Exploring Fabrication Characterization and Functionalization of Transition Iron Oxide, Rare Earth Erbium Oxide, and Dual Oxides Nanocomposites for Biomedical Assay

In this report, we intend to synthesis the iron oxide (Fe 2 O 3 ), erbium oxide (Er 2 O 3 ) and a composite of erbium oxide/iron oxide (Er 2 O 3 /Fe 2 O 3 ) nanoparticles (NPs) by a microwave irradiation technique. After the synthesis, we explore the various physicochemical properties of the sample with the help of various systematic characterization techniques. First, the prepared sample has been subjected to XRD for determining the crystal structure. Then, we conrm the functional groups of the sample with the help of FTIR. Further, we analyze the absorbance and the band gap by UV-Vis spectrometer. Besides, we also investigate the microbial studies, namely, the anti-bacterial and the anti-fungal. Finally, we also analyze the response of human breast cancer cells when they are exposed to iron oxide (Fe 2 O 3 ), erbium oxide (Er 2 O 3 ) and a composite of erbium oxide/iron oxide (Er 2 O 3 /Fe 2 O 3 ) nanoparticles (NPs) with MDA MB 231 by MTT assay.


Introduction
It is well known that it was Richard Feynman who pondered over the idea of nano-technology in 1959. The technology has penetrated into almost all the elds of science, engineering, medicine, etc. So far, the various types of nanoparticles have been synthesized and they found immense applications, especially, in nano-biotechnology, for instance, drug carriers and drug delivery systems 1, 2 and vaccine administration 3 . In the recent decades, the interest has turned towards the synthesizing the various metal oxide nanoparticles as the metal oxides, in general, play an indispensable role in many areas of physics, chemistry and materials science. Further, these oxides are widely utilized for fabricating piezoelectric devices, fuel cells, and microelectronic circuits, sensors, coating surfaces against corrosion and as catalysts. The metal oxides exhibit metallic, semiconductor or insulator behaviors as they facilitate structural geometries with an electronic structure. To date, several chemical synthesis methods have been proposed, namely, chemical vapor deposition, co-precipitation, sol-gel, and microwave irradiation methods for fabricating the various types of nanoparticles 4,5 . From the recent literature survey, it has been reported that different types of Fe 2 O 3 , Er 2 O 3 and Er 2 O 3 /Fe 2 O 3 nanoparticles have been synthesized and they exhibited outstanding antibacterial properties against various types of bacteria of Bacillus SP, S. marcescens, C. albicans, S. aureus, E. coli, P. aeruginosa, B. subtilis, M. varians, A. avus, etc. These nanoparticles have the ability to induce the membrane stress through the direct contact with the walls of bacterial cells. Hence, they damage and disrupt the cell membranes. Eventually, this leads to the cell death. It should be emphasized that Aspergillus and Mucor commonly affect the plant products materials rather than humans [6][7][8][9] . These postharvest pathogenic fungi have been recovered by using these Fe 2  Therefore, in this work, we intend to synthesis the iron oxide nanoparticles, the erbium oxide nanoparticles and nally the composite of erbium doped iron oxide nanoparticles by microwave irradiation technique, which was introduced in 1980's. It is well established that this technique signi cantly increases yields, reduces reaction time, reduces side reactions energy e cient and greener process. Besides, microwave heating provides several bene ts when compared to the conventional heating methods 11,12

Results And Discussion
Structural Analysis XRD pattern analyzed by the particle size and the crystal structure are shown in Figure.1 Here, the XRD pattern can be indexed well to the rhombohedral structure of Fe 2 O 3 (JCPDS 89-2810) where the diffraction peaks are found at 24.22º, 33.23º, 35.53º, 40.77º, 49.25º, 54.04º and 62.71º with correspond to (220), (311), (110), (400), (202), (311) and (214) plane, respectively. The particle size is calculated by using Scherrer's formula 13 given below, where λ is the x-ray wavelength (Cu Kα = 1.5418Å), k is a constant (0.916), β is the full width at half maximum (FWHM) of the peak and θ is the Bragg angle peak position. The lattice constants for Fe 2 O 3 nanoparticles are a = b = 5.040 Å and c = 13.74650 Å and the average crystal size has been calculated as 12 nm. From JCPDF chart 77-0777, the XRD con rmed that Er 2 O 3 possesses the cubic structure. By applying the Scherrer's formula, the FWHM for the strongest re ection peak of (222) with cell parameters a = 10.53 Å, the particle size was calculated to be 15 nm. Further, XRD pattern was also analyzed for the erbium doped iron composite material. It has been found that the peaks of iron oxide and erbium oxide are shifted to new position when they are used as composite; the particle size was calculated to be 17 nm

