Cadmium acetate (Cd (CH₃COO)₂・2H₂O, Merck, pure), Cerium(III) Nitrate Hexahydrate (Ce(NO₃)₃・6H₂O, Spectrochem, 99% purity), Sodium hydroxide (NaOH, Merck, 99.99% purity) and Polyethylene glycol (PEG) were taken as the initial reagents to synthesize the Cd_(1-x) Ce_xO nanostructure. Deionized water was used as the solvent to prepare solutions of precursors.

In a typical synthesis process, appropriate quantities of Cd(CH₃COO)₂・2H₂O and Ce(NO₃)₃・6H₂O were grounded uniformly, mixed and finally dissolved in 350 ml distilled water with the assistance of continuous magnetic stirring. Measurable amount of Polyethylene glycol (PEG) and NaOH solution was then added into the above solution and the resulting one was kept under sonification for 30 minutes so as to achieve the pH value of 11. The prepared solution was then transferred into a Teflon-lined autoclave and heated at 100˚C for 22 hrs. The various compositions of the Cadmium substituted samples have been represented by a given formula Cd_(1-x)Ce_xO with x being 0, 1.0, 3.0 and 5.0 mole percentage. The final products were obtained after washing and filtering several times with distilled water and anhydrous ethanol. Then it was dried at 100˚C in vacuum atmosphere.

The antibacterial activities of synthesized Cd doped CeO₂ NPs were studied against Gram-positive (G+ve) (S. aureus: Staphylococcus aureus) and Gram-negative (G−ve) (E. col: Escherichia coli, P. aeruginosa: Pseudomonas aeruginosa) strains by disk diffusion method. In brief, the bacterial strains were cultured in nutrient broth at 37˚C until the culture reached 1.5 × 10⁸ colony forming units (CFU) per milliliter. About 20 mL of autoclaved molten nutrient agar was poured into the Petri dishes and allowed to cool. All of the bacterial cultures were swapped over solidified agar medium. Later, disks were loaded with Cd doped CeO₂ NPs solution of 20 μg/5μL through micropipette. The plates were incubated at 37˚C for 24 hours and the zones of inhibition (ZOIs) around the disks were measured.

Breast cancer cells (MCF-7), Human Lung cancer cells (A549) and Human Larynx Carcinoma cancer cells (Hep-2) were obtained. Then, these cell lines were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM: Himedia Laboratories, Mumbai, India), medium which was supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin (Hi Media Laboratories Mumbai, India) cells grown at 37˚C in incubator under 5% CO₂ with high humidity [18] [19] [20] .

The anticancer activity of samples on MCF-7, A549 and Hep-2 cells was determined by the MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay was used to assess the cytotoxicity by Mosmann [21] [22] . There Cells (1 × 10⁵/well) were plated in 0.2 ml of the cells with concentration of 1 × 10⁵ cells/ml were plated in well 96-well plates. The plates were incubated for 24 hrs in 5% CO₂ incubator for cytotoxicity. After incubation, normal breast (MCF-7, A549 and Hep-2) cells were cultured in 1:1 mixture of dimethyl sulfoxide (DMSO). Then, they were added to each well and mixed well by micropipette [23] . The percentage of viable cells was visualized by the development of purple color due to formation of formazan crystals. The suspension was transferred to the cuvette of a spectrophotometer and observed significant variance/instability in the optical density (OD). Measurements were performed and the concentration required for a 50% inhibition of viability (IC₅₀) was determined and used for the bioassays.

The cross sectional morphology of apoptotic cells was carried by AO/EB double staining method which was proposed by Spector et al. [24] . The cells were treated with IC₅₀ Concentration of Cd doped CeO₂ for 24 hours in a humidified atmosphere of 5% CO₂ at 37˚C incubation. The cells were harvested and washed with cold PBS. Cell pellets were diluted with PBS concentration of 5 × 10⁶ cells/mL and mixed with 10 μl of AO/EB solution (3.8 μM of AO and 2.5 μM of EB in PBS). The cross-section morphology of apoptotic was investigated by fluorescence microscope (Carl Zeiss, Axioscope 2 plus) with UV (450 - 490 nm) [25] .

