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JCBPS; Section A; November 2015 – January 2016, Vol. 6, No. 1; E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences

Available online at www.jcbsc.org

Section A: Chemical Sciences

CODEN (USA): JCBPAT ISI (International Scientific Indexing) Research Article

1 J. Chem. Bio. Phy. Sci. Sec. A, November 2015 – January 2016; Vol.6, No.1;

Preclinical Assessment of Zinc Ferrite Nanoparticles Synthesized

Using D-Glucose by Hydrothermal Method

C. S. Vicas1, K. Namratha1, K. Byrappa*1 and H. S. Yathirajan2

1Center for Materials Science and Technology, Vijnana Bhavan, University of Mysore, Mysore, India

2D.O.S. in Chemistry, University of Mysore, Mysore, India

Received: 07 November 2015; Revised: 20 November 2015; Accepted: 27 November 2015

Abstract. Eloquent hydrothermal synthesis of Zinc ferrite nanoparticles was carried

out using dextrose as a reducing agent. Nanoparticles were characterized using

dynamic light scattering technique, scanning electron microscopy, Fourier transform

infrared spectroscopy, powder X-ray diffraction and energy dispersive spectroscopy.

The nanoparticles obtained were of an average size of ~47 nm. Microbial toxicology,

Haemolysis of blood, teratology and embryotoxicology on chick embryos were

performed. The material was found to be non haematotoxic and non-teratogenic.

Determination of total protein content using Bradford’s assay showed that the level

remained to be insignificantly altered in the treated embryos. Synthesized ZnFe2O4

also demonstrated an excellent antioxidant property of ~90% when studied using

DPPH free radical scavenging assay. The nanoparticles synthesized have shown

significant inhibition of skin and soft tissue infection (SSTI) causing bacteria such as

Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Bacillus

subtilis. Based on the current evidences compared with the known ferrites, it was

inferred that the biocompatibility and the ability of zinc ferrite nanoparticles to inhibit

the growth of the selected SSTI causing bacteria, opens the doors to a new arena for

the usage of zinc ferrite nanoparticles as potent antibacterial agents for treating skin

infections. They can also be considered for use as ointments and antibacterial creams.

Keywords: Skin and soft tissue infections (SSTIs); Hydrothermal synthesis; Zinc

ferrite; Teratology; Embryo toxicology; Free radical scavenging; Haemolysis;

Ointments.

http://www.jcbsc.org/

Preclinical … Vicas et al.


INTRODUCTION

Magnetic nanoparticles are one of the commonly researched materials of the modern scientific era for

their various biomedical applications such as immunoassay, minimally invasive surgery, cell

separation, radionuclide therapy, magnetic resonance imaging, drug and gene delivery, hyperthermia

and artificial muscle applications1. Among several magnetic materials, magnetite is one such material

on which most of the research has been carried out for biomedical applications2, 3. Apart from

magnetite, nickel ferrite, cobalt ferrite and zinc ferrite materials are also known to possess potential

biomedical applications. Zinc ferrite, the source of present study, has inverse spinel structure, with

isometric symmetry, and is usually represented by the formula [Fe3+]tet[A2+, Fe3+]octO42- (A= Zn)4.

Zinc-ferrite has been studied earlier extensively in the paint and corrosion protection application. The

present study aims at investigating the transcendency of zinc ferrite nanoparticles over other

biocompatible ferrites like magnetite, nickel ferrite, copper ferrite, etc. 5. Possibility of biomedical

applications of zinc ferrite synthesized by hydrothermal route using D-glucose as reducing agent was

investigated6,7. Staphylococcus aureus is a gram positive coccal bacterium that is said to be a major

contributor for skin and soft tissue infections (SSTIs). SSTIs caused by these bacteria are very

common8. Pseudomonas aeruginosa is a gram negative bacterium which is one of the most common

air borne pathogens, which is also known to cause skin infections along with lung related diseases9.

Bacillus subtilis is gram positive bacterium which is known to infect immunocompromised patients10

and Escherichia coli is researched to be one of the predominant bacteria that causes SSTIs in

hospitalized patients11. Antibacterial activity of the zinc ferrite was tested against the selected

bacteria. Toxicology of the material was assessed using chick embryos by inspecting the

teratogenicity caused12 and also by analysing the total protein content of the embryos 13, 14. In addition,

haematotoxicity15 of the material was analysed and antioxidant nature was estimated by DPPH (2, 2-

Diphenyl-1-picrylhydrazyl) free radical scavenging assay16 and microbial toxicity by antibacterial

assay17. The schematic sketch of the current research is as shown in Fig 1.

