Hydrothermal Synthesis of β-Nb2ZnO6 Nanoparticles for Photocatalytic Degradation of Methyl Orange and Cytotoxicity Study

β-Nb2ZnO6 nanoparticles were synthesized by a hydrothermal process and calcined at two temperatures, 500 °C and 700 °C, and assigned as A and B, respectively. X-ray diffraction, together with transmission electron microscopy, revealed that the β-Nb2ZnO6 nanoparticles calcined at 700 °C (B) were more crystalline than the β-Nb2ZnO6 calcined at 500 °C (A) with both types of nanoparticles having an average size of approximately 100 nm. The physiochemical, photocatalytic, and cytotoxic activities of both types of β-Nb2ZnO6 nanoparticles (A and B) were examined. Interestingly, the photodegradation of methyl orange, used as a standard for environmental pollutants, was faster in the presence of the β-Nb2ZnO6 nanoparticles calcined at 500 °C (A) than in the presence of those calcined at 700 °C (B). Moreover, the cytotoxicity was evaluated against different types of cancer cells and the results indicated that both types of β-Nb2ZnO6 nanoparticles (A and B) exhibited high cytotoxicity against MCF-7 and HCT116 cells but low cytotoxicity against HeLa cells after 24 and 48 h of treatment. Overall, both products expressed similar EC50 values on tested cell lines and high cytotoxicity after 72 h of treatment. As a photocatalyst, β-Nb2ZnO6 nanoparticles (A) could be utilized in different applications including the purification of the environment and water from specific pollutants. Further biological studies are required to determine the other potential impacts of utilizing β-Nb2ZnO6 nanoparticles in the biomedical application field.


Introduction
Nanotechnology has inspired the design of diverse nanoscale materials to fulfill the growing material requirements for environmental, biological, and medical applications [1][2][3][4][5][6]. Environmental pollution has become a serious global concern due to the rapid increase in population and industries. Most industrial wastes contain high concentrations of toxic substances that accumulate and negatively affect living creatures and the environment [7][8][9][10]. Therefore, designing nano-photocatalysts is needed to develop stable and effective photocatalysts for different types of organic pollutants. Recently, semiconductor photocatalysts have been extensively used for the purification of organic pollutants in water. TiO 2 , for example, is used as a catalyst for the photodegradation of organic pollutants, but with customized application only as it has a wide band gap, poor catalyst recovery, and a tendency to agglomerate [11,12]. Previously, we reported the catalytic degradation of volatile chlorinated compounds by heterostructured nanoparticles which showed enhanced catalytic activity [8,[13][14][15][16][17][18]. Nanomaterials have also been used in the field of medicine and

Characterization of β-ZnNb 2 O 6 Nanoparticles
The morphology of the β-Nb 2 ZnO 6 (A) and (B) nanoparticles was determined by TEM. Figure 1a,b displays representative images of β-Nb 2 ZnO 6 nanoparticles (A and B) calcined at 500 • C (A) and 700 • C (B), respectively. The average size of the β-Nb 2 ZnO 6 (A) and (B) nanoparticles was 100 nm with nanoplate-like morphology. XRD was employed to find out the crystal structure of the β-Nb 2 ZnO 6 (A) and (B) nanoparticles. Figure 1c shows the XRD configuration of β-Nb 2 ZnO 6 calcined at 500 • C (A) and 700 • C (B). The results indicate that β-Nb 2 ZnO 6 nanoparticles calcined at 700 • C (B) have more crystalline features than β-Nb 2 ZnO 6 calcined at 500 • C (A). This further suggested that crystallinity can be achieved by calcining the sample at a high temperature. The peaks are well-matched with ICDD card no. 00-028-1477 with an unknown crystal system.
