Journal of Magnetism and Magnetic Materials 323 (2011) 237–243
Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials
journal homepage: www.elsevier.com/locate/jmmm
Development of citrate-stabilized Fe3O4 nanoparticles: Conjugation and
release of doxorubicin for therapeutic applications
Saumya Nigam a, K.C. Barick b, D. Bahadur b,n
a
b
IITB-Monash Research Academy, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India
Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India
a r t i c l e in fo
abstract
Article history:
Received 20 April 2010
Received in revised form
15 August 2010
Available online 17 September 2010
We demonstrate a single-step facile approach for the fabrication of citric acid functionalized
(citrate-stabilized) Fe3O4 aqueous colloidal magnetic nanoparticles (CA-MNP) of size 8–10 nm using
soft chemical route. The surface functionalization of Fe3O4 nanoparticles with citric acid was evident
from infrared spectroscopy, thermal and elemental analyses, and zeta-potential measurements. The
drug-loading efficiency of CA-MNP was investigated using doxorubicin hydrochloride (DOX) as a model
drug to evaluate their potential as a carrier system. The quenching of fluorescence intensity and
decrease in surface charge of drug loaded CA-MNP strongly suggest the interaction/attachment of drug
molecules with CA-MNP. More specifically, the present investigation discusses a method for entrapping
positively charged drugs onto the surface of negatively charged CA-MNP through electrostatic
interactions and suggests that bound drug molecules will be released in appreciable amounts in the
mild acidic environments of the tumors. Furthermore, the aqueous colloidal stability, optimal
magnetization, good specific absorption rate (under external AC magnetic field) and cytocompatibility
with cells suggested that CA-MNP is appropriate candidate for biomedical applications.
& 2010 Elsevier B.V. All rights reserved.
Keywords:
Nanoparticle
Iron oxide
Surface functionalization
Drug release
Hyperthermia
1. Introduction
Magnetic nanoparticles have received a great deal of attention
due to their potential biomedical applications such as hyperthermia
treatment of cancer, contrast agent for magnetic resonance imaging,
magnetic separation and sorting of cells and proteins (bio-recognition), controlled and targeted drug delivery [1–6]. In recent past,
among vast varieties of magnetic nanoparticles, superparamagnetic
iron oxide nanoparticles have emerged as an excellent candidate for
biomedical applications due to their better chemical stability and
biocompatibility compared to other metallic magnetic nanoparticles
[7,8]. Many methods have been developed to prepare superparamagnetic iron oxide nanoparticles [9]. The thermal decomposition
of organometallic precursors in organic solvent (high boiling point)
at elevated temperatures in presence of surfactants has been
successfully used for the synthesis of monodisperse iron oxide
nanoparticles [3,9]. However, the iron oxide nanoparticles prepared
by these methods are highly hydrophobic in nature, which hampers
their biomedical applications especially in drug delivery, hyperthermia treatment of cancer and magnetic resonance imaging. Although
many ligand-exchange processes have been established to offer
them hydrophilic surface characteristic for aqueous stability, their
n
Corresponding author. Tel.: + 91 22 2576 7632; fax: + 91 22 2572 3480.
E-mail address: [email protected] (D. Bahadur).
0304-8853/$ - see front matter & 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jmmm.2010.09.009
magnetic field responsiveness has not been effectively improved
[3,9]. Therefore, much effort has been focused on the fabrication
of biocompatible aqueous stable superparamagnetic iron oxide
nanoparticles of controllable sizes having good magnetic response
by soft-chemical routes [2,9–14]. Furthermore, for most of these
biomedical applications, the first significant challenge is to avoid
undesirable uptake of iron oxide nanoparticles by the reticuloendothelial system (RES). The next step is to achieve selective
targeting of the system to the site of interest for the in vivo studies.
In order to overcome these problems, iron oxide nanoparticles
should be engineered with desired functionality.
