Pharmaceutical nanotechnology [615998]

Pharmaceutical nanotechnology
Synthesis and in vitro localization study of curcumin-loaded SPIONs in
a micro capillary for simulating a targeted drug delivery system
Mohammed Anwara,*, Mohammed Asferb, Ayodhya P. Prajapatib,
Sharmistha Mohapatraa, Sohail Akhterc, Asgar Alia, Farhan J. Ahmada,**
aNanoformulation Research Lab, Faculty of Pharmacy, Hamdard University, New Delhi, India
bDepartment of Mechanical Engineering, IIT Kanpur, Uttar Pradesh, India
cDepartment of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, Utrecht 3584 CG, The Netherlands
A R T I C L E I N F O
Article history:
Received 17 February 2014
Received in revised form 13 April 2014
Accepted 15 April 2014
Available online 18 April 2014
Keywords:
CurcuminSuperparamagnetic iron oxide
nanoparticles
SPIONs
Drug localization
Drug targeting
ImagingA B S T R A C T
Nano-sized curcumin-loaded super-paramagnetic iron oxide nanoparticles (CUR-OA-SPIONs) were
synthesized chemically by co-precipitation method using oleic acid as a stabilizer and Myrj 52 as a
surfactant. The synthesized nanoparticles were characterized for their shape, size, surface morphology,
electrokinetic potential, magnetic properties, crystalinity, chemical interactions and thermal transitions.
The synthesized CUR-OA-SPIONs were spherical, mono-dispersed, physically stable and super-
paramagnetic in nature. In vitro localization study and aggregation dynamics of CUR-OA-SPIONs were
studied with a flow of blood inside a square glass capillary (500 /C2 500 mm2cross section) in the presence
of an externally applied magnetic field (Ms = 1200 mT). This research which is first of its kind showed the
fluorescent imaging of CUR-OA-SPIONs with respect to time to understand the aggregation dynamics of
magnetic nanoparticles in a micro capillary simulating the case of targeted drug delivery system. The size
of the aggregation increases with respect to time (t = 0+s to t = 500 s), while no significant change in the
size of the aggregate was observed after time t = 500 s.
ã 2014 Elsevier B.V. All rights reserved.
1. Introduction
Among various smart drug delivery strategies, superpara-
magnetic iron oxide nanoparticles (SPIONs) comprising of
magnetite (Fe3O4) and maghemite (g-Fe 2O3) prove its therapeu-
tic as well as theranostic efficiencies in targeted drug delivery
systems. Properties, such as non-toxicity, biocompatibility, high
degree of saturation magnetization, small size and an ease of
appropriate surface modi fication with different polymers, make
SPIONs the best choice for site-speci fic drug delivery systems
(Mahmoudi et al., 2011; Veiseh et al., 2010 ). Different stabilizers,
such as oleic acid, lauric acid, alkane sulphonic acids, and alkane
phosphonic acids, have been used significantly for the stabiliza-
tion of small-sized SPIONs (Sahoo et al., 2001 ). Among the
stabilizers, an exception within the monosaturated fatty acids isthe oleic acid, which exerts anti-tumorigenic effects by
suppressing the over-expression of human epidermal growth
factor receptor-2 (HER2) without any chronic adverse effects and
toxicity (Colomer and Menendez, 2006; Simopoulos, 2001; Tran
et al., 2010 ). In recent years, there are an increasing number of
research publications on the use of SPIONs for tumor targeting
applications by loading anticancer agents to them. Anticancer
drug-loaded SPIONs can be guided to a target site by the
application of an external magnetic field as in the case of
magnetic drug targeting (MDT).
Curcumin, a natural diphenol (Fig. 1(a)), extracted from the
rhizomes of Curcuma longa , is traditionally used as an ayurvedic
medicine for the prevention of various clinical disorders like
Alzheimer ’s disease, arthritis, diabetes, HIV replication, myocar-
dial infarction, and wound healing, etc. (Aggarwal and Sung,
2009; Boaz et al., 2011; Bright, 2007; Gregory et al., 2008;
Henrotin et al., 2010; Mishra and Palanivelu, 2008 ). But in the last
decade, curcumin had been the major focus of research for the
prevention and therapy of various cancers, such as melanoma,
head and neck, breast, colon, pancreatic, prostate and ovarian
cancers (Aggarwal et al., 2003; Lin et al., 1997, 1998; Mahady et al.,
2002; Wilken et al., 2011 ). The yellow pigment of curcumin also
imparts fluorescent activity, which enables visualization of* Corresponding author at: Nanomedicine Research Lab, Jamia Hamdard
(Hamdard University), Hamdard Nagar, New Delhi 110062, India. Tel.: +91
9313841616.