Fig. 1: Schematic sketch of the current research



EXPERIMENTAL

Hydrothermal synthesis of zinc ferrite: Hydrothermal synthesis of zinc ferrite particles was carried

out using General purpose autoclaves made of stainless steel (SS 316) and Teflon liners under mild

conditions (T = 160 °C and P = autogenous). The reaction mixture was prepared in a beaker using

ultrapure water (Elga Option Q7, UK). Aqueous solution of 0.25 M zinc nitrate hexahydrate

(Zn(NO3)2.6H2O, Sigma-Aldrich) and 0.5M ferric chloride hexahydrate (FeCl3.6H2O, Molychem)

were prepared in 1:2 ratio of Zn:Fe. 1.5 M sodium hydroxide (Rankem) was slowly added drop wise

into the mixture under continuous stirring (magnetic stirrer), until pH 9 was obtained. The precipitate

formed was then washed using ultrapure water several times until pH 7 was reached. The resultant

precipitate after washing was transferred into Teflon liners. 0.5 ml of 0.5 M D-Glucose (Rankem,

India) was added to it and then stirred well. D-Glucose is used as reducing agent in the present

experiment18. Liners were then placed in stainless steel autoclaves and kept in the furnace at 160°C

for 14 hr. After the experimental run, the autoclaves were allowed to cool down and the products

obtained were collected in a separate beaker for washing and washed several times using ultrapure

water with the help of a neodymium magnet (NdFeB magnet). The particles were later freeze dried.

Characterization techniques: Phase purity of the synthesized particles was measured by powder

XRD analysis using Rigaku Mini Flux II (Japan) instrument. Powder XRD analysis was performed

using Cu as target material (1.5406 Å). The average crystallite size of the particles was determined

using Scherrer’s formula as shown in Eqs. (1).

cos

9.0D (1)

Where,

θ is the Bragg’s angle in degrees.

β is the angular line width of half maximum intensity

λ is the wavelength of the X-ray used,

D is the average crystallite size,

Presence of D-glucose coating and functional groups was conformed using Fourier transform infrared

spectroscopy (Jasco FTIR- 460 plus, Japan). Particle size of the nanoparticles was conformed using

Dynamic light scattering (DLS) technique (Nanotrac wave, Microtrac®, USA). Morphological and

elemental composition characterizations were done using scanning electron microscope (SEM) and

energy dispersive spectroscopy (EDS) respectively using Hitachi S-3400N.

Assessment of Antioxidant Activity Using DPPH Assay: Standard protocol was followed to

determine antioxidant property of the Zinc ferrite nanoparticles synthesized using 1, 1-diphenyl 2-

picrylhydrazyl (DPPH) 19-21.

Colour change from violet to pale yellow was observed during DPPH (0.004% solution) free radical

scavenging. Absorbance reading was taken at 517 nm using UV-Vis spectrophotometer (ELICO® SA

165, India). The experiment was performed in a dark room condition, since DPPH is a

photodegradable material.

Methanol was used as reference and DPPH stock solution (0.004%) was used as control. Percentage

of DPPH-free radical scavenging activity was calculated using Eqs. (2):



% Free radical scavenging =

100

c

sc

A

AA (2)

Where, (As) is the absorbance of the sample and (Ac) is the absorbance of the control.

Haemolysis Assay: Fresh chicken blood was collected and stock solution was prepared using 1

DPBS to ensure the cell count of ~5108 RBC/ml 22, 23.

Stock solution was then subjected to serially diluted zinc ferrite nanoparticles concentrations (in

normal saline or 1x DPBS) for a period of 3 hrs. After incubation, the mixtures were centrifuged at

14000 rpm for 5-10 min. Supernatant from each vial was collected and absorbance reading was taken

at 541 nm using UV-Vis spectrometer.

Ultrapure water was taken as positive control and 1 DPBS as negative control.

Percentage of haemolysis was calculated using Eqs. (3):

% Haemolysis =

100

np

ns

AA

AA (3)

Where, (As) is the absorbance of the sample, (An) is the absorbance of the negative control and (Ap) is

the absorbance of the positive control.

