Superparamagnetic iron oxide nanoparticles (SPIONs): Green preparation, characterization andtheir cytotoxicity effects Majid Darroudi, Mohammad… [615994]

Author's Accepted Manuscript
Superparamagnetic iron oxide nanoparticles
(SPIONs): Green preparation, characterization andtheir cytotoxicity effects
Majid Darroudi, Mohammad Hakimi, Elham Good-
arzi, Reza Kazemi Oskuee
PII: S0272-8842(14)00945-6
DOI: http://dx.doi.org/10.1016/j.ceramint.2014.06.051Reference: CERI8736
To appear in: Ceramics International
Received date: 30 May 2014
Revised date: 7 June 2014Accepted date: 8 June 2014
Cite this article as: Majid Darroudi, Mohammad Hakimi, Elham Goodarzi, Reza Kazemi
Oskuee, Superparamagnetic iron oxide nanoparticles (SPIONs): Green preparation,characterization and their cytotoxicity effects, Ceramics International, http://dx.doi.org/
10.1016/j.ceramint.2014.06.051
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1 
 Superparamagnetic iron oxide nanoparticles (SPIONs): Green
preparation, characterization and their cytotoxicity effects
Majid Darroudi1,2,*, Mohammad Hakimi3, Elham Goodarzi3, Reza Kazemi Oskuee4,5
1Nuclear Medicine Research Cent er (NMRC), Ghaem Hospital, School of Medicine , Mashhad University of Medical Sciences,
Mashhad, Iran
2Department of Modern Sc iences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad,
Iran
3Chemistry Department, Payame N oor University, 19395–4697 Tehran, Iran
4Targeted Drug Delivery Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
5Department of Medical Biotechnology, School of Medicine, Mashhad Univ ersity of Medical Sciences, Mashhad, Iran

Abstract
Superparamagnetic iron oxide nanoparticles (SPIONs; Fe 3O4) were prepared by the covalent
binding of starch onto th e surface of magnetic Fe 3O4 nanoparticles, by a “g reen” co–precipitation
method. The scanning electron microscopy (SEM ) illustrated the regular spheres of Fe 3O4
particles that were coated by st arch molecules to be regular s pheres with a mean diameter of
about 40 nm. Powder X–ray diffrac tion patterns (PXRD) indicated that the prepared samples
were pure Fe 3O4 with a spinel structure while the coat ing of starch did not result in a phase
change. The starch coating onto the Fe 3O4 nanoparticles was also demonstrated by Fourier
transform infrared (FTIR) sp ectra. Magnetic measurements revealed that the saturated
magnetization of the starch–SPIONs reached 42.1 emu/g, having nanoparticles with the
characteristics of superparamagnetism, while a do se dependent toxicity with non–toxic effect of
concentration below 62.5 µg/mL was observed in the studies of In vitro cytotoxicity on neuro2A
cells.
Keywords: Iron oxide; superparamagnetic; na noparticles; starch; neuro2A

*Corresponding author:

Majid Darroudi (PhD),
Nuclear Medicine Research Center (NMRC) , Ghaem Hospital, School of Medicine,
Mashhad University of Medical Sciences, Mashhad, Iran.
Tel.: +98 511 8002286, Fax: +98 511 8002287
Email: [anonimizat], [anonimizat]

2 
 1. Introduction
Nanotechnology is now extensively used throu ghout the medicine, drug delivery systems,
optical, electronics, chemical and mechanical applications. Magnetic nanoparticle is an attractive
research material not only in the field of magnetic recording but also in a variety of topics such
as biomedical, optical, electronics, chemical and mechanical applications [1–3]. For these uses,
critical parameters such as size, size distri bution of magnetic nanoparticles, and its physical
properties (e.g., magnetic, optical, and electronic) are vital information. As a member of
magnetic nanoparticles family, superparamagneti c iron oxide nanoparticles (SPIONs) can avail
in different fields such as pigments [4], catalysts [5], ferrofluids [6], and biomedical applications
such as cancer therapy [7], drug delivery syst em [8, 9], tissue engi neering [10], magnetic
resonance imaging (MRI) [11], and hyperthermia [12, 13]. Recently, various methods have been
developed to prepare SPIONs such as sol–ge l method [14], hydrothermal technique [15], co–
precipitation from the solution of ferrous/ferric– salt mixture in alkaline medium [16], reduction
of hematite ( α–Fe 2O3) by CO/CO 2 [17], oxidation of Fe(OH) 2 by H 2O2 [18], microwave
irradiation [19], laser ablation [20], γ–ray radiation [21], chemical vapor deposition [22], and
thermal decomposition [23]. These methods suffer the inability to cont rol the size and size
distribution of SPIONs due to th e large surface area and high surface energy as well as high
magnetization that results in agglomeration and cl uster formation [24]. In recent years, polymer–
template methods have also be en adopted for controlled si ze synthesis of metal oxide
nanoparticles [25–27] and especially SPIONs [28–30]. However, the research is st ill proceeding
to obtain controllable size and well dispersed SP IONs. In this work, well–fined SPIONs were
synthesized using co–precipitation method while soluble starch was used as a capping agent
and/or stabilizing agent to pr event aggregation among nanoparticle s. Co–precipitation method is

