SNL  Vol.9 No.1 , January 2019
Study on Enhanced Antibacterial and Cytotoxicity of Pure and Cadmium Doped Cerium Oxide against Gram-Positive and Gram-Negative Bacteria
ABSTRACT
Pure and Cadmium (Cd) doped Cerium oxide nanoparticles (CeNPs) have been synthesised by the simple chemical co-precipitation technique. Cadmium ions of concentrations 1, 3 and 5 mol% were doped to investigate their influence on the structural and optical properties of CeO2. The synthesised samples have been subjected to X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis and high-resolution transmission electron microscopy (HRTEM). The XRD and Raman patterns have witnessed the cubic structure of the cerium oxide nanoparticles. The average particle size of CeO2 was found to be around 10 nm. SEM image has also ascertained that the grain size of pure CeO2 appeared is bigger than that of the Cd-doped, which intern indicates the grain growth upon doping. Besides, the antibacterial activity of the cadmium doped cerium oxide nanoparticles against some human pathogens revealed that they have exhibited the maximum zone of inhibition against gram-positive bacteria than the gram-negative species. Further, the cytotoxic effect of Cd-doped CeO2 sample is examined in cultured (MCF-7, A549 and Hep-2) cell.

1. Introduction

Of late, nanotechnology has been the primary focus, especially, on biomedical related research activities. It has been well demonstrated that the nanomaterial’s exhibited several interesting properties and they were exploited in the field of life science research, biology and medicine [1] - [6] . Thus, these nanomaterials find a wide range of applications which include luminescent biomarkers, drug delivery systems, tissue engineering, etc [7] . Recently, owing to the enormous developments in nanoscience and nanotechnology, the field of nanomedicine plays an indispensable role to investigate the novel drugs to augment the conventional therapies, namely, surgical interventions, radiation and cytotoxic chemotherapy which are considerably effective in the cancer treatment [8] [9] [10] [11] [12] . It was reported that the Cerium oxide (CeO2) and many rare earth metal oxides of lanthanide series were found to exhibiting face-centered cubic fluorite-type crystal structures. Cerium oxide nanoparticles (CeNPs) originated from the variable valence states (3+ and 4+) of cerium element. These nanoparticles could be explored for various applications in industrial areas such as the catalysts, polishing agents, fuel cells, ultraviolet absorbents in sunscreen lotions, gas sensors, etc. Its redox activity (Ce3+/Ce4+ redox switch) along with the oxygen vacancies due to surface defects has led to various biological activities, namely, anti-inflammation, antiapoptotic, antioxidant property etc [13] . In literature, CeO2 nanoparticles were prepared by several methods namely; hydrothermal synthesis [14] , Co-precipitation technique [15] and Solvothermal Methods [16] [17] . Of these techniques, co-precipitation method is a quite simple process, low in cost, easy to prepare and industrially viable. In the present work, Cd-doped CeO2 nanoparticles have been prepared using co-precipitation technique. The effect of Cadmium doping on the structural and morphological properties of CeO2 has been studied using X-ray diffraction (XRD), Raman spectroscopy, SEM and HRTEM analysis. The biological activity against certain human pathogen was also analysed.

2. Materials and Method

2.1. Materials

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) CexO nanostructure. Deionized water was used as the solvent to prepare solutions of precursors.

2.2. Synthesis Procedure

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)CexO 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.

2.3. Antibacterial Activity of Cd Doped CeO2 NPs

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.

2.4. Cell Culture and Cell Line Maintenance

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] .

2.5. MTT Assay Method for Evaluation of Cell Viability and Cytotoxicity

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.

2.6. Morphological Cross Section of Apoptotic Cells by Acridine Orange (AO) and Ethidium Bromide (EB) Staining

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] .

2.7. Morphological Cross Section of Apoptotic Cells by DAPI Staining

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] .

2.8. Characterization of Cd Doped CeO2 NPs

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.

3. Results and Discussion

3.1. X-Ray Diffraction (XRD) Analysis on Pure and Cd Doped CeO2

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

Figure 1. XRD patterns of the CeO2 nanoparticles containing: (a) 0%, (b) 1.0%, (c) 3.0%, and (d) 5.0% of Cd.

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.

