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Introduction

The term Nanotechnology is applied to the various concepts such as fabrication, characterization, explored comprehensively for multifunctional behavior and technological application of nanomaterial’s sized and more overplay an important role in controlling the size shape of the properties of the material. Since fabricated nanomaterials have enormous uses in many fields [1-5] worldwide, due to their unique size and shape [6,7], this allows the large development in the Nanotechnological world.

Commercially a large volume of CeO₂ NPs are fabricated because they act as a cracking catalyst in petroleum products, flash memory in electronic devices, gas leakage detecting sensors, polishing chemicals to polish mirrors in many electronic devices and wafers, and additive fuel cells in diesel to increase combustion efficiency or mileage ((HEI, 2001; Park et al., 2008a). Since CeO₂ NP donates or stores oxygen which is based on the ecological system. If the environment is rich with oxygen, then CeO₂ itself acts as a catalyst but in the case of hydrocarbon combustions, it donates oxygen.

Among the many metal oxide NPs, the synthesized CeO₂ provides beneficial properties such as attractive morphology, highly reactive and strongly oxidizing agents. Since nanoparticles can bind with the animal’s cancer cell line, further these bindings prevent the growth of cancer cells and therefore nanoparticles have the major application of cancer prevention in the biological field [12-14]. The abnormal growth of cancer cells easily spreads to other parts of the body causing damage to organs for cancer patients. The available chemotherapeutic agents may provide the side effects and therefore it is necessary to prepare bio-compatible compounds in the place of drugs to cure cancer disease and further the cost of treatment should be less possible for cancer patients to cure cancer cells and to achieve these benefits, the nanoparticles have been introduced in this recent decade which provides the effective treatment for cancer patients. In this research, we show that the prepared CeO₂ NP is successfully active against the MDA-MB- 231 cell line[15-16]. Further, in this research, the important biological application such as the effective antimicrobial activity using the synthesized CeO2 has been studied.

Experimental

Preparation of Cerium Oxide (CeO₂) Nanoparticles

The Microwave irradiation technique was employed to synthesize CeO₂NPs. All reactants were purchased from Sigma Aldrich. The precursors used were 1g of Cerium (III) nitrate hexahydrate and 2 g Urea are added in 50 ml of distilled water along with AR grade CeO₂NPs and mixed well; these powders are converted to disk-shaped pellets with the help of a hydraulic handheld and then this pallet sample was kept at 800ºC for 30 minutes in a microwave generating furnace. This heating process on the pellet sample which contains CeO₂NPs allows for the reduction of CeO₂NPs. After half an hour of the heating process, the pellet sample (containing CeO₂NPs) has been burnout to finish the process. These samples were ground for 1hr in an agate mortar to make it into fine powder possessing uniform particle size. The obtained material was subjected to different characterization to confirm the appropriateness of device application.

Characterizations techniques

Characterizing the CeO₂ NPs is very essential which provides the structural information (shape, diameter, and average size CeO₂) and many other various predictions such as absorption (or emission) spectrum, bandgap, and functional groups of synthesized CeO₂ NP sample. These various aspects of information can be obtained by using various instruments such as X-ray, UV- Vis spectrometer, FTIR, PL, EDX, and TEM. Among many instruments, TEM alone uses the beam of the electron, to reveal the surface morphological information of the CeO₂ NP sample. Except for TEM, other all instruments use a particular range of electromagnetic radiation to reveal the crystalline nature of the system, CeO₂ NP size, bandgap and functional groups, and the presence of chemical elements in the CeO₂ NPs.

Test Microorganisms

Aspergillus Mucor fungi was used for carrying out the antimicrobial activity studies. These microorganisms were grown for 3 days at 37 °C in Actinomyces Isolation Media (AIM) broth (Himedia, Mumbai, India). The sensitivity of these microorganisms to the reference antibiotics was checked using mycostatin as a positive control.

Antifungal Activity of the Sample

The samples of iron oxide, erbium oxide and iron oxide/erbium oxide nanoparticles were loaded on Potato Dextrose agar plates at three different volumes (10, 20, and 30 μl) and swabbed with fungi such as Aspergillus and Mucor. Antifungal activities of the samples were determined by well diffusion method on Potato Dextrose Agar (PDA) medium [17]. The PDA medium was composed of (gl−1) potato infusion-200, dextrose -20, and agar-15. The PDA medium was poured into the Petriplate; and after solidification, the inoculum was spread on the PDA plates with sterile swab moistened with the fungal suspension. All the plates were incubated at 37 °C for 3 days and finally the inhibition zone was analysed. These fungi were grown in Actinomyces Isolation Media (AIM) broth (HIMEDIA Mumbai). Mycostain was used as the positive control to check the sensitivity against antibiotics by the well diffusion method on PDA medium.

