stirrer set at 800 rpm to ensure thorough mixing. For the synthesis of ZnO NPs, 3 g of zinc acetate dihydrate (Zn(CH3COO)2·2H2O) is dissolved in 250 mL of DI water. Following this, 98 mL of the Nostoc sp. MK-7 biomass filtrate is added gradually to the zinc salt solution in a 1:10 ratio. The mixture is stirred continuously at 500 rpm for 4 h at room temperature to maintain uniform mixing [23]. The formation of MK-7–ZnO NPs is observed by a visible color change in the reaction mixture, turning yellowish brown, which indicates green MK-7–ZnO NPs synthesis [4]. The mixture is then centrifuged at 5000 rpm for 20 min to separate the MK-7 ZnO NPs, as shown in the schematic illustration in Fig. 1. The supernatant is discarded, and the MK-7 ZnO NPs are washed three times with DI water to remove any debris. Finally, the collected pellet of MK-7 ZnO NPs are dried in a hot air oven at 80 °C for 14 h to obtain a dry powder form of MK-7–ZnO NPs, which can then be used for further physio-chemical characterization and biomedical activities evaluation [24].

Physio-chemical characterization

UV–vis analysis
The green synthesis of MK-7–ZnO NPs was analyzed by LAMBDA 850 + UV–vis spectrophotometer at Jeju National University. Spectral scanning of the reaction between zinc salt solution and the aqueous extract of Nostoc sp. MK-7 was performed within the wavelength range of 200–800 nm [25]. A color change in the mixture during the reaction indicated the formation of MK-7–ZnO NPs. UV–vis spectroscopy samples were collected starting at 0 h (when stirring began) and at 2-h intervals throughout the 4-h stirring period, with a total of three samples taken for readings. After the 4 h of continuous stirring, readings were taken from two samples: one from the raw Nostoc sp. MK-7 aqueous extract and one from the aqueous extract of biogenic MK-7–ZnO NPs [26].

Fourier transform-infrared spectroscopy (FTIR)
The functional groups of MK-7–ZnO NPs were analyzed using a Bruker ALPHA II FTIR spectrometer at Jeju National University, and the spectra were recorded in the range of 4000–1000 cm−1 [27].

X-ray diffraction (XRD)
The crystallographic properties and phase composition of the biogenic MK-7–ZnO NPs were characterized using a Rigaku benchtop X-ray diffractometer (XRD) at Jeju National University [28]. The crystallite size of NPs was calculated using Debye–Scherrer equation [29].

Raman analysis
Raman analysis of the MK-7–ZnO NPs was analyzed in a frequency ranging of 200–1600 cm−1 (Raman shift) with intensity (a.u.) on the y-axis using a micro–Raman Horiba XploRA (Horiba Scientific, Kyoto, Japan) instrument at Jeju National University [30].

FESEM, TEM, and EDS analysis
The morphology of MK-7–ZnO NPs was analyzed using Field Emission Scanning Electron Microscope (FESEM) (TESCAN MIRA 3) at Jeju National University and Transmission Electron Microscopy (TEM) (TECNAI F20) at Korea Basic Science Institute (KBSI), Gwangju Center, South Korea. Energy-Dispersive X-ray Spectroscopy (EDS) was used to assess the chemical and elemental composition of the NPs [31].

Antioxidant activity

Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was performed by preparing a reagent consisting of 10 mmol/L 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) and 20 mmol/L ferric chloride in acetate buffer (pH 3.6) [32]. 100 µL of MK-7–ZnO NPs was mixed with 900 µL of the FRAP reagent, and after 24 h of incubation, the absorbance at 593 nm was measured [33]. Results were expressed as micromoles of trolox equivalent (Fe2+) per gram of the three samples. Ursolic acid was used as a positive control [34].

ABTS radical scavenging assay
ABTS radical scavenging activity was evaluated by preparing a 7 mM 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ABTS solution and combining it with 2.45 mM potassium persulfate to generate ABTS·  + radicals [35]. The mixture was incubated in the dark at room temperature for 16 h. In a 96-well microplate, 20 µL of MK-7–ZnO NPs was mixed with 180 µL of the ABTS· + solution, and the mixture was incubated at room temperature for 10 min in the dark [36]. Absorbance was measured at 734 nm using a microplate reader. Ascorbic acid was used as a positive control in ABTS and CUPRAC assays [37].

Cupric-reducing antioxidant capacity (CUPRAC) assay
The working reagent for the CUPRAC assay was prepared by mixing equal volumes of 10 mM copper (II) chloride, 7.5 mM neocuproine, and 1 M ammonium acetate buffer (pH 7.0) [38]. In a 96-well microplate, 20 µL of MK-7–ZnO NPs was added to 180 µL of the working reagent and incubated at room temperature for 30 min. Absorbance was measured at 450 nm [39].