Embryotoxicity Assay: Embryotoxicity assay was performed using 4 days old fertilized hen eggs.

About 18 eggs of approximately equal weights were collected from National Hatcheries Ltd

(Gundlupete, Mysore).

The eggs were divided into three sets: set A, set B and set C, with each set containing 6 eggs.

Experiment was performed in triplicates in order to confirm the results. The eggs from each group

were labelled as follows: X for control group and Z1, Z2, Z3, and Z4 for each concentration of the

dispersed zinc ferrite nanoparticles and B for blank. In eggs that were labelled as X, 20 µl of sterilized

1 DPBS was injected. Eggs that were labelled as B were left blank without any inoculation. In those

that were labelled as Z1 to Z4, 20 µl of serially diluted zinc ferrite samples (6 mg/ml to 0.75 mg/ml)

were injected.

Inoculation was done through a very small hole made on the egg shell using a sterile needle and with

the help of a Borosil micropipette. After inoculation, the opening was sealed using candle wax.

The eggs were then incubated at 37 °C until the 12th day. On the 12th day of incubation, eggs were cut

open and embryos were collected to check the growth of embryo and teratogenicity24-26. Heart and

livers from the embryos were collected for total protein assay.

Bradford protein assay: After the duration of incubation, embryos were dissected; heart and liver

were isolated from the embryos carefully and washed with PBS to remove excess blood. Isolated

samples were homogenised in 1 DPBS at 1:5 (w/v) ratios and centrifuged to collect the supernatant.

Bradford assay protocol27 (Bradford, 1976) was performed on the supernatants collected after

centrifuging the tissue homogenates of hearts and livers of the embryos28,29. The contents were read at

595 nm using UV-Vis spectrometer for absorbance readings.

Bovine serum albumin stock solution was used as positive control and 1 DPBS with of the Bradford

reagent (4:1 v/v) was used as negative control. The results obtained were tabulated and calculation

was done using Eqs. (4)



% of total protein =

100

np

ns

AA

AA (4)

Where, (An) is the absorbance of the negative control, (Ap) is the absorbance of the positive control

and (As) is the absorbance of the sample.

Antibacterial Assay: Antibacterial property of the Zinc ferrite nanoparticles synthesized was

assessed using pure cultures procured from Microbial Type Culture Collection and Gene Bank,

Chandigarh, India, such as, Escherichia coli (MTCC 1698), Staphylococcus aureus (MTCC 6908),

Pseudomonas aeruginosa (MTCC 4673), Bacillus subtilis (MTCC *121).

Experiment was performed in well plates30-32 and plates were scanned using microplate reader

(Thermo scientific, MultiskanTM) at 580-600 nm after the incubation period. Percentage antibacterial

activity was calculated from the absorbance reading, using the following formula Eqs. (5).

100

np

bg

CC

SSP (5)

Where, Pg – percentage growth, S – absorbance of the sample with inoculum, Sb – absorbance of the

sample blank, Cp – absorbance of the positive control and Cn – absorbance of the negative control.

Percentage inhibition (Pi) can be calculated from Eqs. (6):

gi PP 100 (6)

RESULTS:

Material Characterization

Powder XRD analysis: The graph obtained was matched with JCPDS card NO. 22-1012 as the graph

shows the major peaks (220), (311), (301), (422), (511) and (440) as evident from Fig 2. These peaks

correspond to cubic inverse spinel structures of zinc ferrite. The lattice parameters were found to be

a = b = c and α = β = γ = 90°, showing that the material synthesized belongs to cubic symmetry.

Crystallite size of the material was calculated to be 32 nm using Scherer’s formula.

Fig. 2: powder XRD analysis of the synthesized Zinc ferrite material



FTIR analysis: FTIR spectrum of the title compound is shown in the Fig 3. The stretching vibrations

of Fe+3–O-2 and Zn+2-O-2 bonds in octahedral sites was observed from the band at ~400 cm-1. The

stretching vibrations of Fe+3–O-2 in tetrahedral sites were identified as ~580 cm-1. The presence of

organics due to the addition of D-glucose was evident from C=C bonds and C-O bending vibrations

were noted at ~1600 cm-1 and ~1150 cm-1 locations respectively. Presence of O-H bond at ~3400 cm-1

was probably because of the absorption of atmospheric water by KBr during the making of pellets.