Antibacterial Activity of the Sample

The antibacterial activity of iron oxide, erbium oxide and iron oxide/erbium oxide nanoparticles were tested against Escherichia coli, and Bacillus sp using disc diffusion method. The iron oxide, erbium oxide and iron oxide/erbium oxide nanoparticles were prepared in appropriate concentration of 1 mg/ml with dimethylsulfoxide solution for this process. Then, the dispersed nanoparticles were impregnated to each sterile disc by using micropipette. After that the discs were kept on culture swapped Mueller Hinton Agar medium using sterile force and allowed to incubate for 24 hrs. The average zone of inhibition diameter was measured in millimeter (mm).

Cell Culture and Cell Line Maintenance

The human breast cancer cell lines MDA MB-231 were obtained. Then, these cell lines were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM: Hi Media Laboratories, Mumbai, India), 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].

MTT Assay Method for Evaluation of Cell Viability and Cytotoxicity

The anticancer activity of samples on human breast cancer cell lines MDA MB-231 was determined by the MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay [20-21]. These cells (1 × 105/well) were plated in 0.2 ml of the cells with concentration of 1 × 105 cells/ml. The plates were incubated for 24 hrs in 5% CO₂ incubator for cytotoxicity. After incubation, normal breast (MDA MB-231) cells were cultured in 1:1 mixture of dimethyl sulfoxide (DMSO). Then, they were added to each well and mixed well by micropipette [22]. The percentage of viable cells was visualized by the development of purple color due to the 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.

Results and Discussion

PXRD of Cerium oxide (CeO₂) metal oxide nanoparticle:

The PXRD pattern of CeO₂ NPs, thus contains the particle size information and crystalline nature, which was synthesized by Microwave irradiation technique were present in Figure 1a. The peaks in fig.1a are assigned to the cubic structure of CeO₂ with lattice points a=b=c=5.412 Å and matched with the JCPDS No. 81-7092. Using Debye - Scherer’s formula, the average CeO₂ crystallite size is found as 49 nm[23]. Several Bragg reflections with 2θ values of 28.5°, 33°, 47.5°, and 56.4° are observed corresponding to (111), (200), (220), (311), (222) and (422) (Figure 1).. Further, the crystallinity nature, particle size distribution, and phase formation have been confirmed by HRTEM

TEM

The electrons passing in a TEM instrument through the potential barrier of the synthesized sample afford the topography [Lin P-C 2014, Hinterdorfer P 2011] information of the CeO₂ NPs. In fig.2 (TEM image) can be observed from CeO₂ NPs structured size between 45.112 to 48.556 nm which gives an average crystallite of 46.8 nm (Table 2), and further these crystalline nature [24-25] of the CeO₂ NPs has been confirmed by SsAED pattern (Figure 2d). The results from the TEM image are agreed with the result of XRD data having crystal planes (111) and (200).

EDX Analysis

EDX analysis of CeO₂ NPs was shown in Figure 3, which confirms that the major chemical element such as Cerium and oxygen were present with strong absorption range and homogeneous distributions, further this spectrum proves that the cerium atom uniformly combined with the element oxygen which is summarized in Table.3.

FTIR

FTIR spectrum of CeO₂ NP is shown in Figure 4. The band at 425 cm-1 shows the presence of metal oxide, thus predicts the presence of a strong bond and stretching between metal cerium and oxygen (i.e. O-Ce-O) [26]. The major peak at the point of 706 cm-1 corresponds to the stretching vibration of doubly coordinated oxygen due to corner shared element oxygen to two cerium atoms. The peak at the point of 1466 cm-1 is assigned to the C-N stretching vibration of urea which was added in the synthesis process. The peaks at the point of 1467 and 3428 cm-1 correspond to stretching vibration of c=c and O-H respectively.

UV-Vis spectroscopy

UV-Vis affords the bandgap as well as the stability of the CeO₂ NPs. The UV-Vis of CeO₂ NPs is present in fig. 5a and 5b. The optical property of CeO₂ NP allows them to absorb with a particular range of wavelengths of UV-Vis radiations and maximum absorption and cut- off wavelengths were found at 270 nm and 430 nm respectively. In Figure 5c, it was observed that the bandgap energy of CeO₂ NPs be around 3eV. This shows that the synthesized CeO₂ NPs could be useful in few medical applications [27,28].