Optical Absorption Spectral Studies
An optical absorption spectrum of the Fe 2 O 3 , Er 2 O 3 and Er 2 O 3 /Fe 2 O 3 NPs was carried out between from 300 to 900 nm using UV visible spectrometer and the results are shown in Fig. 3 obeying the following relation for high photon energies (hν); (αhν) 2 against of hν is shown in Figure. 3(b). The E g is evaluated by the extrapolation of the linear part and the band gap for Fe 2 O 3 , Er 2 O 3 and for Er 2 O 3 /Fe 2 O 3 NPs was found to be 2.07, 4.85 and 2.33 eV, respectively 18,19 Photoluminescence Spectra Photoluminescence (PL) spectroscopy is a suitable technique to determine the crystalline quality and the exciton ne structure. Figure  show good antibacterial activity 23 . The mechanism of transition of Fe 3+ and Er 3+ results in the generation of more reactive and potent oxygen species 24,25 .
Sample with Fe 2 O 3 nanoparticles of 100 μl demonstrated higher activity against Bacillus sp and the inhibition zone was 12 mm, followed by E. coli whose inhibition zone was found to be 19 mm, respectively. The iron oxide nanoparticles of 200 μl and 300 μl demonstrated increasing tendency of the inhibition zone and the details are presented Table1. Similarly, the antibacterial activity of Fe 2 O 3 nanoparticles against the bacterial such as Bacillus sp, and E. coli is shown in Figs. 5(a) and 5(d).
From Figs.5(b) and 5(e) and Table1(a), it can be observed that the sample with Er 2 O 3 nanoparticles of 100 μl were active against Bacillus sp, and the inhibition zone was 19 mm, followed by E. coli whose inhibition zone is 13 mm. When the concentration of sample increases with the 200 μl, the sample was active against bacterial but the inhibition zone increases to 22 mm against Bacillus sp and 15 mm against E. coli. Finally, the inhibition zone is 23 mm in 300 μl extract for Bacillus sp which is higher to the standard value. In the case of E. coli, the inhibition rises to 15 mm was observed.
From Figs. 5(c) and 5(f) and Table1(a), it has been found that the sample (Fe 2 O 3 /Er 2 O 3 nanoparticles) of 100 μl was active against Bacillus sp, and the inhibition zone was 21 mm, followed by E. coli whose inhibition zone was 21 mm. When the concentration of sample is increased to 200 μl, the sample was active against bacterial but the inhibition zone increases to 23 mm against Bacillus sp and 22 mm against E. coli. However, for the sample with 300 μl, the sample was active and inhibition zone is 27 mm for Bacillus sp which is higher than the standard value.
In the case of E. coli, the enhanced inhibition of 23 mm was observed.
It is envisaged that there exists an electrostatic interaction between positive charged nanoparticles and negative charged bacteria. Owing to this, the small-sized nanoparticles penetrate inside the cell wall and eventually they cause the cell damage. During the generation of reactive oxygen species (ROS), small-sized particles of constituent ions are released through re ux mechanism 26 . Therefore, the antibacterial activity of the Fe 2 O 3 , Er 2 O 3 and Fe 2 O 3 /Er 2 O 3 was found to be enhanced rate.

Anti-Fungal Studies
The antifungal activity of Fe 2 O 3 , Er 2 O 3 and Er 2 O 3 /Fe 2 O 3 NPs is carried out. The nanoparticles were also subjected to the evaluation of their antifungal activities against the different strains such as Aspergillus and Mucor with different concentrations.
Sample with Fe 2 O 3 nanoparticles of 100 μl demonstrated higher activity against Aspergillus and the inhibition zone was 11 mm, followed by Mucor whose inhibition zone was found to be 18 mm. The iron oxide nanoparticles of 200 μl and 300 μl demonstrated increasing tendency of the inhibition zone and the detailed results are presented in Table.1(b). Similarly, the antifungal activity of iron oxide nanoparticles against the fungi such as Aspergillus, and Mucor is shown in Figs. 6 (a) and 6(d).
From Figs. 6 (b) and 6 (e) and Table1 (b), it has been observed that the Er 2 0 3 nanoparticles with 100 μl were active against Aspergillus and the inhibition zone was 18 mm and the Mucor's inhibition zone was 13 mm. When the concentration of sample is increased to 200 μl, the sample was also active against fungi but the inhibition zone is increased to 21 mm against Aspergillus but 14 mm against Mucor. However, for 300 μl extract, the inhibition zone was found to be 22 mm for Aspergillus and this is higher than the standard value. In the case of Mucor, the inhibition has been raised to 17 mm.
From Figs. 6 (c) and 6 (f) and Table1 (b), it is clearly seen that the Er 2 O 3 / Fe 2 O 3 nanoparticles of 100 μl were active against Aspergillus and the inhibition zone was 13 mm, followed by Mucor whose inhibition zone was 15 mm.
When the concentration of sample is increased to 200 μl, still the sample was active against fungi and the inhibition zone was extended to 19 mm against Aspergillus but 16 mm against Mucor. Lastly, for 300 μl, the sample was still active for Aspergillus and the inhibition zone was 21 mm which is higher than the standard value.
In the case of Mucor, the inhibition has been extended to 16 mm.
The size of the bacteria/microbes is typically in the order of micrometer 27,28 . Nearly, thousands of nanopores were found in their outer cellular membranes. At this juncture, it should be emphasized that the size of the particle of the iron oxide and erbium oxide and Fe 2 O 3 /Er 2 O 3 is less than 100 nm, and they are unlikely to enter the cell wall and damage the fungi from the interior. In addition, the liberated metal ions from the doped nanoparticle may also impact the antifungal activity in disrupting the cell membrane and gaining entry 29 .