3 
 the most common route, which yields comparativ ely good control over the size, size distribution,
and morphology of water–based SPIONs at low te mperatures. The different samples of SPIONs
were successfully synthesized by vig ilantly controlling the amount of Fe2+ cations in aqueous
starch solutions. The crystalline structure, si ze and optical propertie s of as–prepared SPIONs
were characterized by XRD, UV–vis and SEM techniques and the magnetic properties were
investigated by VSM at room temperature. The results indicated that the as–prepared SPIONs
illustrate good crystalline structure, small pa rticle size and high saturation magnetization.
2. Materials and methods
2.1. Materials and reagents
Chemicals and reagents that were used for the synthesis of starch–SPIONs were used without
further purification at analytical grad e. Ferrous chloride tetrahydrate (FeCl 2.4H 2O, Merck),
soluble starch (Amylose molecular form, solu ble, Aldrich), and ammonium hydroxide solution
(NH 4OH, 25 vol%; Merck) were used as the starti ng materials. For the evaluation of metabolic
activity, Neuro2A murine neuroblastoma cells (ATCC CCL–131, Manassas, VA, USA) were
grown in Dulbecco's modified Eagle's medium (1 g/L glucose, 2 mM glutamine), supplemented
with 10% FBS, streptomycin at 100 m g/ml, and penicillin at 100 μg/ml. All cells were incubated
at 37 ˚C in a humidified 5% CO 2 atmosphere. The SPIONs were synthesized at a low
temperature, via co–precipitation route from FeCl 2.4H 2O in an aqueous starch solution as a green
substrate at alkaline medium.
2.2. Synthesis of starch –SPIONs
To synthesize the starch–SPIONs, 0.50 g of FeCl 2.4H 2O was dissolved in 40 ml of double
distilled water (DDW); then, hydr ochloric acid was added (Unt il pH=3(±0.01)) and stirred at

4 
 room temperature for 30 min. The pH of precip itation process was increased to about 9.1(±0.01)
after adding 20% aqueous ammonia plus being digested for 60 min at 70°C. Afterwards, an
aqueous starch solution (40 ml; 2% w/w) was adde d to the initial solution as a stabilizing agent
and thus had the final solution stirred for 90 min at 50°C. The precipitate was then washed with
DDW and ethanol twice to remove the excess of ammonia remaining in the precipitate. The
SPIONs were finally collected as dark brow n nanopowders after a drying process at 80°C (S1).
To investigate the Fe+2 contents, similar experiments were applied with different concentrations
of Fe+2 (1 (S2), 2 (S3), and 3 g (S4)).

2.3. Evaluation of neurotoxicity effect of starch–SPIONs
We had the cytotoxicity of the samples evalua ted through the use of 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenylte-trazolium bromide (MTT) assay [ 31]. To be brief, neuro2A cells were seeded
at a density of 1×104 cells per well in 96-well plates and incubated for 24 h. Thereafter, the cells
were treated with various concentrations of nanoparticles in the presence of 10% FBS. The
starch–SPIONs (S1) were suspe nded in a stock solution at 5 μg/ml in dimethyl sulfoxide
(DMSO)/double distilled water solu tion. After 24 h of incubation, 20 μl of 5 mg/ml MTT in the
PBS buffer was added to each well, and the cel ls were further incubated for 4 h at 37 ˚C. The
medium that contained unreacted dye was discarded, and 100 μl of DMSO was added to dissolve
the formazan crystal formed by live cells. Op tical absorbance was measured at 590 nm
(reference wavelength 630 nm) using a micropl ate reader (Statfax–2100, Awareness Technology,
USA), while cell viability was expressed as a per cent relative to untreated control cells. Values
of metabolic activity are presen ted as mean±SD of triplicates.

5 
 2.4. Characterization of starch–SPIONs
The synthesized starch–SPIONs were characterized by using X–ray diffraction (XRD, Philips,
X'pert, Cu K α), Fourier transform Infrared spectrosc opy (FTIR, Shimadzu 8400, Japan), and field
emission scanning electron microscopy (FESEM , Carl Zeiss Supra 55VP), while magnetism
properties of nanoparticles were measured by vi brating sample magnetometer (VSM, Made in
Kashan University, Iran).

