EFFECT OF TEMPERATURES ON STRUCTURAL, MORPHOLOGICAL AND BIO-PROPERTIES OF ZINC FERRITE NANOPARTICLESHTML Full Text
EFFECT OF TEMPERATURES ON STRUCTURAL, MORPHOLOGICAL AND BIO-PROPERTIES OF ZINC FERRITE NANOPARTICLES
B. Jyothish and John Jacob *
Department of Physics, University of Kerala, Mar Ivanios College, Thiruvananthapuram - 695015, Kerala, India.
ABSTRACT: Zinc Ferrite nanoparticles were prepared by chemical co-precipitation method, using PEG as the capping agent. The synthesized materials were annealed at different temperatures such as 400, 500, 600, 700 and 800 °C and the structural characterization studies were conducted by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectrometer (FTIR), UV-Visible Spectro-scopy and Field Emission Scanning Electron Microscopy (FESEM). On heat treatment of the as prepared samples, crystallite size was observed to increase from 5 nm-22 nm. XRD pattern confirmed the single crystalline phase of developed particles. It was observed that the particle size and X-ray density of the annealed ZnFe2O4 nanoparticles linearly increased with increase in annealing temperature and decrease in lattice parameters. The calculated micro strain values were found to be decreasing with increasing annealing temperatures. The nanoparticle size was confirmed using SEM measurements. Furthermore FTIR mapping confirmed the presence of ZnFe2O4 nanoparticles. The calculated band gap (Eg) of zinc ferrite nanoparticles decreases with increase in temperature. The antibacterial activity of ZnFe2O4 nanoparticles was studied against gram negative E-coli pathogenic bacteria. DPPH assay was used to determine the antioxidant activity of the compound. In-vitro anticancer activity was screened by MTT assay against MCF-7 (Human breast adenocarcinoma) cell and A549 (Lung Cancer) cell line. It was observed that the increase in particle size and annealing temperature influence the inhibition rate of these bio properties. The investigation points out that ZnFe2O4 nanoparticle possess less viability with increase in concentration towards MCF-7 cell than A549 cell.
Ferrites, Nanoparticles, Annealing, Capping agents, Co precipitation method
INTRODUCTION: Ferrites have a major role in scientific and technological areas, because of its wide range applications such as magnetic reso-nance imaging, magnetic recording, catalyst, high-frequency coils and radar absorbing materials while comparing with pure metals, Ferrites are superior magnetic materials due to their high resistivity, magnetic properties, fair cost etc.
Zinc ferrite (ZnFe2O4) nanoparticles exhibit excellent magnetic permeability, low eddy current, non-toxicity, high stability etc. 1, 2 Zinc Ferrite nanoparticles can be prepared by various methods including hydrothermal, sol-gel, electrochemical, co precipitation method, high energy ball milling etc. 3, 4
Among various methods, co precipitation technique was the efficient method because of its control over the surface morphology, simplicity of the procedure, low reaction temperature and eco-friendliness 5. Zinc ferrite normally exhibits spinel structure. It can be represented as [A][B]2O4 where A and B are the cations Zn2+ and Fe3+ in tetrahedral and octahedral sites of the FCC structure, respectively 6, 7. The prepared samples were subjected to 5 different calcination temperature such as 400, 500, 600, 700 and 800 °C for 4 h. The characterization of the synthesized nano particles were carried out by P- XRD, UV-Visible Spectro-scopy, FESEM and FTIR 8, 9. In addition to the characterization studies, the antibacterial, anti-fungal, antioxidant activity and in-vitro anticancer activity against MCF -7 (Human breast cancer, adenocarcinoma) and A549 (Lung cancer) of the synthesized ZnFe2O4 nano-particles were analyzed. It is widely applied for Bio-medical applications because of the ease of implementation and less hazardous materials 10, 11.