Anti-wrinkle (elastase inhibition) assay
20 µL of MK-7–ZnO NPs is mixed with 10 µL of a 6.25 mM N-succinyl-ala-ala-p-nitroanilide solution and 120 µL of 0.1 M Tris–HCl buffer (pH 8.0) in a 96-well microplate [40]. Then, 50 µL of elastase enzyme solution (0.1 mg/mL) is added to each well, and the mixture is incubated at room temperature for 15 min. Absorbance is measured at 405 nm [41]. Ursolic acid is used as a positive control.

Anti-inflammatory assay

MTT assay and nitric oxide (NO) production test
RAW 264.7 cells (murine macrophage-like cell line) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and incubated at 37 °C with 5% CO2 [42]. Different concentrations of MK-7–ZnO NPs were added to the cells for 24 h. A diluted MTT solution (0.2 mg/mL) was exposed to the medium, and the cells were incubated for 3 h. After this, dimethyl sulfoxide (DMSO) was added to evaluate the cell viability [43]. The inhibition of NO production was evaluated by incubating cells treated with non-cytotoxic concentrations of MK-7–ZnO NPs and 1 µg/mL lipopolysaccharide (LPS) for 24 h [44]. Cell viability and NO production inhibition were then measured by assessing absorbance at 570 nm [45].

Anticancer activity

Cytotoxicity assay
MDA-MB-231 human breast cancer cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, and maintained in an incubator at 37 °C with 5% CO2 [4]. The cells were plated in both 96-well and 6-well plates. A concentration of 250 µg/mL of the samples was used to treat the MDA-MB-231 cells [46]. The cytotoxicity of MK-7–ZnO NPs was evaluated using a two-fold serial dilution method to prepare concentrations ranging from 0 to 1000 µg/mL. MDA-MB-231 cells were plated at a density of 2 × 106 cells per well in 96-well plates [47]. Cell viability was assessed using the EZ-Cytox Kit (DogenBio, Seoul, Korea), based on a water-soluble tetrazolium salt (WST) assay. Plates were incubated for 2 h at 37 °C, and absorbance was measured at 450 nm using a microplate reader [48].

Flow cytometry analysis (FACS)
Apoptosis and necrosis were assessed by exposing cells to a 250 µg/mL concentration of MK-7–ZnO NPs and staining with Annexin V and propidium iodide (PI) for 30 min using the Annexin V FITC Apoptosis Detection Kit. Flow cytometry (FACS–Fluorescence-Activated Cell Sorting) was performed using an Accuri™ C6 Flow Cytometer (BD, NJ, USA) in the Bio-Health Materials Core-Facility, Jeju National University [49].

Cytokine secretion and mRNA expression analysis
IL-6 secretion in the culture supernatants of MDA-MB-231 cancer cells treated with MK-7–ZnO NPs was assessed using ELISA kits [50]. For IL-6 mRNA expression analysis, total RNA was extracted from the treated cells with an RNA extraction kit [51]. cDNA synthesis was performed, followed by Quantitative Polymerase Chain Reaction (qPCR) using the SYBR Green RT-qPCR Kit, with RNA as the template, to quantify IL-6 expression levels [52].

Statistical analysis
The experimental results are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Prism Inc.) [46]. Data were analyzed using one-way ANOVA, and differences were considered significant if the p value was less than 0.05. The significance thresholds were as follows: *p < 0.1, **p < 0.01, ***p < 0.001 [53].

Results and discussion

UV–vis spectrum analysis
A color change occurred upon the addition of the Nostoc sp. MK-7 extract to the Zn(CH3COO)2·2H2O) solution, indicating the formation of MK-7–ZnO NPs [2]. The reaction mixture exhibited a shift in color from white to yellowish-brown over 0-, 2-, and 4-h intervals. The color change is due to the reduction of zinc ions (Zn2+) to zinc atoms (Zn0). The gradual increase in color intensity suggests the formation of MK-7–ZnO NPs, resulting from the reduction of Zn2+ ions by phytochemicals present in the Nostoc sp. MK-7 extract [54]. These findings align with previous reports on the synthesis of ZnO NPs using various plant extracts, where color changes were attributed to the reduction of Zn2+ ions by bioactive compounds [28]. The phycocyanin in the Nostoc sp. MK-7 extract play a key role in the synthesis of MK-7–ZnO NPs [47]. The UV–vis spectrum showed increased absorbance over time intervals, confirming the presence of MK-7–ZnO NPs, as depicted in Fig. 2A. A distinct hump at 312 nm was observed in the UV–vis spectrum of MK-7–ZnO NPs, showing MK-7–ZnO NPs formation, as ZnO typically exhibits absorption in the UV region due to its wide band-gap energy as depicted in Fig. 2B. A previous study showed that the same 312 nm absorption peak in the UV–vis spectrum of ZnO NPs synthesized with Ipomoea hederifolia is a characteristic signature of their synthesis, often indicating a blue shift from the bulk material's band gap, which can be influenced by factors like particle size, with smaller particles causing a further blue shift [47]. The slight shift in the absorption peak compared to the typical ZnO band edge (around 360–380 nm) is attributed to the overlapping absorption from phytochemicals present in the MK-7 extract. The phytochemicals compounds act as reducing and stabilizing agents, facilitating the synthesis of MK-7–ZnO NPs [55].