where α represents absorption coefficient, h the Plank's constant, v the light frequency, A an energy independent proportionality constant characteristic of the material, and Eg the optical band gap. The exponent n defines the nature of the optical transitions. The direct transition band gaps were determined from the Tauc plots of (α hv) 2 vs. hv, (Figure 1f,g). The optical band gap Eg values for β-Nb2ZnO6 nanoparticles (A and B) were 3.58 and 3.66 eV, respectively. The composition of the product was further confirmed by EDX analysis. The represented EDX spectrum and elemental mapping are shown in Figure 2. It is clear from Figure 2 that the product contains Nb, Zn, and O. K and L correspond to the amount of energy possessed by the X-ray emitted by an electron in the K and L shells, respectively. Physical characterization of the β-Nb 2 ZnO 6 nanoparticles. TEM images of β-Nb 2 ZnO 6 nanoparticles calcined at 500 • C (A) and 700 • C (B) (a,b), XRD pattern of β-Nb 2 ZnO 6 (A and B) (c), N2 adsorption-desorption isotherm of β-Nb 2 ZnO 6 (A and B) (d), UV-Vis spectra of β-Nb 2 ZnO 6 (A and B) (e), Tauc plots of β-Nb 2 ZnO 6 (A and B) (f,g). N 2 -adsorption-desorption isotherm examination was completed to review the pore size and surface area of the β-Nb 2 ZnO 6 nanoparticles. Figure 1d shows the N 2 sorption isotherms and porosity of the β-Nb 2 ZnO 6 nanoparticles. The typical type-IV isotherm with a narrow H3-type hysteresis loop in Figure 1e suggested that the particles were mesoporous [40]. The BET surface area of β-Nb 2 ZnO 6 calcined at 500 • C (A) was calculated to be 22.67 m 2 /g (pore size: 18.95 nm; pore volume 0.130 cm 3 /g), while the sample calcined at 700 • C (B)was found to have a surface area of 20.81 m 2 /g (pore size: 19.06 nm; pore volume 0.135 cm 3 /g). The slightly smaller surface area of β-Nb 2 ZnO 6 nanoparticles calcined at 700 • C (B) is due to the improvement in the crystalline structure of the β-Nb 2 ZnO 6 nanoparticles at high temperature. Figure 1e displays the UV-visible absorption spectrum of β-Nb 2 ZnO 6 nanoparticles (A and B) recorded in the range of 200-700 nm. The band gap can be calculated by the Tauc formula: where α represents absorption coefficient, h the Plank's constant, v the light frequency, A an energy independent proportionality constant characteristic of the material, and E g the optical band gap. The exponent n defines the nature of the optical transitions. The direct transition band gaps were determined from the Tauc plots of (α hv) 2 vs. hv, (Figure 1f,g).
The optical band gap E g values for β-Nb 2 ZnO 6 nanoparticles (A and B) were 3.58 and 3.66 eV, respectively. The composition of the product was further confirmed by EDX analysis. The represented EDX spectrum and elemental mapping are shown in Figure 2. It is clear from Figure 2 that the product contains Nb, Zn, and O. K and L correspond to the amount of energy possessed by the X-ray emitted by an electron in the K and L shells, respectively. Zeta potential is an imperative technique for determining the surface charge and stability of nanoparticles and can tell us the state of nanoparticle surface nature. The zeta potential of β-Nb2ZnO6 nanoparticles (A and B) is shown in Figure 3; both β-Nb2ZnO6 nanoparticles (A and B) have a less variation in the zeta potential and it was observed at −12.9 and −13 mV, respectively, while the polydispersity index (PDI) of β-Nb2ZnO6 nanoparticles (B) (PDI: 0.518) was slightly decreased as compared to β-Nb2ZnO6 nanoparticles (A) (PDI: 0.690). Similarly, the particle size distribution of β-Nb2ZnO6 nanoparticles (A and B) was observed at 517 and 453 nm, respectively. Results indicated almost similar stability of β-Nb2ZnO6 nanoparticles (A and B) in the deionized water. Zeta potential is an imperative technique for determining the surface charge and stability of nanoparticles and can tell us the state of nanoparticle surface nature. The zeta potential of β-Nb 2 ZnO 6 nanoparticles (A and B) is shown in Figure 3; both β-Nb 2 ZnO 6 nanoparticles (A and B) have a less variation in the zeta potential and it was observed at −12.9 and −13 mV, respectively, while the polydispersity index (PDI) of β-Nb 2 ZnO 6 nanoparticles (B) (PDI: 0.518) was slightly decreased as compared to β-Nb 2 ZnO 6 nanoparticles (A) (PDI: 0.690). Similarly, the particle size distribution of β-Nb 2 ZnO 6 nanoparticles (A and B) was observed at 517 and 453 nm, respectively. Results indicated almost similar stability of β-Nb