Preparation of Zinc Ferrite Nano-particles: ZnFe2O4 nano particles were synthesized by co precipitation method by using ferric nitrate (Fe(NO3)9H2O), zinc nitrate (Zn(NO3)2) and ammonium carbonate (NH4)2CO3 as reagents, PEG as the capping agent and NaOH as the precipitating agent to precipitate the required sample.10.1 g of 0.5MFe(NO3)2, 7.43 g of 0.5 M (Zn(NO3)2) were accurately weighed and dissolved in 100 mL distilled water. The evenly mixed solution was filled in a burette. The weighed of 20 g of 0.1 MPEG, 1 g of 0.5M NaOH and 3.92 g of 0.5M (NH4)2CO3 were taken and were uniformly dissolved in 150 mL distilled water. The solution was poured into a conical flask and was subjected to magnetic stirring by adding the solution taken in burette drop by drop. Magnetic stirring was continued till the whole solution in burette was added to the mixture. The formed brown coloured precipitate was filtered out after centrifugation (4000 rpm) for 15 min. The precipitate was washed several times with distilled water. The separated precipitate was heated to 70-80 °C for drying and after that the precipitate was grinded. The prepared samples were annealed at different temperatures.
In-vitro Anti-cancer Effect Determination by MTT Assay: MCF – 7 (Human breast adenocarcinoma) cell was collected from NCCS (National Center for Cell Sciences), Pune, India. The viability of cancer cells were measured by direct observation of cells inverted phase contrast microscope and adopted by MTT assay, which can be used to identify the viability of nano-particle treated cells 12, 13.
The cell line was cultured in 25 cm2 tissue culture flask with Dulbecco’s modified Eagle medium accumulated with 10% of PBS, sodium bicarbonate L-glutamine. The antibiotic solution containing amphotericin B (2.5 µg/mL), penicillin (100 µg/mL) and streptomycin (100 µg/mL). The cultured cell lines were kept at 37 ºC in a humidified 5% carbondioxide incubator. 1mg of sample was weighed and dissolved in 1 mL Dulbecco’s modified, Eagle medium using a cyclomixer. The sample solution was filtered through 0.22 µm syringe filter to ensure the sterility.
Anticancer Evaluation: The growth medium was removed after 24 h and freshly prepared each compound in 5% Dulbecco’s modified Eagle medium was 5 times continuously diluted by two fold dilution (100 µg, 50 µg, 25 µg, 12.5 µg, 6.25 µg in 500 µL of 0.05 DMEM) and each concen-tration of 100 mL was mixed in triplicates at 37 °C in a humidified 5% carbon dioxide incubator. Non treated control cells were also maintained.
Anticancer Assay by MTT Method: 15 mg of MTT (sigma – M 5655) was reassembled in 3 mL PBS until dissolved and sterilized by filter sterilization. The sample content in wells was removed after 24 h of inhibition period and 30 µL of the reconstituted MTT solution was added to all test solution and cell control wells, the plate was shaken well, after that the sample was incubated at 37 ºC in a humidified 5% CO2 incubator for 4 h. The supernatant was ignored after the incubation period and 100 µL of MTT solubilization solution was mixed and the wells were mixed gently by pipetting up and down in order to solubilize the formazan crystals. Micro plate reader at a wave length of 540 nm was used to measure the values of absorbance. The percentage of growth inhibition was calculated using equation 1.
Percentage of viability = (Mean Optical Density of Samples) / (Mean Optical Density of Control group) × 100 ------------ (1)
Antioxidant Activity (Evaluation of DPPH Free radical Scavenging Activity): Antioxidant are substance which can protect our cells against the effect of free radicals which causing heart diseases, cancer and other diseases 14. Hence, it is necessary to design certain materials, which possess anti-oxidant capability 15.
DPPH assay can be used to determine the radical scavenging activity of the compounds. Here ascorbic acid at 10 mg/mL and DMSO were used as the reference material. After the addition of sample the solution was analyzed at 517 nm which was compared with the reference material. 11-diphenyl -2-picryl hydroxyl (DPPH) which appeared as pink colour when existed as a free radical and when get scavenged (DPPH-H) its colour changes to yellow. When the anti-oxidant was introduced the following reaction will occur,
DPPH+[H-A] → DPPH – H + (A) ---------------- (2)
Where DPPH gets reduced to DPPH-H thereby the absorbance decreases. The variation in decoloration shows the scavenging potential of antioxidant present in the sample.