Fig. 3: FTIR analysis of the synthesized Zinc ferrite material

SEM analysis: Morphology of the material synthesized was studied by scanning electron microscopy.

The images show the formation of cubic zinc ferrite nanostructures. Uniform morphology range of the

particles was also observed as evident from the images in Fig 4.

Fig. 4: SEM micrographs of zinc ferrite

C-O

C=C

O-H

Fe-Otet

M-Ooct

Fe-Ooct

Wavenumber (cm-1)

%T



EDS analysis: The EDS analysis graph is shown in the Fig 5. The resultant graph confirms the

presence of zinc, iron, oxygen and carbon, attributing to the formation of Zinc ferrite. The presence of

carbon was due to the presence of D-glucose.

Fig. 5: EDS elemental analysis of Zinc ferrite

DLS analysis: Average size of the synthesized zinc ferrite nanoparticles was measured using dynamic

light scattering technique. The analysis results show an average particle size distribution of 67.4 nm,

with 95 percentile of particles being at 47.91 nm size range Fig 8. The graph obtained is as shown in

Fig 6.

Fig. 6: Particle size analysis

DPPH free radical scavenging assay: The results obtained show an excellent free radical scavenging

activity of the synthesized zinc ferrite, as plotted in Fig 7. The material showed 94% of excellent

DPPH-free radical quenching at 6 mg/ml concentration. Upon reducing the concentration of zinc

ferrite to half (3 mg.ml), the activity reduced slightly, to 89% and the activity reduced gradually upon

further reduction in the concentration of the material synthesized. At 0.375 mg/ml concentration, zinc

ferrite showed 59% of the activity.



Haemolysis assay: Haemolysis results are as shown in. About 0.57% of haemolytic activity was

observed for 0.375mg/ml concentration of the synthesized zinc ferrite nanoparticles. It was also

observed that there was only slight increase in haematotoxicity with increase in the concentration of

the material. At the concentration of 6mg/ml, the material showed about 5% of haemolysis. From this

observation we infer that, zinc ferrite material synthesized is not toxic to the blood.

Fig. 7: Percentage DPPH free radical scavenging by Zinc ferrite

Fig. 8: Percentage haemolysis activity of Zinc ferrite

Teratogenicity assay: The embryos were observed for morphological defects. Treated embryos were

compared with that of the blank. No evidence of growth inhibition and teratogenicity were observed

in the embryos at all the concentrations of zinc ferrite considered for the experiment. There were no

evidences of hepatic and cardiac edema found and the breast plate formation was proper. There were

no deformations in beak, limbs and wings, as evident from Fig 9.This shows that the injected

nanoparticles did not cause any teratogenicity in the embryos.

Total protein assay: Total protein assay was performed using Bradford’s reagent taking Bovine

serum albumin as standard. Absorbance readings were taken at 595 nm using UV-Vis spectrometer

and percentage of protein content was calculated. The results obtained were as shown in the graph in

Fig 10. The protein content in both heart and liver of the embryos reduced slightly upon exposure to

zinc ferrite material, when compared to that of blank. At lower concentrations (1.5 mg/ml and 0.75

mg/ml) the material synthesized did not show a significant variation. In the presence of 3 mg/ml and 6



mg/ml concentrations, it showed a decrease in the total protein content to 39% and 35% in liver and

heart respectively. This states that the synthesized zinc ferrite nanoparticles caused a minimal amount

of protein degradation and are not embryotoxic in nature.

Fig. 9: Teratogenicity test – showing the healthy embryos even after the treatment

39%42%

48%52%

56%

35% 37% 38%43%

47%

6mg/ml 3mg/ml 1.5mg/ml 0.75mg/ml B

Total Protein in Liver Total Protein in Heart

Fig. 10: Total protein content in liver and heart of the chick embryos

Antibacterial assay: The experiment was performed in triplicates for proper confirmation. Zinc

ferrite nanoparticles showed consistent inhibition of the selected bacteria. Nanoparticles caused

96.78% of growth inhibition of E.coli, 94.36% inhibition of S. aureus, 85% inhibition P.aeroginosa,

Conc. of the Zinc ferrite



and 73.43% inhibition of B.subtilis growth. The results obtained were plotted in a graph as seen in Fig

11.