** Corresponding author at: Nanomedicine Research Lab, Jamia Hamdard
(Hamdard University), New Delhi 110062, India. Tel.: +91 9810720387.
E-mail addresses: md.anwar2008@gmail.com (M. Anwar),
farhanja_2000@yahoo.com (F.J. Ahmad).
http://dx.doi.org/10.1016/j.ijpharm.2014.04.038
0378-5173/ ã 2014 Elsevier B.V. All rights reserved.International Journal of Pharmaceutics 468 (2014) 158 –164
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journa l home page : www.e lsevier.com/loca te/ijpharm

curcumin in vitro and in vivo, providing a platform for their
theranostic application. Thus, curcumin-loaded SPIONs, along
with oleic acid as a stabilizer, provide a novel, innovative
approach for use in cancer treatment (Khopde et al., 2000;
Kurien et al., 2012 ). The SPIONs should be present in a dispersed
form in blood in order to transport the loaded drug to the target
site. The process of localization of SPIONs at the target sites can be
evaluated on the basis of competition between the forces due to
blood flow and applied external magnetic field acting on SPIONs.
The magnetic force acting on a nanoparticle dispersed in a
moving fluid can be written as
Fmag¼ VpDxrB2
2m0 !
(1)
where Vpis the volume of nanoparticle, Dx is the difference of
magnetic susceptibility between the nanoparticle and the fluid,
B2=2m0is the magnetostatic field energy density and m0is the
permeability of free space. For a nanoparticle to experience a
magnetic force (Eq. (1)) there must be a non-uniform magnetic
field as the magnetic force equals to zero in case of uniform
magnetic field. For a nanoparticle to remain in a magnetic trap
and not to be flushed away by the fluid flow, the magnetic force
Fmagmust be larger than the hydrodynamic drag force Fdragacting
on the nanoparticle. The drag force acting on the nanoparticle is
given by
Fdrag¼ 6phrpDv (2)
where Dv is the velocity difference between the nanoparticle and
the surrounding medium, h is the viscosity of the medium, and rp
is the radius of the nanoparticle.
Though a sound scienti fic literature is available on SPIONs, none
of it exactly explains the transport phenomena and subsequent
localization of SPIONs in a bio-fluid like blood at micro scale. The
detailed hydrodynamics during magnetic drug targeting (MDT) is
essential for the optimization of the process before in vivo
applications of SPIONs for drug targeting applications. To the best
of our knowledge, no other experimental studies have been
investigated on in vitro localization of curcumin loaded fluorescentSPIONs in a micro capillary in presence of applied external
magnetic field.
2. Materials and methods
Ferric chloride anhydrous (FeCl 3) and ferrous sulphate hepta-
hydrate (FeSO 4/C17H2O) were purchased from Fluka Chemicals, India.
Ammonium hydroxide (28–30% (v/v)), n-hexane (98%), polyoxy-
ethylene (40) stearate (Myrj 52), oleic acid (90%) and curcumin
were purchased from Sigma –Aldrich, and used as received. The
animal protocol to carry out in vitro study was reviewed and
approved by the Institutional Animal Ethics Committee, Jamia
Hamdard (Approval No: 823) and their guidelines were followed
for the studies.
2.1. Formulation of curcumin-loaded SPIONs
SPIONs were synthesized chemically by the co-precipitation
reaction from an aqueous mixture of Fe3+/Fe2+in the molar ratio
of 2:1 using concentrated ammonium hydroxide solution in an
inert atmosphere of nitrogen (Fig. 1(b)) (Gupta and Gupta, 2005 ).
After the co-precipitation of magnetic nanoparticles, oleic acid
(5–50% (w/w), related to the weight of SPIONs) was added as a
stabilizer for the synthesis of OA-SPIONs. For the loading of
curcumin into OA-SPIONs, curcumin-solubilized oleic acid
(1 mg/mL) was added to the SPIONs suspension at a temperature
of 70/C14C and after 30 min, the temperature was elevated to 110/C14C
in order to remove excess of water and ammonium hydroxide.