FTIR analysis
The FTIR spectrum of Nostoc sp. MK-7 extract showed peaks at 3512, 2964, 2410, 2083, 1640, 1332, and 1054 cm−1, corresponding to O–H, C–H, C = O, C≡C/C≡N, C = O, C–N, and C–O, respectively. These functional groups indicate the presence of bioactive compounds in the MK-7 extract as illustrated in Fig. 3A [56]. The FTIR analysis of MK-7 ZnO NPs shows distinct absorption bands at 3393, 1662, 1473, 1415, and 906 cm−1. The broad band at 3393 cm−1 corresponds to the O–H stretching vibration, indicating the presence of hydroxyl groups on the MK-7 ZnO NPs surface [1]. The broadness of this peak suggests hydrogen bonding, which result from surface hydroxyl groups [57]. The absorption band at 1662 cm−1 is attributed to C = O stretching vibrations of carbonyl groups from organic molecules associated with the MK-7 ZnO NPs surface, implying the presence of biomolecules that have acted as capping agents during green synthesis of NPs [4]. The bands at 1473 cm−1 and 1415 cm−1 correspond to C–O stretching and bending vibrations of carboxylate and phenolic groups, further confirming the association of organic functional groups with the ZnO NPs [10]. A band observed at 906 cm−1 is assigned to the Zn–O stretching vibration, which confirms the formation of MK-7 ZnO NPs as indicated in Fig. 3A. A recent study demonstrated that zinc ions from salt precursors interact with oxygen-rich functional groups in Nostoc sp. extracts, leading to the biosynthesis of ZnO NPs [47]. The presence of these characteristic functional groups indicates that organic residues from the biogenic green synthesis process contribute to the surface stabilization and functionalization of the MK-7 ZnO NPs [58].

X-ray diffraction (XRD) analysis
The XRD patterns of Nostoc sp. MK-7 extract and MK-7 ZnO NPs are shown in Fig. 3B, covering a 2θ range of 0–90°. The Nostoc sp. MK-7 extract exhibits diffraction peaks at 29.12°, 31.6°, 35°, 38.7°, 42.3°, 47.6°, and 55.3°, which can be attributed to the crystalline components present in the Nostoc sp. MK-7 extract, likely arising from biomolecules and residual inorganic salts [47]. These peaks are relatively broad, suggesting low crystallinity of the extract. After the green synthesis with Nostoc sp. MK-7 extract, the MK-7 ZnO NPs show distinct peaks at 31.7°, 34.3°, 36.2°, 47.4°, 56.4°, 62.7°, and 67.8°, corresponding to the (100), (002), (101), (102), (110), (103), and (200) planes, respectively, as shown in Fig. 3 (B). These peaks confirm the formation of hexagonal wurtzite MK-7–ZnO NPs, which is the characteristic crystalline phase of ZnO NPs [59]. The intense peak at 36.2° (101 plane) indicates the preferred growth orientation, while the (002) plane suggests anisotropic growth along the c-axis [60]. The sharp peaks reflect the high crystallinity of the MK-7–ZnO NPs, in contrast to the broader peaks observed in the Nostoc sp. MK-7 extract. A similar diffraction peak at 36.2° was observed in a study on ZnO nanoparticles synthesized using aqueous fruit extracts of Myristica fragrans [47]. No additional peaks were observed in the MK-7–ZnO NPs pattern, indicating phase purity with no detectable impurities from the Nostoc sp. MK-7 extract during the synthesis process. The differences in peak positions and intensities between the extract and the MK-7 ZnO NPs suggest that the Nostoc sp. MK-7 extract acted as a stabilizing and capping agent, facilitating the formation of uniform MK-7–ZnO NPs while interacting with their surface [8]. The Debye–Scherrer equation is used to determine the average crystallite size of the MK-7 ZnO NPs. The (101) diffraction peak at 36.2° 2θ, the prominent peak in the MK-7 ZnO NPs XRD pattern, was selected for the calculation. The Bragg angle was calculated as 18.1°, and the full width at half maximum (FWHM) of the peak is 0.2° [61]. The crystallite size was then calculated using the following formula:where:D = crystallite size (nm). K = (0.9 shape factor). λ  = 0.15406 nm (Cu Kα wavelength). β = 0.2 (FWHM, converted to radians). θ  = 18.1 (Bragg angle, half of 2θ, in radians)
Using the above values, the crystallite size was calculated to be approximately 42 nm. This result indicates that the MK-7 ZnO NPs have uniform size distribution, confirming the effectiveness of the green synthesis method using Nostoc sp. MK-7 extract. A study demonstrated that in Sargassum muticum, ZnO NPs were synthesized using zinc acetate dihydrate, resulting in 42 nm hexagonal, agglomerated NPs [59]. In Myristica fragrans, the same method produced 41.23 nm semi-spherical ZnO NPs [62]. This research on Nostoc sp. also used zinc acetate dihydrate, yielding 41.9 nm hexagonal, agglomerated MK-7 ZnO NPs with a crystalline structure.