The strategies used for surface functionalization comprise
grafting of or coating with organic species, including surfactants
or polymers, or coating with an inorganic layer, such as silica or
gold [11–20]. Further, the presence of these biocompatible layer
on the surface not only stabilizes the iron oxide nanoparticles,
but also provides accessible surface for routine conjugation
of biomolecules through the well-developed bioconjugation
chemistry for a number of biomedical applications. Thus, the
stability of the bonding between functional molecules and iron
oxide nanoparticles is crucial from the application point of view.
The small molecule targeting groups are predominantly attractive
due to their ease of preparation and simple conjugation chemistry
[16,21–23]. Further, multiple grafting or coating of small
molecules can provide multivalent systems, which exhibit
significantly enhanced efficacy towards drugs and biomolecules
238
S. Nigam et al. / Journal of Magnetism and Magnetic Materials 323 (2011) 237–243
[24]. On the other hand, some binding affinity may be lost
through steric hindrances by large surfactant molecules or long
polymer chains, which could be easily overcome with the use of
small molecules having multiple functional groups such as
carboxyl (COOH), amine (NH2), thiol (SH), etc. Furthermore, the
presence of large number of uncoordinated functional groups on
the magnetic nanoparticle surface can be used for linkage of
various biomolecules as well as drugs. Among various small
molecules, citrate moiety has been extensively used for the
preparation of aqueous stable iron oxide nanoparticles as well as
their conjugation to biomolecules and drugs by exploiting
the uncoordinated carboxyl groups present on the surface of
nanoparticles. Recently, Liu et al. [25] fabricated highly waterdispersible magnetite particles with uniform size by solvothermal
reaction at 200 1C by reduction of FeCl3 with ethylene glycol in
presence of trisodium citrate as a stabilizer and found that these
magnetite particles with surface citrate groups can effectively
enrich peptides at trace level. Munnier et al. [10] developed a
new method for reversible association of drug (DOX) to citratestabilized iron oxide nanoparticles prepared by agitating bare
nanoparticles in citric acid solution. Khosroshahi and Ghazanfari
[15] fabricated citrate modified Fe3O4 nanoparticles by stirring
bare Fe3O4 nanoparticles in trisodium citrate solution as intermediate to obtain silica-coated magnetite core-shell nanoparticles. However, most of the these works on fabrication of aqueous
stabilized iron oxide nanoparticles are achieved at either elevated
temperatures [2,3,25] or involved multiple synthesis steps
[10,11,13].
Herein, the goal of this work was to develop a facile single-step
process for preparation of highly biocompatible citric acid functionalized (citrate-stabilized) Fe3O4 aqueous colloidal magnetic
nanoparticles (CA-MNP) with optimal magnetization at low
temperature for potential drug carriers, which can also be used as
effective heating source for the hyperthermia treatment of cancer.
More specifically, in the present study, the drug loading efficiency of
about 90% (w/w) was achieved by electrostatic interactions of drug
molecules (DOX) with CA-MNP, and the DOX conjugated CA-MNP
(DOX loaded CA-MNP) exhibited a sustained release profile.
2. Material and methods
Ferric chloride, ferrous chloride, doxorubicin hydrochloride
and sulphorhodamine-B were purchased from Sigma-Aldrich,
USA. Citric acid (CA), 25% ammonia solution and acetic acid were
procured from Thomas Baker, India. Trichloroacetic acid was
obtained from Loba Chemie, India. All chemicals are of analytical
grade and used as received. In a typical synthesis, 4.44 g of FeCl3
and 1.732 g of FeCl2 were dissolved in 80 ml of water in a round
bottom flask and temperature was slowly increased to 70 1C in
refluxing condition under nitrogen atmosphere with constant
mechanical stirring at 1000 rpm. The temperature was maintained at 70 1C for 30 min and then 20 ml of ammonia solution
was added instantaneously to the reaction mixture and kept at
the same temperature for another 30 min. Then 4 ml of aqueous
solution of citric acid (0.5 g/ml) was added to the above reaction
mixture and reaction temperature was slowly raised up to 90 1C
under reflux and reacted for 60 min with continuous stirring. The
black coloured precipitates were obtained by cooling the reaction
mixture to room temperature, which was then thoroughly rinsed
with water. During each rinsing step, samples were separated
from the supernatant using a permanent magnet.