Cytotoxicity and Cell viability Analysis of MDA MB-231 Cell Line
In the last two decades, there has been immense interest in the pharmacological effects of bioactive compounds on cancer treatments and prevention. The research investigations reveal that there are numerous anti-cancer activities in various cancer cells through different forms of cytotoxic effects without exhibiting considerable damage to normal cells [30][31][32] . In vitro cytotoxic potential of Fe 2 O 3 , Er 2 O 3 and Er 2 O 3 /Fe 2 O 3 NPs and human breast cancer MDA MB-231 cell line and viability of tumor cells were con rmed using MTT assay. When the cells were treated with various concentrations (0, 5, 10, 25, 50, 75, and 100 µg/mL) of iron oxide, erbium oxide and Fe 2 O 3 /Er 2 O 3 NPs for 24 hrs, a signi cant decrease in cell viability was observed when compared to untreated cells whose viability is assumed to be 1 (i.e.,100 %). In this study, various percentages of cell viability were examined in cultured cells. The results showed that Fe 2 O 3 , Er 2 O 3 and Er 2 O 3 /Fe 2 O 3 NPs induced signi cant potential cytotoxic response which is clearly depicted in Figs.7 (a-d). Further, all these details are also clearly presented in Table 2 Fe(NO 3 ) 2 .9H 2 O with Er(NO 3 ) 3 .5H 2 O as a composite both were taken in equal ratio 1:1 with their stoichiometry prepared from the raw materials. The purchased materials were weighed and mixed according to the stoichiometric ratio. The powders were pressed into the disk-shaped cakes using the hydraulic press. These were pre-sintered at a temperature of 800˚C for 30 min. in a microwave furnace (V.B. Ceramics India, 2.45 GHz frequency with a power output of 2.2 kW). The pre-sintered cakes were removed from the microwave furnace and crushed into powder. These samples were ground for 1hr in an agate mortar to make it into ne powder possessing uniform particle size. The obtained material was subjected to different characterization to con rm the appropriateness of device application.

Characterization Techniques
Powder X-ray diffraction (XRD) measurements were carried out for iron oxide, erbium oxide and erbium doped iron oxide 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 20-80° with a step size of 0.02 degrees and a scan speed of 0.1 seconds per step. The FT-IR analysis has been done with Perkin Elmer set of BXII model in range of 4000 -400 cm -1 . The optical spectra of the samples were recorded by UV-Vis spectrometer of Jasco model no-V -670. The PL measurements were carried out at room temperature using 275 nm wavelength as excitation wavelength with a Hitachi f-4500 FL spectrophotometer where xenon lamp was used as an excitation source.

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 myostatin 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 33 . The PDA medium was composed of (gl −1 ) potato infusion-200, dextrose -20, and agar-15. The PDA medium was poured into the Petri plate; and after solidi cation, 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 nally the inhibition zone was analyzed. These fungi were grown in Actinomyces Isolation Media (AIM) broth (HIMEDIA Mumbai). Myostatin 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 dimethyl sulfoxide 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 modi ed 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% CO 2 with high humidity 33 .

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 34,35 . These cells (1 × 10 5 /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% CO 2 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 36 . 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 signi cant variance/instability in the optical density (OD). Measurements were performed and the concentration required for a 50 % inhibition of viability (IC 50 ) was determined and used for the bioassays.