Photocatalytic Activity of β-Nb2ZnO6 Nanoparticles
The photodegradation potential of β-Nb2ZnO6 nanoparticles (A and B) was tested using methyl orange as a typical environmental pollutant under UV-Vis light irradiation. As evident in Figure 4a, the absorption peak at 465 nm, which indicated the presence of methyl orange, decreased in the presence of β-Nb2ZnO6 nanoparticles (A) with increased reaction time. However, for sample B, the decrease in the absorption peak at 465 nm was small, even after long reaction times (Figure 4b). The photodegradation of methyl orange proceeded faster with β-Nb2ZnO6 nanoparticles calcined at 500 °C (A) (Figure 4c) compared to β-Nb2ZnO6 nanoparticles calcined at 700 °C (B). The photocatalytic degradation of methyl orange without β-Nb2ZnO6 nanoparticles was also studied; these results indicated that there was no degradation of methyl orange without β-Nb2ZnO6 nanoparticles, demonstrating that photocatalytic degradation of methyl orange can be achieved with β-Nb2ZnO6 nanoparticles (A) (Figure 4c). The photocatalytic efficiency of β-Nb2ZnO6 nanoparticles (A) was higher as compared to MO and β-Nb2ZnO6 nanoparticles (B) (Fig-Figure 3. Zeta potential of β-Nb 2 ZnO 6 nanoparticles (A and B).

Photocatalytic Activity of β-Nb 2 ZnO 6 Nanoparticles
The photodegradation potential of β-Nb 2 ZnO 6 nanoparticles (A and B) was tested using methyl orange as a typical environmental pollutant under UV-Vis light irradiation. As evident in Figure 4a, the absorption peak at 465 nm, which indicated the presence of methyl orange, decreased in the presence of β-Nb 2 ZnO 6 nanoparticles (A) with increased reaction time. However, for sample B, the decrease in the absorption peak at 465 nm was small, even after long reaction times ( Figure 4b). The photodegradation of methyl orange proceeded faster with β-Nb 2 ZnO 6 nanoparticles calcined at 500 • C (A) (Figure 4c) compared to β-Nb 2 ZnO 6 nanoparticles calcined at 700 • C (B). The photocatalytic degradation of methyl orange without β-Nb 2 ZnO 6 nanoparticles was also studied; these results indicated that there was no degradation of methyl orange without β-Nb 2 ZnO 6 nanoparticles, demonstrating that photocatalytic degradation of methyl orange can be achieved with β-Nb 2 ZnO 6 nanoparticles (A) (Figure 4c). The photocatalytic efficiency of β-Nb 2 ZnO 6 nanoparticles (A) was higher as compared to MO and β-Nb 2 ZnO 6 nanoparticles (B) (Figure 4d). The high photocatalytic activity of the β-Nb 2 ZnO 6 nanoparticles (A) could be credited to the highly effective separation of carrier charges as well as the slowed recombination of electron-hole pairs during the photocatalysis process causing high photocatalytic activity [41,42]. It has been noticed that various constraints such as pore dimension and formation and surface features and crystallinity affect the photocatalytic ability. The lesser photocatalytic ability of sample B may be caused by a decline in surface area, destruction of pores at high temperature, crystal growth, and shielding of active sites on the surface of sample B. This may result in a decrease in surface-active sites on the surface and a reduction in charge separation/transfer, which in turn causes low photocatalytic activity [40]. Furthermore, the kinetics of methyl orange degradation was evaluated to establish the rate constants and was found to follow pseudo-first order reactions: where C 0 and C are the initial and the time-dependent concentrations of methyl orange, t is the time (minutes) and k is the rate constant (min −1 ). As it is clear from Figure 4e), β-Nb 2 ZnO 6 