X-ray diffraction (XRD) pattern was recorded on a Philips
powder diffractometer PW3040/60 with Cu Ka radiation. The
infrared spectra were recorded in the range 4000–400 cm 1 on a
Fourier transform infrared spectrometer (FTIR, Magna 550, Nicolet
Instruments Corporation, USA). The transmission electron micrographs were taken by Phillips CM 200 transmission electron
microscope (TEM) for particle size determination. The thermal
analyses were performed by TA Instruments SDT Q600 analyzer
under N2 atmosphere from room temperature to 700 1C with a
heating rate of 10 1C/min. The elemental analysis was carried out
by FLASH EA 1112 series CHNS (O) analyzer (Thermo Fennigan,
Italy). The hydrodynamic diameter and zeta-potential were
determined by dynamic light scattering (DLS) and Zeta PALS,
respectively (BI-200 Brookhaven Instruments Corp.). The magnetic
measurements of CA-MNP (dried samples) were carried out using
vibrating sample magnetometer (VSM, LakeShore, Model-7410).
The Curie temperature was measured in an applied field of 100 Oe.
In order to evaluate the specific absorption rate and cytocompatibility, amount of Fe in suspension of CA-MNP was obtained by UV
measurement (Cecil, Model No. CE3021) using the phenanthroline
spectrophotometric method [26].
The anticancer agent, doxorubicin hydrochloride (DOX) was
used as a model drug to estimate the drug release behavior of the
CA-MNP. In order to investigate the interaction of drug molecules
with CA-MNP, we have studied the fluorescence spectra of pure
DOX and DOX loaded CA-MNP in addition to zeta-potential
measurements. The aqueous dispersion of different amounts of
CA-MNP (0, 20, 40, 60, 80 and 100 mg from a stock suspension of
2 mg/ml) were added to a 1 ml of DOX solution (10 mg/ml) and
mixed thoroughly by shaking at room temperature for 15 min.
The fluorescence spectra of the supernatant (obtained after
magnetic sedimentation of drug loaded CA-MNP) were then
recorded using Hitachi F 2500 fluorescence spectrophotometer.
The fluorescence spectra of 1 ml of pure DOX (10 mg/ml) were also
taken at different time intervals for comparative studies. The
fluorescence intensities of supernatants (washed drug molecules
were also taken into consideration for calculations) against that of
pure DOX solution were used to determine the loading efficiency
(binding isotherm of DOX with CA-MNP). The loading efficiency
(w/w%) was calculated using the following relation:
% Loading efficiency ¼
IDOX IS IW
100
IDOX
where, IDOX is the fluorescence intensity of pure DOX solution, IS the
fluorescence intensity of supernatant and IW the fluorescence
intensity of washed DOX (physically adsorbed DOX molecules).
The drug release study was carried out under a reservoir–sink
condition. For release study, we have quantified the amount of
DOX loaded CA-MNP according to the binding isotherm. The
loading was carried out, at increased scale, by incubating 2 ml of
aqueous solution of DOX (2 mg/ml) with 1 ml of the aqueous
suspension of CA-MNP (10 mg/ml) for 1 h in dark (however, no
decrease in fluorescence intensities was observed after 15 min of
incubation). The drug loaded CA-MNP (10 mg) was immersed
into 5 ml of acetate buffer—pH 5, and then put into a dialysis
bag. The dialysis was performed against 200 ml of phosphate
buffered saline (PBS)—pH 7.3 under continuous stirring at 37 1C
to mimic the cell environment. 1 ml of the external medium
was withdrawn and replaced with fresh PBS at fixed times to
maintain the sink conditions. The amount of doxorubicin released
was determined by measuring the fluorescence intensity at
lexcitation ¼490 nm and lemission ¼535 735 nm using Perkin Elmer
1420 multilabel counter against the standard plot prepared under
similar condition. Each experiment was performed in triplicates
and standard deviation was given in the plot.