3.2. Raman Studies on Pure and Cd Doped CeO2 NPs

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 cm1. The CeO2 exhibited a strong Raman spectrum at 455 cm1 because of the F2g Raman active mode of the fluorite structure [32] . It also exhibits a shoulder like scattering vibrations at ≈ 602 cm1 and 1050 cm1 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 cm1 and a weak band 602 cm1, 1043 cm1. The band at 452 cm1 represents the triply degenerate F2g 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 cm1 could be attributed to a non-degenerate longitudinal optical (LO) mode of CeO2 [35] .

3.3. SEM Analysis on Pure and Cd Doped CeO2 NPs

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.

Figure 2. Raman spectra of the CeO2 nanoparticles containing: (a) 0%, (b) 1.0%, (c) 3.0%, and (d) 5.0% of Cd.

3.4. HRTEM Analysis on Pure and Cd Doped CeO2 NPs

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.

3.5. Antibacterial Analysis of Cd Doped CeO2 NPs

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

Figure 3. SEM images of pure and doped CeO2 (a) Pure CeO2, (b) 1% Cd, (c) 3% Cd, (d) 5% Cd.

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

3.6. Cytotoxicity Analysis of MCF-7, A549 and Hep2 Cell Line

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] .

3.7. Morphological Cross Section of Apoptotic Cells by DAPI Staining and AO/EtBr Double Staining

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

Figure 4. EDX analysis of 5% Cd doped CeO2.

Figure 5. HRTEM images of pure and doped CeO2. (a) Pure CeO2, (b) SAED pattern of pure CeO2, (c) 3% cd, and (d) SAED pattern of cd doped CeO2.

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

Figure 6. Antibacterial activity of CeO2 NPs containing: (a) Pure, (b) 1.0%, (c) 3.0%, and (d) 5.0% of Cd.

Figure 7. Zone of inhibition values for different concentration doped CeO2 NPs containing: Gram-positive [S. aureus (a)] and Gram-negative [E. coli (b), P. aeruginosa (c)] Pure (P), 1.0% (1), 3.0% (2), and 5.0% (3) of Cd.

Figure 8. Vero and HBL 100 cell lines: (A) Control Cells. (B) Cd doped CeO2 NPs Treated at the cell lines.

Figure 9. Cells treated with (A) control (B) Cd doped CeO2 NPs at the respective IC50 concentrations and the morphologies Observed after staining with DAPI and Ao/EtBr.

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

Table 1. Cytotoxicity of Cd-doped CeO2 in MCF-7, A549 and Hep2 cells.

have the capacity of reducing cell proliferation.

4. Conclusion

Nanocrystalline forms of cerium oxide and cadmium doped cerium oxide have been successfully synthesized using chemical precipitation method. From XRD analysis, it was ascertained that the incorporation of cadmium ions did not alter the unit cell structure of CeO2 for all concentrations of dopant’s viz 1%, 3% and 5%. As per Debye-Scherrer’s calculations, the average particle size of Cd-doped CeO2 was estimated to be in the range 8 - 10 nm. Raman spectrum of Cd-doped CeO2 showed that no new bands formed due to the dopant ions. This implied that the possible modes of scattering vibrations for fluorite type of cubic structure could also be seen in the Cd-doped CeO2 which in turn reciprocates the results obtained from XRD. SEM-EDAX measurements revealed the actual compositions of cadmium ions in the CeO2 matrix. Dopant has also influenced in transforming large aggregate particles into fine ones. However, agglomeration is also observed in doped samples as that of pure CeO2. HRTEM analysis on pure and Cd-doped CeO2 brings out the fact that the decrease in the particle size upon an increase in the dopant concentration. SAED patterns provide additional confirmation of restoration crystal structure by CeO2 matrix even after doping. The minimal inhibitory concentration (MIC) behaviour of Cd-doped CeO2 nanoparticles has been assessed using gram-positive and gram-negative bacteria.

Cite this paper
Govindarasu, K. , Gnanasekaran, K. , Balaraman, S. , Iruson, B. , Krishnamoorthy, S. , Balaraman, S. , Padmaraj, B. , Manikandan, E. , Dhananjayan, S. (2019) Study on Enhanced Antibacterial and Cytotoxicity of Pure and Cadmium Doped Cerium Oxide against Gram-Positive and Gram-Negative Bacteria. Soft Nanoscience Letters, 9, 1-16. doi: 10.4236/snl.2019.91001.
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