nanoparticles (A) revealed a high rate constant (0.0351 min −1 ) in the degradation of methyl orange as compared to β-Nb 2 ZnO 6 nanoparticles (B) and blank methyl orange. ure 4d). The high photocatalytic activity of the β-Nb2ZnO6 nanoparticles (A) could be credited to the highly effective separation of carrier charges as well as the slowed recombination of electron-hole pairs during the photocatalysis process causing high photocatalytic activity [41,42]. It has been noticed that various constraints such as pore dimension and formation and surface features and crystallinity affect the photocatalytic ability. The lesser photocatalytic ability of sample B may be caused by a decline in surface area, destruction of pores at high temperature, crystal growth, and shielding of active sites on the surface of sample B. This may result in a decrease in surface-active sites on the surface and a reduction in charge separation/transfer, which in turn causes low photocatalytic activity [40]. Furthermore, the kinetics of methyl orange degradation was evaluated to establish the rate constants and was found to follow pseudo-first order reactions: where C0 and C are the initial and the time-dependent concentrations of methyl orange, t is the time (minutes) and k is the rate constant (min −1 ). As it is clear from Figure 4e), β-Nb2ZnO6 nanoparticles (A) revealed a high rate constant (0.0351 min −1 ) in the degradation of methyl orange as compared to β-Nb2ZnO6 nanoparticles (B) and blank methyl orange. Photocatalysis experiments were further conducted to understand the role of reactive species during the photocatalysis process. Scavenging agents such as isopropanol, p-benzoquinone, and ethylenediaminetetraacetic acid (EDTA) in the presence of β-Nb2ZnO6 nanoparticles (A) were used applying the same reaction conditions ( Figure  5a). It was perceived that when EDTA was introduced in the reaction, there was a slight Photocatalysis experiments were further conducted to understand the role of reactive species during the photocatalysis process. Scavenging agents such as isopropanol, p-benzoquinone, and ethylenediaminetetraacetic acid (EDTA) in the presence of β-Nb 2 ZnO 6 nanoparticles (A) were used applying the same reaction conditions (Figure 5a). It was perceived that when EDTA was introduced in the reaction, there was a slight decline in the photocatalytic activity, suggesting the limiting role of the hole (h + ) in photocatalysis. Additionally, when p-benzoquinone was used in the reaction, there was more decline in the photocatalytic activity, indicating that reactive superoxide radical (•O 2 − ) performs an important role in photocatalysis process. Similarly, an isopropanol addition in the reaction resulted in a further decline in the photocatalytic activity, signifying that hydroxyl (•OH) plays a significant role in the photocatalytic degradation of methyl orange [43]. decline in the photocatalytic activity, suggesting the limiting role of the hole (h + ) in photocatalysis. Additionally, when p-benzoquinone was used in the reaction, there was more decline in the photocatalytic activity, indicating that reactive superoxide radical (•O2 − ) performs an important role in photocatalysis process. Similarly, an isopropanol addition in the reaction resulted in a further decline in the photocatalytic activity, signifying that hydroxyl (•OH) plays a significant role in the photocatalytic degradation of methyl orange [43]. The recycling/reusability of a photocatalyst is important in the photocatalysis process, so we have tested the stability of β-Nb2ZnO6 nanoparticles (A) by recycling experiments. The same experimental conditions were used as in the photocatalytic experiment, except that after each run β-Nb2ZnO6 was separated, and fresh methyl orange was used. We performed three recycling experiments to check the stability of the β-Nb2ZnO6 nanoparticles (A). As it is clear from Figure 5b, the differences in photocatalytic activity after each recycling were minor, indicating the stability of β-Nb2ZnO6 nanoparticles.