Raman analysis
The Raman spectrum of MK-7–ZnO NPs exhibited several distinct peaks across the Raman shift range of 200–1600 cm−1, confirming the formation of ZnO with contributions from MK-7 organic components. The low-frequency peak at 98 cm−1 corresponds to the E2 (low) phonon mode, characteristic of lattice vibrations in the wurtzite ZnO structure [47]. The peak at 326 cm−1 is assigned to the E2 (high) mode, a prominent fingerprint of the wurtzite ZnO crystal structure as shown in Fig. 3C. The band observed at 434 cm−1 also corresponds to E2 (high) vibrations, reinforcing the crystalline nature of the NPs [30]. The peak at 715 cm−1 is attributed to second-order or combination vibrational modes, possibly influenced by interactions between the ZnO lattice and organic molecules from the Nostoc sp. MK-7 extract. A peak at 1117 cm−1 is similarly assigned to higher-order phonon modes or surface vibrations involving capping biomolecules [63]. A previous study showed similar results for ZnO NPs in the antifungal potential of ZnO and MoS2 NPs against Fusarium oxysporum and Fusarium graminearum. The Raman spectra of ZnO NPs exhibited sharp peaks at 330 cm−1, and 437 cm−1 [30]. The presence of higher-order and organic-related peaks suggests that phytochemicals from the MK-7 extract participate in surface stabilization and functionalization of the MK-7–ZnO NPs, supporting the efficacy of the green synthesis approach [64].

FESEM, TEM and EDS analysis
The morphological and structural characteristics of MK-7–ZnO NPs were investigated using FESEM, TEM, SAED, and EDS analyses. The FESEM images show that the MK-7–ZnO NPs are irregular in shape, with particles appearing to have a roughly hexagonal structure as shown in Fig. 4A [61]. The MK-7–ZnO NPs are in the size range of 500 nm to 2 µm, with significant variation in their agglomeration and surface roughness. The TEM and High-Angle Annular Dark-Field (HAADF) images provide more detailed information on the size and internal structure of the MK-7–ZnO NPs, confirming that the majority of the particles are within the nanometer range as shown in Fig. 4B, C. Some of the particles appear to be spherical, while others are more elongated, with sizes varying between 100 and 200 nm [61]. This size distribution is consistent with the findings from the XRD analysis, which suggests a crystalline structure with an average particle size of approximately 41.9 nm based on the Scherrer equation. The selected area electron diffraction (SAED) pattern shows distinct diffraction rings corresponding to specific crystal planes, including (100), (002), and (110), confirming the crystalline nature of the MK-7–ZnO NPs as indicated in Fig. 4D [65]. These peaks indicate that the NPs have a hexagonal wurtzite structure, which is typical for ZnO NPs. EDS confirmed the presence of zinc (Zn), oxygen (O), and carbon (C).The pronounced peaks for Zn and O validate the formation of ZnO, while the significant carbon content indicates the presence of organic residues from Nostoc sp. MK-7 extract, which acted as a reducing and stabilizing agent during synthesis as shown in Fig. 4E–I [8]. The EDS analysis of MK-7–ZnO NPs shows 5.94 wt% and 19.88 at% carbon (C), 11.74 wt% and 29.50 at% oxygen (O), and 82.32 wt% and 50.62 at% zinc (Zn). The net intensities were 130.90 for C, 334.10 for O, and 1346.10 for Zn, with low net errors (C: 1.12%, O: 0.73%, Zn: 0.31%) as showed in Supplementary Table 1. A recent study demonstrated that zinc (Zn) and oxygen (O) constitute the primary components of ZnO NPs biosynthesized by the cyanobacterium Nostoc sp. [47]. Furthermore, another study identified Zn and O as the essential elements in ZnO NPs synthesized using strawberry waste extract [66]. These results confirm synthesis of MK-7–ZnO NPs, with Zn and O as the main components and enhancing stability and surface functionalization [33]. The particles display a wurtzite structure, and with some spherical particles, suggesting potential for improved surface area, bioactivity, and biomedical applications [67].