One gram of CUR-OA-SPIONs as received above was added to
6.0 g of n-hexane to form an organic dispersion (Fig. 1(c)). This
dispersion was added to an aqueous solution (20 mL) of Myrj 52
(10–60% (w/w), related to dispersed phase (CUR-OA-SPIONs +
n-hexane)) for the aqueous phase transfer (Anwar et al., 2013 ).
After mixing for 1 h at 500 rpm, the mixture was subjected to
sonication twice for 2 min at 90% amplitude with a Branson
Sonifier W450 digital in an ice-cooled bath followed by the
evaporation of n-hexane under continuous stirring. 2 mL of water
was added every 30 min to compensate the loss of water along
with evaporation of n-hexane at 80/C14C for 3 h. After the complete
evaporation of n-hexane, a stable water-based ferrofluid of CUR-
OA-SPIONs was obtained (Fig. 1(d)).
2.2. Characterization of SPIONs
Particle size distribution and zeta potential were measured
using Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK). Prior
to all DLS measurements, samples were diluted and ultrasonicated
for 15 min at 25/C14C at a detection angle of 90/C14(n = 3). Morphology of
SPIONs was confirmed by transmission electron microscopy (TEM;
Morgagni 268D SEI, USA), where appropriate dilution of samples
were made and deposited on the copper grid.
The magnetic properties of the SPIONs were measured by
vibrating sample magnetometry (VSM, LDJ9600-1, LDJ, USA). The
applied field was in the range of 0–10 kOe. The physical form of the
SPIONs and CUR-OA-SPIONs were determined by powder X-ray
diffraction (XRD, X’pert pro, Pan Analytical, Netherlands) over a
range of 5–70/C14in 2 h with Ni-filtered Cu-Ka radiation. The scan
speed was 3 min. FT-IR measurements were carried out by
TENSORTM 37, Bruker in the range of 4000 –400 cm/C01. Pellets for
FT-IR analysis were prepared by mixing a small amount of
nanoparticles powder with KBr powder. Transition temperature
analysis was studied by differential scanning calorimetry (DSC)
(PerkinElmer, USA). A small amount of dried SPIONs and curcumin
was sealed in a 40-mL aluminum pan and an empty aluminum pan
was taken as the reference. The temperature of the pans was raised
from 30/C14C to 450/C14C, at a rate of 10/C14C/min.
Fig. 1. (a) Chemical structure of curcumin. (b) SPIONs attracted by a magnet. (c)
Dispersion of OA-SPIONs in n-hexane. (d) Aqueous CUR-OA-SPIONs. (e) Experi-
mental setup for in vitro localization study of nanoparticles.M. Anwar et al. / International Journal of Pharmaceutics 468 (2014) 158–164 159

2.3. Magnetic field characterization
It is desired to localize the magnetic field over a short length of
the capillary so that SPIONs can be localized at a speci fic location
inside the capillary. The localization of SPIONs at a speci fic site
during drug targeting is essential in order to minimize the side
effects and maximize the bioavailability of loaded drugs at
the target sites. As discussed earlier, a high magnetic field gradient
is required for obtaining a large trapping force on the SPIONs; a
permanent magnet (1.5 mm /C2 3 mm, Ms = 1200 mT) was placed
near the right side wall of the capillary as shown in Fig. 2(a). The
non-uniform magnetic field B generated by the cylindrical
permanent magnet, is given by
B ¼Bre
2d ț lffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðd ț lȚ2ț r2q /C0dffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
d2ț r2q2
643
75 (3)
where Breis the remanence or residual induction of the magnet, r is
the radius of the magnet, l is the length of the magnet and d is the
distance between the magnet and the speci fic point.