The mechanism of charge transfer on β-Nb2ZnO6 nanoparticles is illustrated in Figure 6, that is, the generation of electron-hole pairs (e − /h + ), under UV light irradiation, in the conduction and valence bands of β-Nb2ZnO6 nanoparticles, respectively. The electrons in the conduction band of β-Nb2ZnO6 nanoparticles reduce the molecular oxygen to •O2 − and holes on the valence band interact with − OH to produce •OH radicals, triggering the reduction and degradation of methyl orange [18]. The recycling/reusability of a photocatalyst is important in the photocatalysis process, so we have tested the stability of β-Nb 2 ZnO 6 nanoparticles (A) by recycling experiments. The same experimental conditions were used as in the photocatalytic experiment, except that after each run β-Nb 2 ZnO 6 was separated, and fresh methyl orange was used. We performed three recycling experiments to check the stability of the β-Nb 2 ZnO 6 nanoparticles (A). As it is clear from Figure 5b, the differences in photocatalytic activity after each recycling were minor, indicating the stability of β-Nb 2 ZnO 6 nanoparticles.
The mechanism of charge transfer on β-Nb 2 ZnO 6 nanoparticles is illustrated in Figure 6, that is, the generation of electron-hole pairs (e − /h + ), under UV light irradiation, in the conduction and valence bands of β-Nb 2 ZnO 6 nanoparticles, respectively. The electrons in the conduction band of β-Nb 2 ZnO 6 nanoparticles reduce the molecular oxygen to •O 2 − and holes on the valence band interact with − OH to produce •OH radicals, triggering the reduction and degradation of methyl orange [18].

Cytotoxicity of β-Nb 2 ZnO 6 Nanoparticles
The cytotoxicity of β-Nb 2 ZnO 6 nanoparticles had been recorded by estimating the cell viability via MTT assay [17,18]. MCF-7, HCT116, and HeLa cells were treated with pre-determined concentrations (1, 0.5, 0.25, and 0.125 mg/mL) of β-Nb 2 ZnO 6 nanoparticles (A) and (B) for 24, 48, and 72h, followed by the cells incubating in an MTT solution, which utilizes a formazan, colorimetric reduction of tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by living cells, producing purple crystals which, after dissolving in DMSO, were assayed at 570 nm on Elisa microplate reader. The assay exposed the increased cytotoxic potential of nanoparticles A at 0.5 and 1 mg/mL against MCF-7 and HCT116 cells with 30-47% cell viability and over 50% cell viability at the reduced nanoparticle concentrations following 24 and 48 h of treatment (Figure 7a (Figure 9). Conclusively, the studies showed high cytotoxic action of β-Nb 2 ZnO 6 nanoparticles (A) and (B) for MCF-7 and HCT116 cells and low cytotoxic action for HeLa cells, for which there is a need to explore further biomolecular assays to ascertain reasons for variable cytotoxicity. The present data, however, confirms that β-Nb 2 ZnO 6 (A) and (B) have a close cytotoxic activity and further optimization is required for the enhancement of biocompatibility of β-Nb 2 ZnO 6 for large-scale utilization.