Fig. 11: Percentage inhibition of bacterial growth by Zinc ferrite

DISCUSSION

Eloquent hydrothermal synthesis of cubic symmetric Zinc ferrite nanoparticles using D-glucose as

reducing agent. The synthesized nanoparticles were characterized using DLS, SEM & EDS, FTIR and

XRD. The characterization results showed that the synthesized nanomaterials are with an average

crystallite size of 38 nm, containing Zn, Fe, C and O as its components (attributed to the formation of

ZnFe2O4 coated with D-glucose). DLS results show an average particle size of ~67 nm. The material

was tested against fresh chicken blood (RBC) and against 4-day-old chick embryos to understand its

haemolytic activity and embryotoxicity and teratogenicity. From the results obtained it was evident

that the title compound did cause any rupture to the red blood corpuscles and exhibited no teratogenic

effects. Moreover, the material showed ~90% of DPPH free radical scavenging activity, which is

desirable in wound healing. Foreign objects that enter the body can cause several types of adversities

which include the degradation of proteins. Hence, the chick embryos were exposed to the synthesized

material, by injecting the material at different concentrations, in order to understand the changes in the

level of total protein content in the heart and liver tissues. It was understood that the material did not

alter the amount of total protein content of the hearts and liver of the embryos significantly. In

addition to the above, the synthesized nanoparticles showed good antioxidant nature and caused an

effective inhibition in the growth of Bacillus subtilis, Streptococcus aureus, Pseudomonas aeruginosa

and Escherichia coli bacteria.

Ferrites of metals like nickel, cobalt and copper are known to possess properties that are currently

employed in magnetic storage devices, corrosion resistant coatings, biomedical devices, etc

(Valenzuela 2012)5. Magnetite has been extensively researched as one of the biocompatibility

materials but it lacks antibacterial nature33. Fewer researches have been done in employing these

ferrites for in vivo applications owing to the toxic nature of heavy metals. Based on the results

obtained, we infer that the zinc ferrite nanoparticles synthesized are more advanced than magnetite

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

S. aureus

P. aeroginosa

B. Subtilis

E. coli



and other known ferrites of metals like nickel, cobalt, copper, etc as the synthesized nanoparticles are

completely biocompatible besides possessing excellent antibacterial and antioxidant properties. The

ability of zinc ferrite nanoparticles to inhibit the growth of S.aureus, E. coli, B.subtilis and

P.aeroginosa, which are the most common SSTI causing bacteria, can be employed in preparing

ointments, creams, gels and antibacterial powders for treating skin infections.

CONCLUSION

Facile hydrothermal technique was employed in order to synthesize biocompatible, cubic symmetric

zinc ferrite nanoparticles. Obtained nanoparticles were characterized using FTIR, DLS, XRD, SEM

and EDS and the average particle size was found to be ~47 nm. Toxicological studies revealed that

the synthesized material does not show any toxicity towards blood upon exposure. Embryotoxicity of

zinc ferrite material was tested using 4 days old chick embryos. There were not evidences of

teratogenicity and embryotoxicity observed from the results. Bradford’s assay performed on the

treated embryos show that there was no significant variation in the amount of total protein content of

the hearts and livers. The synthesized material also possesses excellent antioxidant property of ~90%

DPPH free radical scavenging activity. Zinc ferrite synthesized successfully inhibited the growth of

primary SSTI causing bacteria like Escherichia coli, Streptococcus aureus, Bacillus subtilis, and

Pseudomonas aeruginosa. These bacteria are said to cause several types of skin infections, several

pulmonary infections and food poisoning. Based on the evaluated results and in comparison with the

properties of other known ferrites, the present authors conclude that the zinc ferrite nanoparticles

synthesized are ideal for biomedical applications such as in preparing creams, ointments, gels and

antibacterial powders to treat skin infections caused by these bacteria.

ACKNOWLEDGEMENT

This work was supported by University Grant Commission, India, under University with Potential for

excellence Programme (UPE), University of Mysore. We thank Prof. M. S. Thakur, Visiting

Professor, Centre of Material Science and Technology, University of Mysore, for his help in

preparation of the manuscript. We also thank Ms. D. Shanthini Keerthana and Dr. P. Shubha of Centre

for Materials Science and Technology, University of Mysore, for their help in carrying out the toxicity

studies.

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