0.1 mM free radical solution is prepared by dissolving 4 mg of DPPH in 100 mL ethanol. Stock solution (10 mg/mL) of the sample was prepared and from this different volume such as 1.25 µg/mL- 20 µg/mL (12 µg/mL-200 µg/mL) were made up to final volume of 20 µL with DMSO and to this 1.48 mL of DPPH (0.1 mM) solution were added. Control was prepared by adding an equal amount of distilled water as that of sample. The solutions were incubated in dark conditions at room temperatures for 20 min and absorbance was measured at 517 nm. The % inhibition is calculated by equation (3).
% inhibition = (Control – test) / Control × 100 ------------- (3)
Antifungal Activity (Agar Well Diffusion Method): Antifungal activity of ZnFe2O4 samples were analyzed using Agar well diffusion method, in which Aspergillus niger (ATCC 16404) was used as the culture of test organism. This activity was based on the principle that when samples were introduced into the medium, the antifungals present in the sample interact with the test organisms, which were seeded in a plate. The whole process was carried out on petriplates of 100 mm, which were coated with Potato dextrose agar medium prepared by dissolving 39 g of potato dextrose agar medium in 1000 mL distilled water. After that the dissolved medium was autoclaved at 15 lbs pressure at 121 ºC for 15 min.
This will be poured over the petriplates and overnight grown species of Asperigillus niger was swabbed. Samples heated at different temperature were added to the well of 10mm bored using a well cutter at different concentrations say 250, 500 and 100 µg/mL. The zone of inhibition was analyzed by comparing with the standard clotrimazole at a concentration of 10 mg/mL after incubating over-night at room temperature. The resulting zone will be uniformly circular as there will be confluent lawn of growth, and the diameter of zone was measured in millimeter.
Antibacterial Activity (Agar-Well Diffusion Method): The antibacterial activity of zinc ferrite nanoparticles samples was studied against Escherichia coli (ATCC 25922) by using Agar-well diffusion method 16. The experiment was carried out on 100 mm petriplates coated with Muller Hinton Agar medium prepared by dissolving 33.8 g of MHI Agar media in 1000 mL distilled water. Dissolved medium was autoclaved at 15 lbs. pressure at 121 ºC for 15 min. The auto-claved medium was mixed well and poured on to 100 mm petriplates (25-30 mL/plate) while still molten. Nutrient broth was prepared by dissolving 13 g of commercially available nutrient medium in 1000 mL distilled water and boiled to dissolve completely. This medium was dispensed and sterilized by autoclaving at 15 lbs pressure (121 ºC) for 15 min. Streptomycin was used as the anti-bacterial agent at a concentration of 10 mg/mL
Petriplates coated with 20 mL Muller Hinton Agar medium seeded with bacterial culture of E. coli, was growth of culture adjusted according to McFarland standard 0.5%. The antibacterial activity of sample was analyzed by adding different concentration such as 250, 500 and 1000 µg/mL on the wells of approximates 10 mm which was bored using a well cutter. Plates were incubated at 37 ºC for 24 h. Antibacterial activity was measured from the diameter of inhibition zone formed around the well. The resulting zone of inhibition will be uniformly circular as there will be a confluent lawn of growth 17.
RESULTS AND DISCUSSION:
XRD Studies: The XRD patterns of the annealed ZnFe2O4 nano-particles were recorded using a Philips x1 pert pro diffractometer with Cu-kα, (λ = 1.54056 Å) radiation in the range of 20 ~10 - 70º. The source was operating under an accelerating voltage of 40 kV with a tube current of 30 mA.
FIG. 1: XRD PATTERNS OF ZnFe2O4 NANOPARTICLES ANNEALED AT DIFFERENT TEMPERATURES
Crystallite size of the sample at different annealing temperature were calculated using Debye Scherer formula for peaks corresponding to (311) plane,
D = Bλ / (β cosθ )- - - - (4)
Where D is the crystallite size, λ is the wavelength of Cu - k∝, β is the full width of half maximum of the diffraction peaks and θ is the Bragg’s angle. It could be shown from the Table 1 that as the calcination temperature increases there is an increase in the crystallite size (D) from 5 to 22 nm.