Antioxidant activity

Ferric-reducing antioxidant power (FRAP) assay
The FRAP assay measured the ability of antioxidants present in MK-7–ZnO NPs to reduce ferric (Fe3+) ions to ferrous (Fe2+) ions. As antioxidants in the MK-7–ZnO NPs reduce Fe3+-TPTZ complexes to Fe2+-TPTZ complexes, they change color to blue, which allows for quantification of antioxidant activity [68]. Ursolic acid was used as a positive control, and a standard curve was constructed using FeSO₄ to analyze the results. The results showed that both Nostoc sp. MK-7 and MK-7 ZnO NPs exhibited antioxidant activity in a concentration-dependent manner, with MK-7 ZnO NPs having a higher reducing power due to their better ability to reduce Fe3+ to Fe2+ [42]. The FRAP results showed a clear concentration-dependent increase in antioxidant activity for all samples. At lower concentrations (20–40 µg/mL), all samples exhibited low reducing power, which gradually increased with concentration. At 50 µg/mL, the MK-7–ZnO NPs showed a marked rise in reducing activity compared to the Nostoc sp. MK-7 extract, though still below that of ascorbic acid [69]. At higher concentrations (80–100 µg/mL), the MK-7–ZnO NPs displayed significantly greater ferric reducing power than the Nostoc sp. MK-7 extract, indicating enhanced antioxidant potential, while ursolic acid remained the most potent overall, as shown in Fig. 5A.

ABTS radical scavenging assay
Both Nostoc sp. MK-7 and MK-7 ZnO NPs exhibited concentration-dependent ABTS radical scavenging activity. Ascorbic acid was used as a positive control [36]. Within the concentration range of 1.56–100 μg/mL, Nostoc sp. MK-7 showed ABTS radical scavenging rates of 1.85%, 3.69%, 3.76%, 5.42%, 14.16%, 26.30%, and 31.53%, while MK-7 ZnO NPs achieved rates of 6.84%, 9.00%, 14.22%, 16.93%, 27.03%, 46.80%, and 89.04%. The findings indicated that MK-7 ZnO NPs exhibited antioxidant activity with an IC50 value of 35 ± 5 µg/mL, whereas Nostoc sp. MK-7 had an IC50 value greater than 100 µg/mL, as illustrated in Fig. 5B [17]. At the highest concentration (100 μg/mL), Nostoc sp. MK-7 and MK-7 ZnO NPs demonstrated radical scavenging activities of 31.53% and 89.04%, respectively, indicating that MK-7 ZnO NPs exhibit superior ABTS radical scavenging activity compared to Nostoc sp. MK-7 [22].

Cupric-reducing antioxidant capacity (CUPRAC) assay
CUPRAC assay is based on the reaction of copper (II) ions being reduced to copper (I) ions by an antioxidant and the color change due to copper (I) ion formation is used to evaluate the antioxidant capacity quantitatively. Ascorbic acid was used as a positive control, and the results were expressed as absorbance values at 450 nm [22]. The experimental results showed that both Nostoc sp. MK-7 and MK-7 ZnO NPs exhibited antioxidant potential in a concentration-dependent manner, especially MK-7 ZnO NPs showed better antioxidant capacity as compared to Nostoc sp. MK-7 extract as illustrated in Fig. 5C [39].
The antioxidant potential of MK-7–ZnO NPs and Nostoc sp. MK-7 extract was evaluated through three different assays. Data are presented as the mean ± standard deviation (SD) from three independent experiments. (A) Ferric-Reducing Antioxidant Power (FRAP) assay, with ursolic acid as the control. (B) ABTS Radical Scavenging Activity, with ascorbic acid as the positive control. (C) CUPRAC assay, with ascorbic acid as the positive control.

Anti-wrinkle activity

Elastase inhibition assay
The elastase inhibitory activity test assessed the ability of MK-7–ZnO NPs to inhibit elastin degradation, a protein crucial for maintaining the elasticity of human skin, with ursolic acid used as a positive control [40]. At concentrations of 1.56–100 μg/mL, Nostoc sp. MK-7 showed inhibitory activity of 2.42%, 9.45%, 9.51%, 13.79%, 13.87%, 27.91%, and 37.28%, respectively, while MK-7 ZnO NPs exhibited 5.63%, 12.91%, 23.31%, 35.49%, 45.08%, 46.62%, and 50.55% inhibitory activity, demonstrating concentration-dependent inhibition of elastase enzyme activity, as shown in Fig. 6. The IC50 value for Nostoc sp. MK-7 is greater than 100 µg/mL, as it failed to reach 50% inhibition at any concentration tested [3]. In contrast, the IC50 value for MK-7 ZnO NPs was calculated to be 55 ± 5 µg/mL, demonstrating a stronger elastase inhibition effect compared to Nostoc sp. MK-7. At 100 μg/mL, the inhibitory activity of Nostoc sp. MK-7 and MK-7 ZnO NPs was 37.28% and 50.55%, respectively, further confirming the superior enzyme inhibition exhibited by MK-7 ZnO NPs [22].