3. Results and discussion
Fig. 1 shows the PXRD patterns of prepared starch–SPIONs at different contents of Fe+2 in the
precipitating reaction. Six typical peaks for SPIONs (2 θ=30.1 ˚, 35.4 ˚, 43.0 ˚, 53.4 ˚, 57.0 ˚, and
62.6 ˚), marked by their indices ((220), (311), (400), (422), (511), and (440 )), were observed in
all the samples while the relative intensity of th e peaks was consistent. All the prepared samples
exhibited consistent peaks with the cubic inverse spinel structure of Fe 3O4 in the standard data
from JCPDS file # 19–0629. There are no other diffrac tion peaks detected as impurities such as
ferrite nitrite, goethite, and maghemite or amorphous phase. The PXRD patterns display the
broad nature of the diffraction bands, illustrating small crystallite sizes that can be quantitatively calculated by the Debye–Scherrer formula, i.e., (D=K λ/βcosθ) [32, 33], which represents a
relationship between peak broadeni ng in PXRD and crystallite size, where D is the particle size,
K is a constant (Debye–S cherrer constant (0.89)) related to the particle shape and crystalline
plane, λ is the X–ray wavelength (0.15406 nm), β is the full width at half–maximum of
diffraction peak, and θ is the X–ray diffraction angle. Using the x–ray re sults and this equation,
the crystallite size of SPIONs (S1) coated w ith starch was calculat ed to be ~12 nm.

Fig.

The mo r
disperse d
agglome r
relativel y. 1: PXRD p
rphology o f
d, as obser v
rated due to
y porous net w
patterns of p
f nanoparti c
ed in the F E
the interact i
work.
prepared st
cles was al
ESEM ima g
ion between
6 arch–SPIO
most spher i
ges (Fig. 2)
magnetic n a
Ns with dif f
ically regul
of the star c
anoparticles ,
ferent cont e
ar in shap e
ch–SPIONs,
, whereas th e

ents of Fe+2.
e and unif o
but some e
e gel repres e
.
ormly
extent
ents a

The vale n
the size o
in many after the below:

where Fapproac
h
are form e
which i s
temperat u
Fi
nce of the i r
of SPIONs.
co–precipi t
addition of
eO·Fe 2O3 in
hes report o n
ed by the c o
s carried o u
ure, and by
ig. 2: FESE M
ron precurs o
To prepare S
tation meth o
the base sol
ndicates th a
n the prepar a
o–precipitat i
ut by contr o
the additio
M images o
or used in th e
SPIONs wit h
ods, a black
ution to the
at the dival
ation of SPI O
ion method o
olling vario u
n of surfac t
7 of prepared
e synthesis a
h solution o
precipitate
iron precur s
ent and tri v
ONs with di f
of Fe2+ and
us paramete r
tants [34]. H
starch–SPI
appears to p l
f Fe2+ and F
of these p a
sor, reactin g
valent iron
fferent sizes
Fe3+ cation s
rs such as
Howeve r, in
ONs (S1).
lay a key ro
Fe3+ cations (
articles was
g as display e
atoms in m
in the rang e
s at the mol a
base conce n
this work,
le in deter m
(molar ratio
formed ins t
ed in the eq u
magnetite. M
e of below 5
ar ratio of 1
ntration, so l
an aqueous

mining
1:2),
tantly
uation

Many
50 nm
to 2,
lution
Fe2+

8 
 solution alone produced a dark green precipitate immediately after being mixed with the base
solution, and the obtaine d precipitate gradually turned black in color, which indicated the
formation of intermediates prior to the SPI ONs. Refait and Olowe [35, 36] studied the
alkalization reaction of Fe2+ cations as regards to the forma tion of iron hydroxide and iron oxide,
proposing the following reactions as th e mechanism of SPIONs formation:

Feଶା൅2 O Hି՜F e ሺ O H ሻ ଶ ሺ2ሻ
3FeሺOHሻ ଶ൅1 / 2 O ଶ՜F e ሺ O H ሻ ଶ൅2 F e O O H൅H ଶO ሺ3ሻ
FeሺOHሻ ଶ൅ 2FeOOH ՜ Fe ଷOସ൅2 H ଶO ሺ4ሻ