This is because at high temperature diffusion of grains becomes more rapid which causes better organization of the sample. The XRD profile indicates the formation of single phase FCC spinal structure. In the sample the peak intensity was very sharp and narrow in nature which confirmed that the samples were at high quality, fine grain size and with good crystallinity 18. The lattice constant a is measured by using Bragg’s equation, for prominent (311) Peak
A = d_hkl √(h^2+k^2+l^2 )------------------- (5)
The specific surface area (s) can be calculated by the relation,
S = 6/(d_x*t)-------------(6)
TABLE 1: CRYSTALLITE SIZE (NM), PARTICLE SIZE (NM), LATTICE CONSTANT A (Å), X-RAY DENSITY (DX), VOLUME (V), SPECIFIC SURFACE AREA (S), MICRO STRAIN (ԑ)
|Crystallite Size (nm) XRD (Å)||Particle size (nm) SEM||Lattice Constant (a)||X-ray Density ρXRD (g/cm3)||Volume (Å) 3(S) (m2/g)||Specific Surface Area||Micro strain (Ԑ)|
FIG. 2: VARIATION OF CRYSTALLITE SIZE AND MICRO STRAIN WITH CALCINATION TEMPERATURE
From the Table 1 it was understood that the crystalline size and x-ray density (dx) were increased by increasing annealing temperature from 400 to 800 ºC, but the values of lattice parameter (a) decreases by increasing calcination tempera-tures. From the Fig. 1 it was observed that an additional peak at 2θ=33.15° which represents the formation of secondary phase. The calculated value of lattice parameter for ZnFe2O4 was in good agreement with standard data 8.440Å from JCPDS card number 82-1049. It was observed that from the lattice parameters of the crystalline sample X-ray density (ρx) and bond lengths (A-O and B-O) and ionic radii (rA and rB) on A-site and B-site of all the samples can be measured by the following equations 6.
ρ_(x= (8 M)/〖Na〗^3 )---------------(7)
Where N is the Avogadro number and M is the molecular weight and
A-O = (μ-1/4)a√3) ------------(8)
B-O = (5/8-μ)a-------------------(9)
r_A = (μ-1/4)a√3-R_0------------ (10)
r_B = (5/8-μ)a-R_0------------ (11)
Where µ is the oxygen ion parameter (0.381Å) and Ro is the radius of oxygen ion (1.32Å)
To find the crystalline size and micro strain simultaneously in the sample William-Hall (W—H) equation is used 6.
β_hkl cosθ_hkl=kλ/D+4εsinθ_hkl------- (12)
Where D is the average crystallite size from W-H plot and micro strain can be represented as Ԑ. Micro strain is the measurement of the concentration of defects in the sample 19.
TABLE 2: THE VALUES OF BOND LENGTH (Å), IONIC RADII (Å)
|Bond length (Å )||Ionic radii (Å)|
|A-O site||B-O site||rArB|
FIG. 3: WILLIAM-HALL PLOTS FOR ZINC FERRITE ANNEALED AT (A) 400, (B) 500, (C) 600, (D)700 AND (E) 800 °C
FIG. 4: FE-SEM IMAGES OF ZINC FERRITE NANOPARTICLES AT DIFFERENT ANNEALING TEMPERATURES
FE-SEM images at different magnifications of ZnFe2O4 nanoparticles were shown in Fig. 4. The figures revealed the spherical morphology of the particles. Particle sizes were nearly comparable with XRD crystallite sizes in Table 1. The formation small size particles resulted in extensive agglomeration ascribed to high surface energy.
FIG. 5: FTIR SPECTRUM OF ZINC FERRITE NANO-PARTICLES ANNEALED AT DIFFERENT TEMPE-RATURES
FTIR Mapping: In order to confirm the spinal structured ferrites, FTIR studies were conducted at various temperatures. All the samples exhibited a peak near 550 cm-1 which indicates the Fe – Ostretching vibration, and the values found to be shift to lower frequency as temperature increases. Another absorption band was observed within the range of 380–450 cm-1 which corresponds with the Zn – Ostretching modes. These observations concluded that they were the characteristic peaks of ZnFe2O4 nano particles. FTIR analysis also helps to understand the nature of residual carbon in the sample. In the present study as the sample temperature increased the number of absorption band found to be decreasing.