Anti-inflammation activity

MTT assay and NO production test
The cytotoxicity and NO production inhibition effects of Nostoc sp. MK-7 and MK-7 ZnO NPs were evaluated in anti-inflammatory experiments using RAW 264.7 cells [70]. An MTT assay was performed to confirm the cytotoxicity of Nostoc sp. MK-7, and no toxicity was observed at concentrations below 40 μg/mL. Accordingly, the inhibitory effect on NO production was analyzed at concentrations below 40 μg/mL, revealing NO production inhibition rates of 5.96%, 9.20%, 11.11%, and 32.44% at concentrations of 5, 10, 20, and 40 μg/mL, respectively as depicted in Fig. 7A. The MTT assay conducted to identify the cytotoxicity of MK-7 ZnO NPs showed no toxicity at concentrations below 20 μg/mL, so the NO production inhibition effect was analyzed at concentrations below 20 μg/mL [71]. As a result, NO production inhibition rates of 8.90%, 13.41%, 32.57%, and 83.61% were observed at concentrations of 2.5, 5, 10, and 20 μg/mL, respectively, as depicted in Fig. 7B. These results confirmed that MK-7 ZnO NPs exhibited a higher NO production inhibition effect compared to Nostoc sp. MK-7 [72].

Cytotoxic and anti-cancer effects of MK-7 ZnO NPs

Cytotoxicity evaluation
The cytotoxic effects of Nostoc sp. MK-7 and MK-7 ZnO NPs were assessed in MDA-MB-231 cancer cells using a proliferation assay (% of control), as shown in Fig. 8A [16]. The assay revealed that both Nostoc sp. MK-7 and MK-7 ZnO NPs exerted dose-dependent cytotoxic effects, with a more pronounced effect observed in cells treated with MK-7 ZnO NPs. Notably, MK-7 ZnO NPs significantly reduced cell viability compared to Nostoc sp. MK-7 alone, suggesting that the addition of ZnO NPs enhances the cytotoxic potential of Nostoc sp. MK-7 [3]. This was in contrast to the Nostoc sp. MK-7 extract alone, which also showed dose-dependent cytotoxicity but to a lesser extent.

Cellular response and apoptosis analysis
To evaluate the mode of cell death, flow cytometry (FACS) was performed to assess the distribution of live, apoptotic, and necrotic cells after treatment with the different sample treatments (Fig. 8B) [73]. The flow cytometry results demonstrated that treatment with both Nostoc sp. MK-7 and MK-7 ZnO NPs led to an increased percentage of apoptotic and necrotic cells compared to the control group [74]. However, the MK-7 ZnO NPs-treated group showed a significantly higher proportion of apoptotic cells, suggesting that the incorporation of ZnO nanoparticles enhances the pro-apoptotic effects of Nostoc sp. MK-7. This was further corroborated by the Annexin V-FITC and propidium iodide (PI) staining, which highlighted the higher levels of apoptosis and necrosis induced by MK-7 ZnO NPs compared to Nostoc sp. MK-7 alone [7].

Cytokine secretion and mRNA expression
Cytokine secretion analysis was performed to evaluate the effect of Nostoc sp. MK-7 and MK-7 ZnO NPs on IL-6 secretion in MDA-MB-231 cells [75]. As shown in Fig. 8C, cells treated with MK-7 ZnO NPs exhibited a significant reduction in IL-6 secretion compared to those treated with Nostoc sp. MK-7 alone. The data suggest that MK-7 ZnO NPs effectively suppress IL-6 production, a cytokine associated with inflammation and tumor progression [76]. In addition to cytokine secretion, the mRNA expression levels of IL-6 were assessed via RT-qPCR [73]. Figure 8C shows a significant decrease in IL-6 mRNA expression in MDA-MB-231 cells treated with MK-7 ZnO NPs compared to those treated with Nostoc sp. MK-7. This reduction in IL-6 expression suggests that MK-7–ZnO NPs possess superior anti-inflammatory properties and enhance the anti-cancer effects of Nostoc sp. MK-7 [77].

