Biocompatible suspension of nanosized γFe2O3 synthesized by novel
methods
N. K. Prasad, D. Panda, S. Singh, M. D. Mukadam, S. M. Yusuf et al.
Citation: J. Appl. Phys. 97, 10Q903 (2005); doi: 10.1063/1.1849056
View online: http://dx.doi.org/10.1063/1.1849056
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JOURNAL OF APPLIED PHYSICS 97, 10Q903 共2005兲
Biocompatible suspension of nanosized ␥-Fe2O3 synthesized
by novel methods
N. K. Prasad
Metallurgical Engineering and Materials Science, Indian Institute of Technology, Bombay, India,
Mumbai, India
D. Panda
School of BioSciences & Bioengineering, Indian Institute of Technology, Bombay, India, Mumbai, India
S. Singh, M. D. Mukadam, and S. M. Yusuf
Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai, India
D. Bahadura兲
Metallurgical Engineering and Materials Science, Indian Institute of Technology, Bombay, India,
Mumbai, India
共Presented on 9 November 2004; published online 17 May 2005兲
The present work deals with the synthesis of nanosized ␥-Fe2O3 by methods and its suspension in
a biocompatible fluid consisting of cellulose in water whose properties seem different from the
conventional ferrofluid and magnetic rheological fluid. From TEM and x-ray diffractions line
broadening particle size has been estimated in the nanometer range. Mössbauer and magnetic
measurements have been done to investigate the relaxation behavior of these nanoparticles which
consists of significant amount of surface spins. Biocompatibilities of suspensions have been
confirmed using HeLa cell lines and can be suitable for different bioapplications. © 2005 American
Institute of Physics. 关DOI: 10.1063/1.1849056兴
I. INTRODUCTION
␥-Fe2O3 is a biocompatible compound and may be useful for applications like hyperthermia and drug delivery. For
both applications, it is necessary to suspend in biocompatible
media.1 Nano sized ␥-Fe2O3 can be synthesized by several
wet chemical methods, such as, coprecipitation, sol-gel, microemulsion, hydrothermal synthesis, etc.2,3 All these processes have certain limitations. Among these, hydrothermal
process is a better option but its reaction time is longer at
temperatures below 200 ° C. But, an introduction of microwave 共MW兲 heating 共called microwave hydrothermal M-H兲
reduces the processing time and energy cost. Here, two
methods of synthesis of ␥-Fe2O3 nanoparticles are described.
A suspension containing up to 30 mg of ␥-Fe2O3 / ml
have been successfully prepared into cellulose containing
distilled water and biocompatibility for these suspensions
have been tested with HeLa cells.
II. EXPERIMENT
A required amount of anhydrous FeCl3 was dissolved
into ethylene glycol. Different amounts of KOH were added
to the solution for different pH values. The experiments were
done for pH values of 9, 10, 11, and 12 for two processes
used. This solution was then kept on a hot plate at 200 ° C
for 3 h in the first process while solution was refluxed for
45 min in a microwave oven in the second process 共microwave refluxing process兲. A black precipitate of ␥-Fe2O3
settled down which was decanted with water and acetone
several times. Black ␥-Fe2O3 of nanosize was thus obtained
a兲
Author to whom correspondence should be addressed.
0021-8979/2005/97共10兲/10Q903/3/$22.50
which turned to dark brown after drying. Ethylene glycol
acts as a solvent for FeCl3 and as a capping agent, which
inhibits the growth of the size of the particles and also as an
absorber of microwave in the case of second process.4
X-ray diffraction patterns of ␥-Fe2O3 powder were taken
by Philips x-ray diffractometer employing Cu– K␣ radiation
共␭ = 1.54056 Å兲. For particle size determination and electron
diffraction, the transmission electron microscopy 共TEM Philips CM 200兲 was used.
Mössbauer spectra were recorded at 298 and 78 K in
standard transmission geometry and in the constant acceleration mode, using a source of 57Co in Rh matrix. The spectrometer was calibrated with ␣-Fe as standard and the isomer
shift values were given relative to this. The experimental
data were fitted with the help of a least-squares curve fitting
program. The magnetic measurements have been carried out
using a vibrating sample magnetometer over the temperature
range of 5 – 320 K. The hysteresis loops were recorded in a
magnetic field up to 20 kOe
A homogeneous solution of cellulose was prepared in
distilled water in which nanosized ␥-Fe2O3 particles were
added and stirred for one hour. This gave a suspension of the
particles. We prepared suspensions of ␥-Fe2O3 of density 26
and 30 mg/ ml. This suspension seems to have different
properties than ferro-fluid or magnetic rheological fluid.
A. Cell culture and proliferation assay
HeLa cells were grown in minimum essential medium
共Himedia兲 supplemented with 10% v / v fetal bovine serum,
Kanamycin 共0.1 mg/ ml兲, Penicilin G 共100 units/ ml兲 and sodium bicarbonate 共30 mg/ ml兲 at 37 ° C in a 5% CO2 atmo-
97, 10Q903-1
© 2005 American Institute of Physics
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10Q903-2
J. Appl. Phys. 97, 10Q903 共2005兲
Prasad et al.
TABLE I. Size range of ␥-Fe2O3 synthesized by two processes estimated
from XRD and TEM.