Peak observed at 3343, 1645 and 1580 cm-1 at 400 ºC corresponds to OH, C=C and C=O stretching, respectively which disappeared as the temperature of the sample increased, but O-H bending vibration band near the range 1347 cm-1 was found to be present in the sample even at high temperature at almost same frequency. The disappearance of the peaks of residential carbon in sample was burned away during the annealing process.
Optical Analysis: The optical characteristics of ZnFe2O4 nanoparticles was studied by UV-Visible absorption spectrum 20. The Tauc plot band gap Eg values of ZnFe2O4 nanoparticles are nearly 1.64, 1.62, 1.59, 1.53 and 1.47eV. Fig. 6 showed that the band gap energy values decreased with increase in temperature.
FIG. 6: TAUC PLOT OF hʋ vs. (αhʋ)2 FOR ZINCFERRITE NANOPARTICLES WAS ANNEALED AT (A) 400, (B) 500, (C) 600, (D) 700, AND (E) 800 ºC
Antioxidant Activity: The antioxidant activity of ZnFe2O4 nano-particles were determined by using DPPH radical scavenging assay method. In the present study the antioxidant activity at different annealing temperatures were noted. In each temperature, the variation of inhibition corres-ponding to different concentration was analyzed and depicted in Fig. 7. From the graph it was observed that with each annealing temperature, as the concentration of synthesized ZnFe2O4 nano-particles increased, the anti-oxidant activity also increased and it was also observed that at different calcination temperature 500, 600, 700, 800 ºC for 4 h. The lower concentration such as (12.5 mg/mL, 25 mg/mL, 50 mg/mL) with their inhibition rate was more than that of the standard (Ascorbic Acid).The higher concentrations such as (100 mg/mL & 200 mg/mL), the inhibition rate was gradually decreasing as compared with the standard, but at higher annealing temperature 800 ºC for 4 h. the antioxidant activity was suddenly decreased. The antioxidant activity could be associated with the transfer of free electrons from the oxygen atom of nanoparticles to free radicals present in the nitrogen atom of DPPH molecules. The antioxidant activity was mainly attributed to high surface to volume ratio of nano architecture.
Analysis of Cytotoxicity: The cytotoxic activity of ZnFe2O4 nanoparticles at different concentration against the MCF - Cells (Human breast adenocarcinoma Cell line) and A549 (Lung- Cancer Cell) using MTT assay shown in Fig. 8.
FIG.9: (A) UNTREATED LUNG CANCER CELL LINE, (B) SAMPLE CONCENTRATION OF 25 µg/mL ZnFe2O4 (C) 50 µg/mL ZnFe2O4, (D) 100 µg/mL ZnFe2O4
The test outcome revealed that in the case of zinc ferrite nanoparticles against A549 (Lung cancer) Cell, the cancer cell death increases as the concentration of ZnFe2O4 increases. The addition of 6.25 µg/mL of ZnFe2O4 showed the cell viability of 87.12%. 12.5 µg/mL with 81.04% of cell viability, 25 µg/mL with 76% of cell viability, 50 µg/mL with 72.9% of cell viability and 100 µg/mL with 70.67% of cell viability. In MCF – 7 (Human breast adenocarcinoma) cell line investigated that the amount of ZnFe2O4, nanoparticles as well as the cancer cell death increased. The addition of 6.5 µg/mL of ZnFe2O4 showed a the cell viability of 97.28%, 12.5 µg/mL with 89.18% cell viability, 25 µg/mL with 77.61% cell viability, 50 µg/mL with 68.26% cell viability and 100 µg/mL with 49.84% cell viability. From this study it was confirmed that as the concentration of zincferrite nanoparticles increases, the breast cancer cell death increases. The highest cell viability (100%) observed at 0 µg/mL started to decrease gradually with the increased concentration.