Discussion
This study investigates the eco-friendly synthesis, characterization, and biological activities of MK-7–ZnO NPs derived from Nostoc sp. MK-7 extract [33]. The synthesis of MK-7–ZnO NPs was confirmed through a combination of analytical techniques, demonstrating the formation of MK-7–ZnO NPs with physical, chemical, and bioactive properties. The UV–vis spectrum exhibited a characteristic absorption peak at 312 nm, a typical signature for ZnO NPs, confirming their formation [28]. FTIR analysis revealed functional groups in the Nostoc sp. MK-7 extract, interactions between these bioactive compounds and ZnO that contribute to the stabilization of the MK-7–ZnO NPs [19]. Specifically, the O–H stretching vibrations observed at 3290 cm−1 in the extract shifted to 3391 cm−1 in the MK-7–ZnO NPs, indicating potential hydrogen bonding or coordination between the polyphenolic compounds and Zn2+ ions. These interactions are critical for NP stabilization and functionalization [78]. Previous studies reported characteristic peaks at 3535, 3000–3020, 1600, 1175, 1025, and 449 cm−1, corresponding to O–H, CH2/CH3, C = O, C–N, C–O–C, and Zn–O, respectively [13]. These findings confirm that biomolecules in cyanobacterial extracts act as reducing, stabilizing, and capping agents. Collectively, these observations support the green synthesis, stabilization, and functionalization of MK-7–ZnO NPs. XRD and Raman spectroscopy confirmed that the MK-7–ZnO NPs had a hexagonal wurtzite structure, with a crystallite size of approximately 42 nm, underscoring the high crystallinity and nanoscale properties essential for bioactivity [79]. The morphological characteristics, analyzed by FESEM and TEM, revealed spherical MK-7–ZnO NPs, with aggregation into larger clusters, potentially enhancing their surface area for more effective biological interactions [3]. The MK-7–ZnO NPs synthesized using Cyanobacterial Nostoc sp. MK-7 strain exhibit a crystalline structure with a hexagonal shape, as confirmed by the SAED pattern [20]. The particles are predominantly in the nanometer range, with a size distribution from 100 to 2 µm, and they appear to be agglomerated in some regions, due to the nature of the biosynthesis process. The XRD data supports the TEM and SAED observations, with a crystalline size of 41.9 nm. The green synthesis method using Cyanobacterial Nostoc sp. MK-7 appears to result in NPs that are both crystalline and exhibit a mix of spherical and irregular shapes, with a tendency to form aggregates [80]. These properties could be advantageous for applications in areas like drug delivery, where both size and crystal structure play significant roles in the functionality of the NPs [81]. EDS confirmed the presence of zinc and oxygen in the MK-7–ZnO NPs, corroborating their green synthesis [10]. The biological activities of MK-7–ZnO NPs were evaluated in a series of in vitro assays, which demonstrated promising antioxidant, anti-wrinkle, anti-inflammatory, and cytotoxic effects [82]. In antioxidant assays, including FRAP, ABTS, and CUPRAC, MK-7–ZnO NPs exhibited superior radical scavenging and reducing power compared to the Nostoc sp. MK-7 extract alone, suggesting that the incorporation of ZnO NPs enhances the antioxidant potential of the extract. This enhancement is due to the increased surface area and the unique properties of MK-7–ZnO NPs, which facilitate more effective interactions with reactive oxygen species (ROS) [83]. These findings position MK-7–ZnO NPs as a promising candidate for applications in oxidative stress-related conditions. In terms of anti-wrinkle effects, assessed by elastase inhibition, showed that MK-7–ZnO NPs were more effective in preserving skin elasticity and inhibiting wrinkle formation compared to the Nostoc sp. MK-7 extract alone [84]. These results suggest that the incorporation of ZnO NPs potentiates the biological effects of the Nostoc sp. MK-7 extract, expanding their utility in dermatological formulations aimed at both aging and pigmentation disorders [85]. While in anti-inflammatory properties the MTT assay and NO production tests revealed that MK-7–ZnO NPs significantly inhibited NO production in LPS-induced RAW 264.7 macrophages. The anti-inflammatory effects were concentration-dependent, with more pronounced inhibition observed at higher concentrations [86]. This suggests that MK-7–ZnO NPs could play a crucial role in mitigating inflammation-related conditions, which are often linked to various chronic diseases, including cancer [87]. Additionally, cytotoxicity evaluation, cellular response, apoptosis analysis, cytokine secretion and mRNA expression studies in MDA-MB-231 cancer cells revealed that MK-7–ZnO NPs effectively suppressed IL-6 secretion, a cytokine involved in tumor progression and chronic inflammation [23]. The reduction in IL-6 mRNA expression further supports the anti-inflammatory potential of MK-7–ZnO NPs, suggesting that these nanoparticles could have therapeutic applications in conditions characterized by excessive inflammation, such as cancer [88]. MK-7–ZnO NPs demonstrated dose-dependent cytotoxicity, with a more pronounced effect compared to the Nostoc sp. MK-7 extract [44]. Flow cytometry analysis showed that MK-7–ZnO NPs induced higher levels of apoptosis and necrosis in MDA-MB-231 cells, further confirmed by Annexin V-FITC and propidium iodide (PI) staining. These findings suggest that the incorporation of ZnO NPs not only potentiates the pro-apoptotic properties of the Nostoc sp. MK-7 extract but also enhances its ability to induce cell death in cancer cells [89]. Moreover, the superior suppression of IL-6 secretion by MK-7–ZnO NPs, a key cytokine associated with inflammation and cancer progression, was observed [90]. This further emphasizes the dual role of MK-7–ZnO NPs in reducing inflammation while inhibiting cancer cell proliferation, thus positioning them as a promising therapeutic agent for cancer treatment [46]. These results align with earlier research that has emphasized the anti-cancer potential of ZnO NPs, which are recognized for their ability to induce cytotoxicity through apoptosis and trigger changes in the tumor microenvironment. The mechanism of the anticancer potential of ZnO NPs is induced by ROS-mediated oxidative stress, causing mitochondrial dysfunction, altered Bax/Bcl-2 expression, caspase-9 and caspase-3 activation, DNA fragmentation, and apoptosis [91]. This is consistent with recent reports showing that ZnO NPs trigger intrinsic apoptotic pathways and cell cycle arrest in cancer models [92]. In this study, the green synthesis of MK-7–ZnO NPs with Nostoc sp. MK-7 extract enhanced their cytotoxic efficacy, particularly by modulating inflammatory pathways that contribute to cancer progression [93]. The synergistic effect between the Nostoc sp. MK-7 extract and ZnO NPs suggests that the combination could be particularly effective in targeting cancer therapy which are associated with metastasis and resistance to conventional therapies [94]. The suppression of IL-6 by MK-7–ZnO NPs further highlights their potential as an anti-cancer agent by modulating the inflammatory microenvironment, a critical factor in tumor progression. MK-7–ZnO NPs represent a promising multifunctional bioactive agent with potential in various therapeutic areas, including antioxidant therapy, skin care, anti-inflammatory treatments, and cancer therapy [95]. The synergistic effects of Nostoc sp. MK-7 extract and ZnO NPs enhance their biological activities, making them versatile for a wide range of applications [74]. Specifically, MK-7–ZnO NPs can modulate inflammatory responses and inhibit cancer cell proliferation, positioning them as potential candidates for cancer treatment, particularly for targeting cancer cells and reducing chronic inflammation [96]. Similar to MK-7–ZnO NPs, other biologically synthesized nanoparticles show comparable biomedical potential (Halymenia dilatata-derived CuO NPs, crystalline structure, and anticancer activity, HeLa, IC50 = 147.51 µg/mL [97]; Pleurotus djamor-mediated ZnO NPs, antioxidant and anticancer effects, A549, LC50 = 42.26 µg/mL [98]; Ocimum tenuiflorum-derived ZnO NPs, antibacterial and antibiofilm activity with biocompatibility [99]; Pleurotus sajor-caju-mediated ZnO NPs, anticancer, LC50 = 47.42 µg/mL, catalytic activity [100], comparable to MK-7 ZnO NPs inducing apoptosis and IL-6 suppression in MDA-MB-231 cells; ZnO-based heterostructures, ZnO/g-C3N4/V2O5, and CuFe2O4–rGO green nanocomposites, enhanced charge separation and multifunctional antibacterial, anticancer, and photocatalytic activities [101]. EPS-mediated green synthesis emphasizes the role of polysaccharides in nanoparticle stabilization [102], consistent with FTIR analysis of Nostoc sp. MK-7, which confirmed that bioactive compounds facilitate reduction and stabilization, enhancing the therapeutic efficacy of MK-7 ZnO NPs. However, the green synthesis of ZnO NPs faces several challenges in biomedical applications [16]. Variability in particle size and aggregation, due to inconsistencies in biological extracts, can reduce their effectiveness in in vitro, in vivo, and pre-clinical trials [103]. Additionally, the lack of precise control over the green synthesis process may result in batch-to-batch inconsistencies, hindering reproducibility and standardization. While green-synthesized ZnO NPs are more biocompatible than chemically synthesized ones, they can still exhibit toxicity, especially at higher concentrations, which necessitates optimization [104]. Furthermore, scalability remains a significant challenge due to the variable availability and quality of natural extracts, limiting large-scale production for clinical use. To fully evaluate the potential of MK-7–ZnO NPs, future studies should focus on elucidating the molecular mechanisms underlying their synergistic effects, particularly their impact on molecular and cellular pathways [10]. In vivo studies are mandatory to validate the safety and efficacy of MK-7–ZnO NPs in dynamic micro- and macro-physiological settings [105]. Additionally, investigating their effects across various cancer types, assessing their selectivity for cancer versus normal cells, and performing biological assays will be crucial for determining the broader applicability and efficacy of MK-7–ZnO NPs in cancer therapy [106].