FIG. 1. XRD patterns of ␥-Fe2O3 共synthesized by both processes and with
different pH values兲. Here 1 = first process, 2 = second process, 关 兴 = pH
values.
sphere. Cell proliferation was determined using sulforhodamine B 共SRB兲 assay in 96 well plates as described
earlier.5 Briefly, cells 共1 ⫻ 105cells/ ml兲 were grown for 24 h
and treated with different proportion of magnetic particles
suspension. We added 3% and 13% of both the suspensions
into wells where cells were absent to know their effects on
absorbance. After 24 h incubation, cells were processed for
SRB assay to determine cell viability. Wells were washed
thrice with PBS to remove the suspended particles. Cells
were then fixed with a solution of 10% trichloroacetic acid
and stained with 0.4% SRB dissolved in 1% acetic acid. Cell
bound dye was extracted with 10 mM unbuffered Tris base
共pH 10.5兲 and the optical density was taken at 550 nm which
is used to calculate cell viability. Each experiment was done
in triplicates.
Sl. no.
pH values
Process
1
2
3
4
5
6
7
8
9
10
11
12
9
10
11
12
1
2
Size range
from XRD
共nm兲
Size range
from TEM
共nm兲
8–12
8–13
8–15
8–15
9–17
12–18
10–16
12–18
8–18
8–18
8–20
8–20
10–16
12–18
12–18
13–20
with only two sextets. The CS values are 0.286 and
0.324 mm/ s, where as BHf values are 503 and 521 kOe for
the component 1 and 2, respectively. In this case the superparamagnetic nature completely ceases and particles behave
as ferrimagnetic.8 This is attributed to particle size range of
8 – 20 nm.
The reduced values of BHf compared to the bulk are due
to the smaller particles size and their surface spins.6 The CS
values obtained are consistent with that for bulk ␥-Fe2O3.7,8
Figure 4共a兲 shows the zero-field cooled 共ZFC兲 and FC
data. Irreversibility is very large and divergence starts at a
high temperature of 310 K. This kind of large divergence is
expected in nanosized particles with a wide distribution
which have varying concentration of disordered surface
spins. These may progressively freeze resulting in a wide
distribution of relaxation times and hence the divergence.
From Fig. 4共b兲 it is observed that M r and HC values are very
close to zero at 320 K but at 5 K they show hysteresis.9 We
observed M S = 77.6 emug−1 at 5 K which is close to the bulk
values of M S = 80 emug−1. The M r value at 5 K is around
17 emug−1 which is nearly zero at 320 K. This behavior is
typical of superparamagnetic materials. However Mössbauer
III. RESULT AND DISCUSSION
Figure 1 shows typical x-ray diffraction 共XRD兲 patterns
of ␥-Fe2O3 synthesized by both the processes and with different pH values. XRD patterns confirm the formation of
monophasic ␥-Fe2O3 and showed a considerable line broadening for all the samples. The average particle size was estimated from x-ray line-broadening using Scherrer’s equation.
The particle size was also determined by TEM and their
shape was found spheroidal. The size range of the particles
determined by TEM and XRD patterns are given in Table I.
Figure 2 shows a typical TEM micrograph of ␥-Fe2O3 synthesized by first process with pH= 12. The inset shows the
corresponding electron diffraction.
Mössbauer spectra are shown Fig. 3. The spectrum at
ambient temperature consists of two sextet 共tetrahedral and
octahedral兲 components and a paramagnetic component. The
values obtained for CS 共center shift兲 are 0.312, 0.362, and
0.55 mm/ s for three components, respectively. The hyperfine
fields 共BHf兲 observed are 475 and 437 kOe for the component
1 and 2, respectively. The superparamagnetic component is
very little. The spectrum obtained at 78 K could be fitted
FIG. 2. Typical TEM photograph, of ␥-Fe2O3 synthesized by first process at
pH= 12. Inset shows SAED pattern.
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10Q903-3
J. Appl. Phys. 97, 10Q903 共2005兲
Prasad et al.
FIG. 5. Effects of suspension on the viability of HeLa cells. Suspension
A = 26 mg of ␥-Fe2O3 / ml and B = 30 mg of ␥-Fe2O3 / ml. ␥-Fe2O3 synthesized by first process at pH= 12 has been used.
data contradicts. This could be attributed to the different relaxation times for magnetic and Mössbauer studies.
As shown in Fig. 5, there were no change in the cell
viability at low concentration of suspension while at 13% of
suspension, cell viability was decreased by 39% and 32% for
samples A and B, respectively. The absorbance in the cases
where only suspensions were added, were too low to have
any effects on the absorbance value of the cells. From these
data, it was found that 3.6 mg of ␥-Fe2O3 / ml can be added
without significant decrease in viability of the cells which is
much higher compared to the value reported.10
FIG. 3. Mössbauer spectra of ␥-Fe2O3 synthesized by first process at pH
= 12, 共a兲 at 298 K and 共b兲 at 78 K.
IV. CONCLUSIONS
Nanosized ␥-Fe2O3 has been synthesized by two methods. The size of the particles was found to be in the range of
8 – 20 nm. Mössbauer spectrum and magnetic measurements
suggested that the relaxation phenomena of disordered surface spins of nanosized particles may be responsible for the
observed behavior. The particles have been successfully suspended in water containing cellulose which shows a good
degree of biocompatibility. We suggest that this kind of suspension can be used for applications such as hyperthermia
treatment of cancer or drug delivery, etc.
ACKNOWLEDGMENT
The financial supports of DBT and DST Govt. of India
are gratefully acknowledged.
1
FIG. 4. 共a兲 FC/ZFC magnetization curve at 200 Oe and 共b兲 magnetization
versus applied field curve at 5 and 320 K, of ␥-Fe2O3 synthesized by first
process at pH= 12. Inset 共b兲 shows magnetization versus applied field curve
in expanded scale.
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