The heating ability of sample suspensions was obtained from
the time-dependent calorimetric measurements using an RF
generator (Comdel CLF-5000). 1 ml (10 mg/ml of Fe) of Fe3O4
colloidal suspension was taken in a glass sample holder with
suitable arrangements to minimize the heat loss. The AC magnetic
S. Nigam et al. / Journal of Magnetism and Magnetic Materials 323 (2011) 237–243
field of 7.64, 8.2 and 10.0 kA/m, and fixed frequency of 425 kHz
were used to evaluate the specific absorption rate (SAR). The SAR
was calculated using the following equation [3,14]:
SAR ¼ C
DT 1
Dt mFe
where, C is the specific heat of solvent (C ¼Cwater ¼4.18 J/g 1C),
DT/Dt is the initial slope of the time-dependent temperature
curve and mFe is mass fraction of Fe in the sample.
Sulforhodamine-B (SRB) assay was performed to evaluate
cytocompatibility of the CA-MNP with HeLa cells. The cells were
seeded into 96-well plates at densities of 1 104 cells per well for
24 h. Then different concentrations of CA-MNP colloidal suspension (0, 31.250, 15.625, 7.813, 3.906, 1.953, 0.977, 0.488 mg/ml of
Fe) were added to cells and incubated for 24 h at 37 1C and 5%
CO2. Thereafter, the cells were washed thrice with phosphate
buffer saline (PBS) and processed for SRB assay to determine the
cell viability. For this, cells were fixed with a solution of 50%
trichloroacetic acid and stained with 0.4% SRB dissolved in 1%
acetic acid. Cell-bound dye was extracted with 10 mM unbuffered
Tris buffer solution (pH 10.5) and then absorbance was measured
at 560 nm using a plate reader. The cell viability was calculated
using the following formula:
% Viability ¼ ðAbsorbance of sample=Absorbance of controlÞ 100
3. Results and discussion
Fig. 1 shows (a) XRD pattern and (b) TEM micrograph of
CA-MNP. The XRD pattern reveals the formation of single-phase
Fe3O4 inverse spinel structure with lattice constant, a ¼ 8.378 Å,
which is very close to the reported value of magnetite (JCPDS Card
No. 88-0315, a ¼8.375 Å). The presence of sharp and intense
peaks confirmed the formation of highly crystalline nanoparticles.
Fig. 1. (a) XRD pattern and (b) TEM micrograph of CA-MNP (Inset of (b) shows the
selected area electron diffraction pattern of CA-MNP).
239
The crystallite size of CA-MNP is estimated about 8 nm from X-ray
line broadening using the Scherrer formula. From TEM micrograph, it is clearly observed that Fe3O4 nanoparticles are almost
spherical in shape and size of about 8–10 nm (s r10%).
The selected area electron diffraction pattern of this sample
(Inset of Fig. 1b) can be indexed to the reflections of inverse spinel
Fe3O4 structure and shows only diffraction intensity associated
with highly crystalline Fe3O4, which is consistent with the XRD
result.
Fig. 2 shows the FTIR spectra of pure CA and CA-MNP. The
absorption bands for the pure CA are well resolved, but those of
the CA-MNP are rather broad and few. The 1710 cm 1 peak
assignable to the C¼O vibration (asymmetric stretching) from the
COOH group of CA shifts to an intense band at about 1600 cm 1
for CA-MNP revealing the binding of a CA radical to surface of
Fe3O4 nanoparticles by chemisorptions of carboxylate (citrate)
ions [16,27,28]. Carboxylate groups of CA form complexes with Fe
atoms on the surface of Fe3O4 rendering partial single bond
character to the C¼O bond. This results in weakening of C¼O
bond, which shifts the stretching frequency to a lower value.
Furthermore, the vibrational modes appearing at 1400, 1250
and 1065 cm 1 in CA-MNP corresponds to the symmetric
stretching of COO , symmetric stretching of C–O, and OH group
of CA [29]. The strong IR band observed at around 575 cm 1 in
CA-MNP can be ascribed to the Fe–O stretching vibrational mode
of Fe3O4.