Imaging of Nb 2 ZnO 6 Nanoparticles Treated Cells
The morphological impacts of β-Nb 2 ZnO 6 nanoparticles (A and B) on treated cells were indicated by imaging using confocal microscopy. MCF-7, HCT116, and HeLa cells together with 0.25, 0.5, and 1 mg/mL of β-Nb 2 ZnO 6 (A) and (B) nanoparticles were treated for 48 h and fixed with cold absolute methanol prior to the staining procedure. The cells were stained with DAPI, a blue fluorescent dye that binds to adenine/thymine-rich segments on DNA (blue) to visualize the nuclei. Treated MCF-7, HCT116, and HeLa cells showed nuclear fragmented structures at the lowest used concentration as well as a reduction in cell number in comparison to the untreated cells. It was obvious that β-Nb 2 ZnO 6 (A) and (B) were dramatically changing the morphology of treated cells upon increasing the concentration, as shown in Figure 10. Interestingly, β-Nb 2 ZnO 6 (A) and (B) treated HeLa cells showed more DAPI stained cells which is consistent with the cell viability results and estimation EC 50 that HeLa cells were weakly affected by the used concentrations after 48 h of treatment (Figures 10 and 11). The imaging results clearly demonstrated a correlation with the cytotoxicity results of β-Nb 2 ZnO 6 nanoparticles, implicating their ability in inducing morphological changes and nuclear fragmentation which might be associated with a cell death mechanism.
HCT116, and HeLa cells showed nuclear fragmented structures at the lowest used concentration as well as a reduction in cell number in comparison to the untreated cells. It was obvious that β-Nb2ZnO6 (A) and (B) were dramatically changing the morphology of treated cells upon increasing the concentration, as shown in Figure 10. Interestingly, β-Nb2ZnO6 (A) and (B) treated HeLa cells showed more DAPI stained cells which is consistent with the cell viability results and estimation EC50 that HeLa cells were weakly affected by the used concentrations after 48 h of treatment (Figures 10 and 11). The imaging results clearly demonstrated a correlation with the cytotoxicity results of β-Nb2ZnO6 nanoparticles, implicating their ability in inducing morphological changes and nuclear fragmentation which might be associated with a cell death mechanism.

Synthesis of β-Nb2ZnO6
Niobium chloride (0.270 g) and zinc nitrate (0.2974 g) were weighed and shifted to a Teflon-lined autoclave with 20 mL of distilled water. After stirring, urea (0.2402 g) and ammonium fluoride (0.148 g) were added to the mixture. After stirring, the autoclave was heated at 200 °C for 12 h. The obtained precipitation was centrifuged and washed repeatedly using deionized water, followed by washing with ethanol, and dried overnight (60 °C). The samples were calcined at 500 °C and 700 °C in the furnace and assigned as (A) and (B), respectively.

Synthesis of β-Nb 2 ZnO 6
Niobium chloride (0.270 g) and zinc nitrate (0.2974 g) were weighed and shifted to a Teflon-lined autoclave with 20 mL of distilled water. After stirring, urea (0.2402 g) and ammonium fluoride (0.148 g) were added to the mixture. After stirring, the autoclave was heated at 200 • C for 12 h. The obtained precipitation was centrifuged and washed repeatedly using deionized water, followed by washing with ethanol, and dried overnight (60 • C). The samples were calcined at 500 • C and 700 • C in the furnace and assigned as (A) and (B), respectively.
The morphology and size of β-Nb 2 ZnO 6 nanoparticles (A) and (B) were determined by a transmission electron microscope (TEM) (FEI, Morgagni 268, Brno, Czech Republic) and X-ray diffraction (Rigaku, Japan) quantified with Cu-Kα radiation (λ = 1.5418 Å) with a 1 • per minute speed of scanning (range 10-80 • ). Surface area (BET) was determined by Micromeritics ASAP 2020 PLUS (Norcross, GA, USA) by degassing the samples (180 • C) and by employing N 2 adsorption data with a range of relative pressure (P/P 0 ) from 0.0 to 1.0. A diffuse reflectance UV-visible spectrophotometer was used for recording the UV-Visible spectra (UV-Vis, JASCO V-750, Great Dunmow, Essex, UK).