FIG. 10: (A) UNTREATED BREAST CANCER CELL LINE, (B) SAMPLE CONCENTRATION OF 6.25 µg/ml ZnFe2O4, (C) 50 µg/ml ZnFe2O4, AND (D) 100 µG/ML ZnFe2O4
The Fig. 8 clearly pointed out that ZnFe2O4 500 ºC, 4 h. nanoparticles possessed less viability towards MCF – 7 cells (Human breast adenocarcinoma) than A 549 (lung cancer cell) at higher concen-trations. It was also observed that the cell survival capacity decreased promptly in MCF – 7 cell with the increase in concentrations of ZnFe2O4 nano-particles whereas the viability was moderately decreased in A 549 (lung cancer cell) with increase in the concentration of ZnFe2O4 nanoparticles.
Antibacterial and Antifungal Activity: The antibacterial activity of ZnFe2O4 nanoparticles was studied against gram negative E. coli bacterial strain using agar well diffusion method. Significant antibacterial activity was observed against E. coli and displayed nearly uniform inhibition rate (10-12) at different annealing temperature in high concentrations such as 500 µg/mL and 100 µg/mL. The zone of inhibition was represented in mm Fig. 12. The effect of ZnFe2O4 nanoparticles on bacterial linear growth revealed that the bacterial growth was reduced by increasing the concen-tration as well as higher calcinations temperatures Fig. 11A.
The antifungal study demonstrated that the percentage of inhibition was found to be increased up to certain annealing temperature (600 °C), further rise in temperature the antifungal activity of synthesized ZnFe2O4 nanoparticle was decreased and the rate of inhibition was found to be suddenly reduced. The variation of inhibition rate against different annealing temperature is graphically displayed in Fig. 11B.
FIG. 11: PERCENTAGE OF INHIBITION OF SAMPLE AT DIFFERENT CONCENTRATION. 250 µg/ml, 500 µg/ml, 1000 µg/ml AT DIFFERENT ANNEALING TEMPERATURE AGAINST (A) E. COLI (ATCC 25922), GRAM NEGATIVE (B). ASPERGILLUS NIGER (ATCC 16404)
FIG. 12: ANTIBACTERIAL ACTIVITY AND ANTIFUNGAL ACTIVITY OF ZnFe2O4 NANOPARTICLES. FIG (A, B, C) E. COLI GRAM NEGATIVE- ZONE OF INHIBITION 12 MM, 10 MM, 10 MM AND FIG (D, E, F) ASPERGILLUS NIGER – ZONE OF INHIBITION 11, 12 AND 14 MM
CONCLUSION: ZnFe2O4 nanoparticles were simply and successfully prepared by a low cost co precipitation method using PEG as the Capping agent. Structural characteristics and biological properties of prepared samples were well studied. The XRD result confirmed the crystallite size and X-ray density was identified to be increased with increase in annealing temperature whereas lattice constant tent to be decreasing. The crystallite size calculated from XRD data using Debye Scherer method well matches with the calculated particle size from FESEM. Morphological study of FESEM showed that the synthesized ZnFe2O4 particles posessed spherical morphology. Presence of zinc ferrite nano-particles at different annealing temperatures were confirmed from FTIR results and a decrease in band gap energy with increase in temperature was obtained using UV-Visible result analysis.
The results of in-vitro cytotoxic, anti-bacterial, anti-fungal and antioxidant activity showed that the synthesized ZnFe2O4 nanoparticles can be used for bio medical applications. The ZnFe2O4 nano-particles were more reactive towards MCF-7 (Human breast adenocarcinoma) and DPPH free radical scavenging activity. The results revealed that the particle size and annealing temperature strongly influenced the described behaviour.
ACKNOWLEDGEMENT: We are deeply acknowledged to the principal, Mar Ivanios College, Thiruvananthapuram, Kerala for providing necessary facilities for doing this work.
CONFLICTS OF INTEREST: We declare that no conflicts of interest.
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How to cite this article:
Jyothish B and Jacob J: Effect of temperatures on structural, morphological and bio-properties of zinc ferrite nanoparticles. Int J Pharm Sci & Res 2020; 11(12): 6391-01. doi: 10.13040/IJPSR.0975-8232.11(12).6391-01.
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B. Jyothish and J. Jacob *
Department of Physics, University of Kerala, Mar Ivanios College, Thiruvananthapuram, Kerala, India.
18 February 2019
28 March 2019
17 April 2019
01 December 2020