The magnetic field produced by the permanent magnet was
simulated by using Vizimag software (Fig. 2(b)). Fig. 2(c) shows
the distribution of magnetic flux density (B) along the length of
the capillary corresponding to three different locations, i.e.
y = /C00.25 mm (right side wall), y = 0.0 mm (center line) and
y = +0.25 mm (left side wall) of the glass capillary. As expected,
the magnetic flux density is highest at x = 0 mm, while the flux
density decreases rapidly towards the edges of the magnet. This
rapid decrease in magnetic flux density implies a strong magnetic
field gradient along the length of capillary, and hence a large
magnetic force on SPIONs. Based on the results of the simulation it
would be expected that maximum localization of SPIONs would
take place around x = 0.0 mm and at the right side wall of thecapillary (y = /C00.25 mm). Fig. 2(d) shows the magnetic flux density
across the width of the glass capillary (y = /C00.25 mm to
y = +0.25 mm) for stream wise locations; x = 0.0, 1.0 and
/C01.0 mm, respectively. As the magnetic flux density is almost
uniform across the width of the glass capillary, hence there is
negligible magnetic field gradient along the y-direction, as a result
of which, very weak magnetic force compared to blood flow act on
SPIONs in the y-direction.
2.4. In vitro localization study of CUR-OA-SPIONs
For in vitro drug localization study, aqueous solution CUR-OA-
SPIONs was mixed with blood (1:1 v/v) and then passed through a
glass micro capillary in the presence of magnetic field produced by
a permanent magnet. To visualize the localization of CUR-OA-
SPIONs inside the glass capillary in the presence of magnetic field,
fluorescence microscope was used (Fig. 1(e)). The fluorescence
microscope system consisted of an inverted microscope (DMI 6000
CS), combined with a continuous wave laser and a photo multiplier
tube (PMT). The laser beam was illuminated from below the
microscope stage through a 10/C2 objective dry lens (NA = 0.40). The
blood was withdrawn from retro-orbital plexus of rats and mixed
with anticoagulant citrate phosphate dextrose adenine solution
and stored at 1–6/C14C prior to the experiment. The prepared sample
was then passed through a glass capillary of 500 mm /C2 500 mm
square cross-section as shown in Fig. 2(a). A cylindrical NdFeB
permanent magnet was placed close to the right side-wall of the
capillary in order to generate magnetic field inside the glass
capillary. The CUR-OA-SPIONs were illuminated by the laser light,
and emitted light at a peak excitation wavelength of 530 nm.
Fluorescence images of localization of CUR-OA-SPIONs with
increasing time were taken through the PMT and then transferred
to the computer for further processing.
Fig. 2. (a) Expression for the motion of SPIONs inside the glass capillary. (b) Simulation domain. (c) Magnetic flux density perpendicular to the flow direction. (d) Magnetic
flux density along the flow direction.160 M. Anwar et al. / International Journal of Pharmaceutics 468 (2014) 158–164

2.5. Statistical analysis
All the data were processed using Microsoft Excel 2007
software and presented as mean /C6 standard error of the mean.
The Student ’s t test was used to find the statistical significance. A
value of p < 0.05 was considered statistically significant. All the
graphs were plotted using Origin 6.1 software.
3. Results
We have synthesized SPIONs chemically by co-precipitation
method by the following stochiometric equation in an inert
atmosphere of nitrogen:
Fe2țț 2Fe3țț 8OH/C0! Fe3O4ț 4H2O (4)
The size distributions of nanoparticles were shown in Table 1.
From our previous studies, we optimized a very small particle size
of 16 /C6 0.98 nm (PDI = 0.176) after the dispersion of OA-SPIONs
(45% w/w) in n-hexane (Anwar et al., 2013 ). However, in this
experiment, average size of CUR-OA-SPIONs (45% w/w) in
n-hexane was found to be 20.72 /C61.06 nm with a PDI value of
0.240 /C6 0.032. The hydrodynamic size of CUR-OA-SPIONs in
aqueous solution of Myrj 52 (60% w/v) was found to be
194.6 /C6 0.3 nm with a unimodal and narrow size distribution
(PDI = 0.207 /C6 0.019) and remains almost constant on further
increase in surfactant concentration. The colloidal stability of
ferrofluid formulation can be predicted by the zeta potential
measurement. The zeta potential of CUR-OA-SPIONs in distilled
water was found to be /C030.3 mV (Fig. 3(a)). The TEM image of CUR-
OA-SPIONs showed spherically-shaped, monodispersed particles
with an average size of 197 /C6 3 nm showing analogy to DLS
measurement, i.e. 194.6 /C6 0.03 nm (Fig. 3(b)).