In addition to the above study, the morphological cross-section of apoptotic cells was also examined by DAPI staining. 4’,6-diamidino-2-phenylindole (DAPI) staining was carried out according to the method described by Papi et al. with some modifications [26] . Hep-2 cells were grown on sterile glass slides overnight and treated for 48 hours with sulforaphene (in serum free media) concentration of IC₅₀ (33.8 µM). The cells were incubated for 48 hours in a humidified atmosphere of 5% CO₂ at 37˚C. At the end of the incubation, cells were fixed with 4% paraformaldehyde and then phosphate buffered saline (0.1% in PBS). Cells were finally stained using DAPI in PBS (2.5 µg/mL) and allowed to stand for 20 min in a dark condition. Finally, morphological cross-section reforms were observed by fluorescence microscopy. (Magnification × 400) (Zeiss, Oberkochen, Germany) [27] .

Powder X-ray diffraction (XRD) measurements were carried out for pure and cadmium doped CeO₂ samples using a Bruker D8 advance diffractometer with monochromatized Cu Kα radiation (λ = 1.5418 Å). The X-ray source was operated at 40 kV with a current of 40 mA. The measurements were performed by θ/2θ scans in the 2θ range from 20˚ to 80˚ with a step size of 0.02˚ and at a scan rate of 2˚/min. The micrographs of cadmium doped CeO₂ samples were also obtained using the scanning electron microscope (Model: JEOL-JSM 6360) and high-resolution transmission electron microscope-HRTEM (Model: JEOL/JEM 2100). Elemental analysis was carried out for ascertaining the concentrations of cadmium in CeO₂ materials with the aid of energy dispersive X-ray spectroscopy equipped with scanning electron microscopy. Micro-Raman spectra were recorded in backscattering configuration and analyzed using a JobinYvon T64000 spectrometer equipped with the nitrogen cooled charge-coupled-device detector. The normal and apoptotic cells have been visualised using an upright fluorescent microscope (Nikon Eclipse, Inc., Japan) at 40× magnification with the excitation filter at 510 - 590 nm.

XRD patterns of pure and Cd doped CeO₂ nanoparticles at different concentrations of Cd (1, 3 and 5 mol%) are shown in Figure 1. The structural properties of the pure and Cd doped CeO₂ nanoscale materials were determined through XRD measurements. The synthesized product exhibits the diffraction peaks with parallel planes (hkl) which indicate the presence of CeO₂ compound with cubic

NaCl structure (JCPDS card number: 34-0394). The intensities of (111), (200), (220), (311), (400) and (411) peaks in the 1, 3 and 5% Cd added CeO₂ were found to be prominent. It can be seen from the XRD pattern that the positions of the peaks have been shifted towards the higher 2θ value with increase in “Cd” content. In Figure 1, the “d” values are calculated using (111) peak for different concentration of Cd ions doped in CeO₂ matrix. The d₍₁₁₁₎ values are found to decrease with the Cd content. Thus, the decreasing trend of d₍₁₁₁₎ values reflects that lattice parameters decrease with Cd doping which is in good agreement with the earlier reported results [28] [29] [30] . XRD results also provide a clue that the Cd ions replace some of the Ce ions in the CeO₂ matrix and ruled out the formation of any other crystalline phase. The average crystalline sizes of the pure and Cd-doped CeO₂ have been found in the range of 20 - 42 nm using Scherrer’s equation [31] .

D = 0. 89 λ / ( β cos θ ) . (1)

Here θ is the Bragg diffraction angle, β is the peak width at half maxima. The broadening of the diffraction peaks with increase in the concentration of dopant reveals the formation of nanocrystals with the phase CeO₂.

Figure 2 shows the Raman spectra of the pure and Cd (0, 1, 3 and 5 mol%) doped CeO₂ nanoparticles in the frequency range 200 - 1200 cm⁻¹. The CeO₂ exhibited a strong Raman spectrum at 455 cm⁻¹ because of the F_2g Raman active mode of the fluorite structure [32] . It also exhibits a shoulder like scattering vibrations at ≈ 602 cm⁻¹ and 1050 cm⁻¹ owing to the normal Raman inactive (IR active) transverse and longitudinal optical phonon modes, respectively, at the Brillouin zone center [33] . The spectrum of Cd doped CeO₂ showed the prominent peaks at 452 cm⁻¹ and a weak band 602 cm⁻¹, 1043 cm⁻¹. The band at 452 cm⁻¹ represents the triply degenerate F_2g mode and it is identified as a symmetric breathing mode of the O atoms around Ce ions [34] . The weak band observed near 602 and 1043 cm⁻¹ could be attributed to a non-degenerate longitudinal optical (LO) mode of CeO₂ [35] .