Fig. 3 shows the TGA-DTA plots of CA-MNP. A weight loss of
about 7.5% with a sharp endothermic peak at 60 1C can be
ascribed to the removal of physically absorbed water and CA
molecules on the Fe3O4 nanoparticles. The weight loss of about
13.5% with a broad exothermic peak at 260 1C can be associated
with the removal of chemically attached CA molecules from the
surface of Fe3O4 nanoparticles. The weight loss of about 3.0%
beyond 400 1C with a sharp exothermic peak at 500 1C is
associated with the phase transformation of Fe3O4–Fe2O3.
Furthermore, the elemental analysis shows the presence of
organic components (carbon and hydrogen) on CA-MNP. Thus,
the FTIR, TGA and CHNS(O) results confirmed that Fe3O4
nanoparticles have been functionalized with citric acid during
the course of synthesis.
Fig. 2. FTIR spectra of pure CA and CA-MNP.
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S. Nigam et al. / Journal of Magnetism and Magnetic Materials 323 (2011) 237–243
Fig. 3. TGA-DTA plots of CA-MNP.
Fig. 5. Field dependence of magnetization (M vs. H) plot of CA-MNP at 300 K. Inset
shows its temperature dependence of magnetization (M vs. T) measurement.
Fig. 4. Zeta-potential of CA-MNP at different pH values. Inset ‘a’ shows the
hydrodynamic diameter of CA-MNP obtained from DLS measurements and inset ‘b’
shows the possible schematic representation of CA-MNP (CA coating on the
surface of Fe3O4 MNP).
stability to Fe3O4 nanoparticles. Further, the electrostatic repulsive
forces originate among the highly negatively charged CA-MNP
(25.5 mV at pH 6) in aqueous suspension also play an important
role in their water stabilization.
Fig. 5 shows the field dependent magnetization (M vs. H) plot
of CA-MNP at 300 K. The CA-MNP exhibits superparamagnetic
behavior without magnetic hysteresis and remanence. The
maximum magnetization of CA-MNP was found to be 57 emu/g
at 20 kOe. The observed magnetization is comparable to that
of neat Fe3O4 nanoparticles (60.5 emu/g) obtained by the
co-precipitation method [32] and higher than the aqueous stable
Fe3O4 nanoparticles (43.2 emu/g) obtained by the high-temperature thermal decomposition method followed by subsequent
surface functionalization through ligand-exchange strategy [3].
Thus, these aqueous stable Fe3O4 nanoparticles having high
magnetic response could be exploited for magnetic drug targeting, hyperthermia treatment and magnetic resonance imaging.
Further, the temperature dependence of magnetization (M vs. T)
measurement (Inset Fig. 5) shows that the Curie temperature (TC)
of CA-MNP is about 580 1C, which is in agreement with that
reported for Fe3O4 [3,33] whereas the TC of g-Fe2O3 is around
645 1C [34]. These results confirm that the phase formed is Fe3O4
rather than g-Fe2O3. The slight hump observed in the M vs. T curve
at around 300 1C may be assigned to an increase in interparticle
interaction near the surface because of the removal of organic
moiety (citric acid) as indeed suggested by the TGA-DTA analysis.
We have used the fluorescence spectra and zeta-potential
analyses to investigate the interactions of drug molecules with
CA-MNP and their loading efficiency. At low pH (4–6), protonated
primary amine present on the drug induces a positive charge to
the doxorubicin molecule (cationic DOX) [35]. While at low pH,
carboxylic moiety of citric acid (pK1 ¼3.13, pK2 ¼4.76 and pK3 ¼6.40)
is deprotonated and carries a negative charge [36]. The surface
charge of the CA-MNP at pH 6 (the pH at which drug loading
experiment was carried out) was found to be negative ( 25.5 mV)
from zeta-potential measurement. An increase in surface charge
from 25.5 to 10.5 mV is observed for the CA-MNP after drug
loading (100 mg DOX reacted with 200 mg CA-MNP from their stock
solutions of 2 mg/ml) in zeta-potential measurement. This result
strongly suggested that positively charged drug molecules are
bound to negatively charged CA-MNP through electrostatic interactions. The impressive affinity of doxorubicin for negatively charged
molecules such as oleate ions and phospholipids has been the
subject of numerous earlier investigations [37–40]. The interaction
Fig. 4 shows the zeta-potential of CA-MNP at different pH
values. From zeta-potential measurements, it has been observed
that adsorption of CA onto the surface of Fe3O4 nanoparticles
results in highly negative surface charge and isoelectric point (IOP)
is not observed in measured pH range of 3–6 (IOP of bare
Fe3O4 nanoparticles is 6.7 [30]). These high negative values of
zeta-potential for the CA-MNP further confirmed the presence of
negatively charged carboxylate groups on the surface of Fe3O4
nanoparticles. Furthermore, DLS measurements (Inset ‘a’ of Fig. 4)
indicate that these samples render aqueous colloidal suspension
with mean hydrodynamic diameters (almost constant with invariable change in polydispersity index) of about 25 nm (s o10%) due
to the presence of associated and hydrated organic layers [3,31].