Fig. 4(a) shows the X-ray diffraction patterns of the native Fe3O4
nanoparticles. Series of characteristic peaks at 2u = 18.30/C14, 30.11/C14,
35.46/C14, 43.10/C14, 57.00/C14, 62.60/C14and 74.06/C14were observed corre-
sponding to the hkl values of {111}, {2 2 0}, {311}, {4 0 0}, {511},
{4 4 0} and {5 3 3}, respectively. Fig. 4(b) presents the M–H curves
for CUR-OA-SPIONs where, saturation magnetization of CUR-OA-
SPIONs was found to be 45 emu/g. Chemical interactions of SPIONs
with oleic acid and curcumin were interpreted by Fourier
transform infrared (FT-IR) analysis (Fig. 5). The DSC curves of
curcumin showed an endothermic peak at 177.721/C14C, whereas, no
endothermic was observed in case of SPIONs up to 450/C14C (Fig. 6).
For in vitro localization study of CUR-OA-SPIONs the blood
containing SPIONs was passed through the glass capillary at a flow
rate of Q = 5 mL/min. It has been observed that the CUR-OA-SPIONs
were attracted towards the right side wall of the capillary adjacentto the permanent magnet and starts forming aggregate. The
aggregate was observed over a time a period from t = 0+to t = 800 s.
After t = 500 s, no significant growth of aggregate was observed.
4. Discussions
After the synthesis of SPIONs, they are coated with oleic acid
(5–50% w/w) as a stabilizer to prevent the aggregation between the
metal oxide nanoparticles. During this, oleic acid forms ammoni-
um oleate by reacting with ammonium hydroxide, which
decomposes at 70/C14C by releasing ammonia leaving behind oleic
acid for coating. Same procedure was adopted for the synthesis of
CUR-OA-SPIONs, wherein, after the addition of curcumin-solubi-
lized oleic acid, a black lump-like magnetic gum was obtained,
which was washed several times with distilled water, cooled to
room temperature and dried to get CUR-OA-SPIONs. All the
reaction was maintained under inert nitrogen atmosphere to
prevent the oxidation of SPIONs. During aqueous phase transfer, in
the first step, organic dispersion of CUR-OA-SPIONs in n-hexane
was drop-wise added to the aqueous solution of Myrj 52 to form a
stable (o/w) emulsion and in the second step, organic solvent was
evaporated to get an aqueous solution CUR-OA-SPIONs. It is very
important to note that curcumin is sparingly soluble in hexane,
which nullifies the probability of leaching of loaded curcumin from
the OA-SPIONs in organic dispersion medium (Khopde et al.,
2000 ).
We coated SPIONs with curcumin-solubilized oleic acid over
the concentration range from 41% to 45% w/v oleic acid to get a
complete dispersion in n-hexane without any sedimentation.
Below 40% w/w coating, we observed the sedimentation of SPIONs,
which might be due to insuf ficient overall coating of SPIONs,
unable to provide sufficient stability. On increasing the concentra-
tion of oleic acid up to 45% w/w, particle size was found to be
decreased with a unimodal distribution, but on further addition of
oleic acid the particle size tended to increase. This might be
attributed to the excess amount of oleic acid entrapped within
SPIONs and formation of multilayered structure over SPIONs.
Above 50% w/w coating, we observed a bimodal distribution of
particle, which might be due to free oleic acid droplets (/C255% of
total particle intensity). So we performed the aqueous phase
transfer of 45% w/w oleic acid-coated CUR-OA-SPIONs with
Myrj 52. On increasing the concentration of surfactant (Myrj 52)
from 15% to 50% w/w, the hydrodynamic size was found to
decrease from 314.9 nm to 194.6 nm which almost remained
constant on further increasing concentration of Myrj 52. So,
remaining characterization was done with the formulation having
50% w/v of Myrj 52. From the colloidal stability point-of-view, zeta
Table 1
Particle size distribution of CUR-OA-SPIONs in organic and aqueous phase.