Figures 3(a)-(d) represent the overall surface morphology of pure and Cd doped CeO₂ nanoparticles. The figures reveal that the pure CeO₂ compound consists of large aggregates transformed to more fine aggregates up on increasing the concentration of dopant. The images also show the agglomeration of homogeneous particles with a size distribution of around 1µm in diameter. Figure 4 shows the EDX spectrum of Cd doped CeO₂ and it confirms the presence of Cd ions in the CeO₂ matrix. Further, the estimated compositions do exist in the sample in respect of Cadmium, cerium and oxygen elements. The atomic % of Cd, Ce, and O are 1.20, 24.45 and 74.35, respectively, for 3% of Cd doping.

HRTEM images of the samples shown in Figure 5(a) and Figure 5(c) provide the average crystalline size for pure and doped CeO₂ and it confirms that the average crystalline size of Cd doped nanoparticles is smaller than that of pure CeO₂. The particle size of pure and Cd doped CeO₂ is 6 nm and 5 nm, respectively. The results are in good agreement with XRD data. It was observed that size of particles decreases with increase of dopant concentrations. Figure 5(b) and Figure 5(d) depicts SAED patterns of CeO₂ and Cd doped CeO₂ and it confirms the fluorite structure of CeO₂ and the ring patterns showed (111), (200), (220) and (311) planes of cubical unit cell.

The antibacterial assay was performed against G+ve and G−ve bacterial entities using Cd doped CeO₂ NPs sample loaded at a concentration of 20 μg/05μL on disks. Figure 6 and Figure 7 reflect the measurements in size of ZOI around Cd doped CeO₂ NPs poured disks. The synthesized Cd doped CeO₂ NPs have proven efficiency and comparatively low genotoxic and cytotoxic behavior toward healthy cells, when compared to Cd doped CeO₂ NPs synthesized by

various chemical methods [36] [37] [38] .

The cytotoxic effect of Cd doped CeO₂ NPs was examined in cultured (MCF-7, A549 and Hep-2) cell line by exposing cells for 72 hours. The cultured medium of Cd doped CeO₂NPs led to inhibition at the various concentrations (10 to 100 μg/ml as shown in Figure 8. The cancer cell viability is decreased partially with increasing the concentration of Cd doped CeO₂ NPs. The results show the dose-response relationship with tested cells only at higher concentrations, but there is no significant toxicity [39] .

Acridine orange (AO)/Ethidium Bromide (EtBr) staining and DAPI staining

methods were utilized to study morphological evidence of apoptosis on the Cd doped CeO₂ treated cells. From Figure 9, it is very clear that the apoptosis was noticed with the morphological changes in the cell shape. Points at that there morphological change are because of the activation of caspase cascades. We found that the cells were regular in morphology and they have grown fully in patches in the control group. However, after the treatment, the cells started

exhibiting the apoptotic characteristics of nuclear condensation, cell shrinkage and fragmentation [40] . Further, the DAPI staining also revealed an increase in the number of apoptotic treated cells in terms of both nuclear condensation and cell structure loss [41] [42] [43] . Cd-doped CeO₂ nanoparticles possess a fair control of the pathogenic activity. The cytotoxicity activity of Cd-doped CeO₂ was also assessed for the cell lines MCF-7, A549 and Hep2 and the typical IC₅₀ values are tabulated in Table 1. The obtained values have been compared with literature values reported for HT29 and SW620 cells using pure CeO₂ nanoparticles.

The cytotoxic effects of pure and Cd-doped CeO₂ nanoparticles suggest that they can be used for the development of drugs against colorectal cancer. The apoptotic potential CeO₂ nanoparticles upon doping with Cd ions were studied in breast cancer cell line MCF-7. A characteristic change in chromatic condensation and nuclear fragmentation has been observed which indicates the mechanism of cell death induced by the Cd-doped CeO₂ NPs. A similar kind of study made for pure CeO₂ NPs for HT29 cells [44] in the expression levels of Bcl2 and BclxL proteins. In both cases, it has been confirmed that nanoparticles of CeO₂

have the capacity of reducing cell proliferation.