Specifically, some of the carboxylate groups of citric acid strongly
coordinate to iron cations on the Fe3O4 surface to form a robust
coating (a possible schematic representation of CA-MNP is shown
in the inset ‘b’ of Fig. 4), while uncoordinated carboxylate groups
extend into the water medium, conferring a high degree of water
S. Nigam et al. / Journal of Magnetism and Magnetic Materials 323 (2011) 237–243
of DOX molecules with CA-MNP was also evident from the
predominant quenching of DOX fluorescence in presence of
CA-MNP (Fig. 6a), whereas self-quenching (DOX–DOX interaction
due to p–p stacking) of pure DOX is not observed (Fig. 6b).
Furthermore, the fluorescence intensity of DOX decreases (till
saturation loading is achieved) on increasing the concentration of
CA-MNP, which is obvious due to the increase in loading efficiency
of DOX into CA-MNP. The fluorescence spectroscopy (fluorescence
intensity is highly dependent on the state of the molecule, i.e. free or
in attached form) has been successfully used to study interactions
between DOX and its surrounding in the previous literature for
instance when the drug intercalates DNA or penetrates within
membrane models or liposomal drug carriers [41–44]. The loading
efficiency (binding isotherm of DOX with CA-MNP) obtained from
quenching of fluorescence intensities is shown in the inset of Fig. 6a.
From the inset of Fig. 6a, it has been observed that loading efficiency
is strongly dependent on the ratio of particles to DOX in the reaction
solution and a maximum of about 90% (s r5%) drug loading
efficiency (w/w) could be achieved by electrostatic interactions. The
obtained loading efficiency is much higher than that reported (14%)
Fig. 6. Normalized fluorescence spectra of (a) 10 mg/ml of DOX (1 ml) reacted
with different amounts (0, 20, 40, 60, 80 and 100 mg) of CA-MNP for 15 min and
(b) 10 mg/ml of pure DOX (1 ml) at different time intervals. Inset of (a) shows the
loading efficiency (binding isotherm of DOX with CA-MNP) obtained from
quenching of fluorescence intensities.
241
by Munnier et al. [10]. They have discussed that drug molecules
(DOX–Fe2 + complex) were attached to the surface –OH groups of
citric acid treated Fe3O4 nanoparticles via Fe2 + ions. The presence of
citric acid moieties on the surface of Fe3O4 nanoparticles may
provide steric hindrance to the attachment of DOX–Fe2 + complex
with surface –OH groups thereby reducing the loading of the drug
onto the nanoparticles. However, the drug loading in the present
study is attributed primarily to the electrostatic interactions
between positively charged DOX molecules and negatively charged
carboxyl moieties present on the surface of Fe3O4 nanoparticles
(as indeed suggested from zeta-potential measurements) showing
comparatively higher drug loading.