Photoluminescence (PL) spectroscop

PL spectrum of CeO₂ is useful to detect the high-quality crystalline nature and the fine structure of the sample. PL at ambient room temperature was implemented to reveal the optical absorption of Cerium Oxide (CeO₂) nanoparticles. In Figure 6, CeO₂ NP shows the UV emission and green excitations at 284 nm and 550 nm respectively. These results were also reported in previous research [29]. In common, the presence of oxygen vacancies in the sample creates defects, thus are responsible for the creation of broad peaks at larger wavelengths of light.

Anti-Fungal activity

The synthesized metal CeO₂ NPs provides fewer hazards to the outside environment, but its toxic activity against the various fungi is higher [30-32], to find out the antifungal activity of CeO₂ NPs, the disc diffusion technique has been carried out and results are shown in fig.7 and further summarized in Table 5.

The cell viability effect (or zone inhibition) of CeO₂ nanoparticles on the fungi is discussed by explaining the two different mechanisms. First, the creation of H₂O₂ around the CeO₂ NP leads to the possible hydrogen bond development between (OH group) cellulose content of fungi and oxygen atom which causes higher zone inhibition. In second, the release of Ce2+ ion causes damage to the cell membrane which leads to the growth of higher zone inhibition [34-45]. Results reveal that 300 mg of the CeO₂ NP sample provides a higher toxic activity than the toxic activity provided by the lower concentrations of ₂ NPs. The separate 300 mg of the CeO₂ NPs causes the higher zone inhibition as 20 mm and 15 mm respectively for Mucor and Aspergillus. Finally, the results from figure 7 show that the toxicity given by the CeO₂ is higher for Mucor than Aspergillus.

Figure 7 the Anti-fungal activity of Crium oxide nanoparticles. 1) Aspergillus and 2) Mucor at 100 mg, 200 mg, and 300 mg, 3) concentration where the control.

Anti-cancer

The cytotoxicity of CeO₂ NPs with various concentrations (5 μg, 10 μg, 50 μg, 75 μg, and 100 μg /mL) against MDA-MB- 231 cell lines were investigated by the MTT method and its results were present in fig.9, further, the observed values are tabulated (Table 7). In the MTT assay, the cell morphology was captured after 48 hours to obtain the best results. In previous research, the significant results against MDA-MB-231 were observed by adding the 50 μg/mL or 75 μg/mL [52-55]. An advantage of CeO₂ NP is its available lesser size in the sample, so we believed that the synthesized lesser concentrations of CeO₂ NPs in this research, have the higher potential to destroy the MDA-MB-231 cell line. Therefore the chosen concentration of CeO₂ NPs in this research is lesser than the previously published results of the cytotoxicity study. The chosen lowest concentration (5 μg/ml) of CeO₂ NP provided the highest cell destruction with 86% cytotoxicity causing the lesser cell viability (14%.) of the MDA-MB-231 cell line.

Conclusion

In this study, using Microwave radiation the CeO₂ NPs have been prepared and its effective anticancer and antimicrobial studies were successfully executed. The formed CeO₂ crystalline structure and its size range around 45 to 49 nm have been analyzed using XRD and TEM analysis. All the possible major elements and functional groups present in the sample which was synthesized by adding cerium nitrate, urea, and water molecules, have been successfully found using EDX and FTIR studies respectively. The bond between major element cerium and oxygen was identified by noting the absorbance peak at 706 cm-1 in the FTIR spectrum. Antifungal results show that the CeO₂ NP effectively kills higher the number of Mucor than Aspergillus, further the possible mechanism that causes damage to the fungi by using CeO₂ NP was discussed. The synthesized CeO₂ NPs were also found effective against BACILLUS SP and E. COLI bacteria. The results of the MTT assay using the synthesized sample against MDA-MB 231 cell lines indicated the higher percentage of toxicity of CeO₂ NPs. Therefore this study was successfully performed towards biological applications.

Funding

The technical support of this project has been provided by the Tamilnadu State Council for Science and Technology under grant number, C.No.TNSCST/STP-PRG/AR/2018-2019/9333 is gratefully acknowledged.

Conflict Of Interest

The authors declare that they have no conflict of interest

Availability of Data and Material

The authors confirm that the data supporting the findings of this research are available within the article and its supplementary materials.