Conclusion
In this study, MK-7–ZnO NPs were successfully synthesized using a green approach with Nostoc sp. MK-7 extract, producing stable, crystalline nanoparticles with nanoscale characteristics confirmed by UV–Vis, FTIR, XRD, Raman, FESEM, TEM, and EDS analyses. The MK-7–ZnO NPs exhibited enhanced biological activities, including strong antioxidant, anti-wrinkle, anti-inflammatory, and anti-cancer effects. Notably, the incorporation of ZnO NPs significantly potentiated the bioactivity of the Nostoc sp. MK-7 extract, with superior radical scavenging, enzyme inhibition related to skin aging, inhibition of pro-inflammatory cytokines, and induction of apoptosis in MDA-MB-231 breast cancer cells. These findings demonstrate the therapeutic and cosmetic potential of MK-7–ZnO NPs. The study highlights the synergistic effect of combining cyanobacterial bioactive compounds with ZnO NPs, offering a multifunctional nanomedicine platform for antioxidant therapy, skin care, anti-inflammatory applications, and cancer treatment. This work also emphasizes the eco-friendly and sustainable potential of Nostoc sp. MK-7 in biogenic nanoparticle synthesis. Future in vivo studies are warranted to validate the molecular mechanisms, biocompatibility, and clinical applicability of MK-7–ZnO NPs, paving the way for environmentally sustainable, bioactive nanomedicines with broad therapeutic and cosmetic implications.

Supplementary Information
Below is the link to the electronic supplementary material.