Fig. 7 shows the drug release profile of DOX loaded CA-MNP in
cell mimicking environment (reservoir: pH 5 and sink: pH 7.3 at
37 1C). It has been observed that drug molecules release slowly
over a period of 50 h and the shape of the release profile suggests
that the complete release of drug was not attained. The drug
loaded CA-MNP released about 60% of loaded drug in acetate
buffer (pH 5) against PBS (pH 7.3) after 50 h. The release of DOX
could be attributed to the weakening of the electrostatic
interactions between the drug and the partially neutralized
carboxyl groups on the nanoparticle surface, due to an increase
in the protons in the colloidal solution. Munnier et al. [10]
discussed that release of DOX molecules (released gradually over
a period of 1 h and thereby attaining a plateau) from loaded
particles (achieved by chelation of the DOX–Fe2 + complex
with –OH groups on the surface of citric acid treated Fe3O4
nanoparticles) is primarily due to stimulated hydrolysis of drug
molecules. However, in the present study, weakening of the
electrostatic interactions is a slower process, which leads to
the sustained release of drug molecules over a period of 50 h.
These results indicate that the bound drug molecules will be
released in appreciable amounts in the mild acidic environments
of the tumors.
Time-dependent calorimetric measurements were performed
on suspension of CA-MNP to investigate their heating efficacy
(Fig. 8) as additional functionality. The specific absorption rate
(SAR) of CA-MNP were found to be 32.26, 38.63 and 49.24 W/g of
Fe with an applied field (H) of 7.64, 8.82 and 10.0 kA/m,
respectively (at a fixed frequency of 425 kHz). It has been
observed that the time required to reach 43 1C (hyperthermia
temperature) decreases with increase in field (inset of Fig. 8),
which is obvious as the heat generation is proportional to the
square of applied AC magnetic field. These SAR values should not
Fig. 7. Drug release profile of DOX loaded CA-MNP in cell mimicking environment
(reservoir: pH 5 and sink: pH 7.3 at 37 1C).
242
S. Nigam et al. / Journal of Magnetism and Magnetic Materials 323 (2011) 237–243
absorption rate, which could find promising applications in drug
delivery and hyperthermia treatment of cancer.
4. Conclusions
Fig. 8. Time-dependent calorimetric measurements of CA-MNP.
A simple facile approach for the preparation of citratestabilized Fe3O4 aqueous colloidal nanoparticles of size 8–10 nm
using a soft chemical approach is described. The detailed
structural analyses by FTIR, TGA-DTA, CHNS(O) and zeta-potential
confirmed the functionalization of Fe3O4 nanoparticles with citric
acid. These nanoparticles also show good colloidal stability,
optimal magnetization, cytocompatibility with cells and good
specific absorption rate (under external AC magnetic field) for
magnetic hyperthermia treatment of cancer. It is well evident that
the positively charged drug molecules such as DOX could easily
bound to the negatively charged CA-MNP through electrostatic
interactions. The drug release profile in cell mimic environment
indicates that the bound drug molecules will be released in
appreciable amounts in the mild acidic environments of the
tumors. Thus, CA-MNP can be used as a potential carrier for
effective magnetic drug targeting and hyperthermia treatment of
cancer. Further, the citrate groups on Fe3O4 surface can provide
accessible surface for routine conjugation of biomolecules
through the well-developed bioconjugation chemistry for a
number of other biomedical applications, such as magnetic
biolabeling, efficient bioseparation and contrast enhancement
for magnetic resonance imaging etc.
Acknowledgement
The financial support by nanomission of DST, Govt. of India is
gratefully acknowledged.
References
Fig. 9. Viabilities of HeLa cells incubated with medium containing CA-MNP.
be viewed in terms of performances, but only as the demonstration that these nanoparticles are effective heating source for
hyperthermia treatment of cancer.
Fig. 9 shows the viabilities of HeLa cells incubated with
medium containing CA-MNP. The SRB assay results indicate that
the viability of the HeLa cells is not affected by the mere presence
of CA-MNP, registering normal growth in the cells, suggesting that
nanoparticles are reasonably biocompatible and do not have toxic
effect for further in vivo use. The percentage of cell viability is
slightly above 100% at lower concentration may be due to the
presence of iron (iron oxide nanoparticles), which sometimes
facilitates in cell growth [3,31]. The present study discussed the
formation of aqueous stable, highly crystalline, biocompatible
citric acid functionalized Fe3O4 nanoparticles having an optimal
magnetization, higher drug loading efficiency and good specific
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