Thus, in this synthesis procedure with Fe2+ cations alone, SPIONs are formed as a result of the
dehydration reaction of Fe(OH) 2 and FeOOH (reaction (4)) in which the latter compound is
produced by the partial oxidation of Fe(OH) 2 using oxygen gas that dissolved in air (reaction
(3)). This is the mechanism that controls the transformation of Fe(OH) 2 phases to the final phase
of SPIONs.
Fig. 3 shows the FTIR spectra of the as–synthesi zed SPIONs in starch media. The peaks at 1458
(bending mode) and 3448 cm–1 (stretching mode) observed in FT IR spectrum relates to the –OH
group. The characteristic peaks of SPIONs at around 443, 582, and 667 cm–1 could be observed
in the spectrum, which relates to the Fe–O bond [37], while th e FTIR of remained starch
molecules can be preserved in Fi g. 3. The main absorption peaks of starch molecules can appear
in a range of 900–1200 cm–1, corresponding to the C–O bond stretching of starch. The absorption
peaks at 1076 and 1141 cm–1 are related respectively to the C–O bond stretching of C–O–C and
C–OH groups in starch molecule s [38]. Also, there are two absorption peaks at about 2900 and

3750 c m
well.

Magneti c
Accordi n
superpar a
emu/g),
(MRI) f o
100 em u
particles ,
cytotoxi c
line, as s h
62.5 and
m–1, which a r
Fig. 3: F
c characteri z
ng to the vi b
amagnetic b
meaning th e
or medical d i
u/g) [40], th e
, surface di s
city studies c
hown in Fi g
125 µg/mL )
re attributed
Fourier tra n
zation of th e
brating sam p
behavior an
e synthesiz e
iagnosis an d
e decrease o
sorder and
clarifies tha t
g. 5, tested t h
) as indicate
to C–H an d
nsform infr a
e starch–SP I
ple magneto
d have lo w
ed starch–S P
d cell separa t
of Ms is c a
cation distr i
t, starch–SP I
hrough usin g
d by the M T
9 d O–H nor m
ared spectr u
IONs at ro o
metry (VS M
w saturation
PIONs are f
tion. Comp a
aused by th e
ibution [41 ]
IONs did n o
g different d o
TT result. T h
mal vibratio n
um of starc h
om tempera t
M) results, t h
magnetizat
facilitating m
ared to the M
e nature of m
]. After 24
ot have any t
oses (0.97, 1
he prepared s
n modes, re s
h–SPIONs (
ture is repr e
he starch–S P
ion (Ms) v
magnetic re s
Ms value of b
monodisper s
h of treat m
toxic effect t
1.95, 3.91, 7 .
sample (S1)
spectively [ 3
(S1).
esente d in F i
PIONs illus t
alues (17.5 –
sonance im a
bulk Fe 3O4 (
sed and ult r
ment, the in
to a neuro2 A
.81, 15.63, 3
demonstrat e
39] as

ig. 4.
trated
–42.1
aging
(~85–
rafine
vitro
A cell
31.25,
ed no

significa n
prepared
the conc e

Fig. 4nt toxicity e
nanoceria i
entration de c
: Ma gnetiz a
ven in conc
s well toler a
creased as t h
ation plots o
entrations u p
ated by Neu r
he concentra t
of prepared
10 p to 62.5 µ g
ro2A cells. T
tion decreas
starch–SP I
g/mL in the
The level o f
ed.
IONs with d
MTT assay
fcytotoxicit y
different co n
meaning th a
y as a functi

ntents of F e
at the
on of
e+2.

Conc l
In sum m
method
nanopartstrongly were in
t
properti e
cytotoxi c
applicati olusion
mary, Fe 3O4
using starc h
icles are w
depen dant o
the range o f
es with Ms
c effect on
ons.
Fig. 5:
nanoparticl e
h as templ a
well–dispers e
on the amou n
f 30–40 n m
of 17.5–4 2
Neuro2A c
: MTT assa y
es have bee n
ates in mo d
ed as the m
nt of iron so u
m and the pr e
2.1 emu/g.
cell lines, m
11 y of starch –
n successful
derate temp
magnetic pr o
urce (Fe2+).
epared star c
In this stu
making it s u
–SPIONs (S
ly prepared
erature (50°
operties of
The mean d
ch–SPIONs
dy starch– S
uitable can d
1).
via a gree n
°C). It is f o
the prepar e
diameters of
exhibited s u
SPIONs ex h
didate for v a

n/co–precipi t
ound that F
ed samples
the nanopa r
uperparama g
hibited ver y
arious biol o
tation
Fe3O4
were
rticles
gnetic
y low
ogical

12 
 Acknowledgments
The authors gratefully acknowle dge the financial support for this work provided by Mashhad
University of Medical Sciences (MUMS) a nd Payam Noor Universi ty, Mashhad Branch
(Mashhad, Iran) based on the M.Sc. thesis of Mrs. Elham Goodarzi. We kindly thank Dr. Leila
Gholami for excellent technical assistance.

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