Photocatalytic Activity
The aim of evaluating the photocatalytic action of the β-Nb 2 ZnO 6 nanoparticles (A) and (B) was realized by achieving photodegradation of methyl orange under visible light irradiation using a Xenon lamp (300 W, with > 400 nm cut-off filter). Each experimental set consisted of 0.050 g of β-Nb 2 ZnO 6 nanoparticles (A) and (B) dispersed in 50 mL methyl orange (aqueous 10 mg/L). For the establishment of an adsorption-desorption balance between the photocatalyst and methyl orange, the solution was continuously stirred for a specific period in the dark followed by illumination with the Xenon lamp. After 15 min, 3 mL of the sample was removed and centrifuged. The degradation of methyl orange was calculated at 465 nm using UV-Visible spectrophotometry. The extent of degradation was calculated by Efficiency (%) = (C 0 − C)/C 0 × 100 where C 0 is the initial methyl orange concentration and C is the time-dependent concentration of methyl orange following irradiation with β-Nb 2 ZnO 6 nanoparticles (A and B).

Cell Culture and Cytotoxicity of β-Nb 2 ZnO 6 Nanoparticles
The human cell lines (American Type Culture Collection, Manassas, VA, USA) applied to test the cytotoxicity of β-Nb 2 ZnO 6 nanoparticles (A and B) against were: MCF-7 (breast cancer), HCT116 (human colon cancer), and HeLa (cervical cancer), the cell lines attained were as (MCF-7-ATCC ® HTB-22™, HCT116-ATCC ® CCL-247™, and HeLa-ATCC ® CCL-2™, preserved with Dulbecco's Modified Eagle's Medium (DMEM) accompanied with 1% Lglutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin (Gibco) at 37 • C with a humidity of 5 percent CO 2 . Cells (trypsinized with Trypsin-EDTA 0.25%, TFS) were rested (5 min) at 5 percent CO 2 humidity before defusing with 1:1 of DMEM and finally placed under 1000× rpm in a centrifuge (5 min). Following three to six treatments, cells were situated in 96-well plates at 10 4 cells/well and maintained in DMEM for 24 h. The cells were independently reacted with β-Nb 2 ZnO 6 (A) and (B) nanoparticles (at concentrations 1, 0.5, 0.25, and 0.125 mg/mL) with a dilution of 1 mL DMEM and 100 mL of each of the above was used in replicates of two post removal of precultured media and incubation period of 24, 48, and 72 h.
The viability check was done two times by addition of MTT solution (Sigma) prepared as 5 mg/mL in 1 × phosphate-buffered saline. MTT (10 µL solution) was placed into each well as well as positive controls with 0.5 mg/mL concentration. The assay was carried out for 4 h at 37 • C at the end of which, 100 µL of dimethyl sulfoxide (DMSO) was introduced to bring about the conversion of tetrazolium salts to formazan by metabolically active cells. Spectra at 570 nm were noted with the help of SYNERGY Neo2 multi-mode microplate reader, Biotek, to compute cell viability as Cell viability (%) = Abs sample /Abs control × 100 MTT cell viability results were stated as the mean ± standard deviation (SD) of two objective experiments. The results were subjected to the ordinary two-way ANOVA test which was carried out by GraphPad Prism Software (GrapPad, La Jolla, CA, USA). In all cases, p-value ≤ 0.05 was considered significant (GP: 0.1234 (ns), 0.332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****).

Imaging by Confocal Microscopy
MCF-7, HCT116, and HeLa cell lines were seeded in 8-well Nunc™ Lab-Tek™ Chamber Slide System (Thermo Fisher Scientific) at 35 × 10 4 cells/well together with 0.25, 0.5, and 1 mg/mL of each β-Nb 2 ZnO 6 (A) and (B) nanoparticles for 48 h. The cells were fixed using cold absolute methanol for 10 min, then treated with ProLong™ Gold Antifade