Organic size distributionaCUR-OA-SPIONs Hydrodynamic size distributionaof CUR-OA-SPIONs
% of oleic acidbcoating (% w/w) Mean size /C6 SD PDI /C6 SD Concentration of Myrj 52c
(% w/w)Mean size /C6 SD PDI /C6 SD
41 35.38 /C6 1.01 0.125 /C6 0.009 15 314.9 /C6 2.1 0.120 /C6 0.022
42 31.41 /C6 1.21 0.223 /C6 0.013 20 300.9 /C6 2.3 0.133 /C6 0.012
43 22.74 /C6 0.13 0.316 /C6 0.019 25 272.1 /C6 1.3 0.233 /C6 0.019
44 22.42 /C6 0.21 0.170 /C6 0.043 30 265.0 /C6 1.0 0.196 /C6 0.017
45 20.72 /C6 1.06 0.240 /C6 0.032 35 246.9 /C6 1.3 0.195 /C6 0.015
46 27.40 /C6 0.46 0.245 /C6 0.018 40 233.1 /C6 1.9 0.282 /C6 0.011
47 28.20 /C6 0.19 0.313 /C6 0.053 45 221.8 /C6 1.5 0.137 /C6 0.013
48 31.75 /C6 0.17 0.331 /C6 0.034 50 198.7 /C6 0.9 0.212 /C6 0.021
49 31.85 /C6 0.13 0.133 /C6 0.009 55 195.2 /C6 0.5 0.150 /C6 0.018
50 39.71 /C6 1.76 0.148 /C6 0.010 60 194.6 /C6 0.3 0.207 /C6 0.019
aEach value represents the (mean /C6 SD) of three determinations.
bRelated to the weight of SPIONs.
cRelated to dispersed phase (CUR-OA-SPIONs + n-hexane).M. Anwar et al. / International Journal of Pharmaceutics 468 (2014) 158–164 161

potential was measured, which was found to be /C030.3 mV. For an
absolute value of zeta potential higher than 25–30 mV, it is
generally accepted that the particles are electrostatically stable
(Xu, 2000 ).
A “positive ” image was seen by TEM, where CUR-OA-SPIONs
appeared dark (Fig. 3(b)) along with lighter dark surrounding,
which might be attributed to oleic acid coating. From the powder
XRD analysis, information about the crystallographic structure,
chemical composition, and physical properties of the materials can
be illustrated. Bragg reflection showed a good coincidence with
standard magnetite (Fe3O4) XRD patterns, which depict that Fe3O4
nanoparticles have a cubic spinel structure. Exact peaks corre-
sponding to Fe3O4were observed in the XRD analysis of CUR-OA-
SPIONs, which proved the presence of a uniform coating over theSPIONs surface without any change in its crystalline nature. In
Fig. 4(b), the hysteresis loop had negligible coercivity at room
temperature due to the small size of superparamagnetic iron oxide
nanoparticles and no magnetization was induced after the
withdrawal of magnetic field.
We compared the FT-IR spectrum of SPIONs, OA-SPIONs and
CUR-OA-SPIONs in Fig. 5. SPIONs and OA-SPIONs showed the wide
band at 3124 –3624 cm/C01and 3169 cm/C01is assigned to O/C0/C0H
vibrations. In case of OA-SPIONs, the sharp bands at 2924 cm/C01and
2851 cm/C01are attributed to asymmetric and symmetric C/C0/C0H
vibrations of the methylene groups of oleic acid. The band at
1624 cm/C01and 1404 cm/C01can be ascribed to the asymmetric and
symmetric COO/C0stretches. FT-IR analysis of CUR-OA-SPIONs
revealed peaks at 3119 cm/C01and 3027 cm/C01corresponding to
alkenyl C/C0/C0H stretching and aromatic C/C0/C0H stretching of benzene
ring, respectively. The characteristic absorption peak of Fe/C0/C0O is
observed at 566 cm/C01, 613 cm/C01and 611 cm/C01for SPIONs,
OA-SPIONs and CUR-OA-SPIONs, respectively. Based on the FT-IR
spectra, oleic acid and curcumin-solubilized oleic acid gave their
confirmation of coating over SPIONs. During co-precipitation
reaction, temperature was elevated up to 110/C14C, so there might be
a chance of curcumin pigment degradation, consequently reducing
its fluorescent activity. To analyze this, a DSC study was carried out
to observe any endothermic peak associated with it. Endothermic
peak at 177.721/C14C, corresponding to the pigment melting point
proved its stable use in co-precipitation reaction (Pan et al., 2006 ).
SPIONs were found to be stable up to 450/C14C without any thermal
transition peak (Fig. 6).
With the applied external magnetic field, the CUR-OA-SPIONs
experienced a high magnetic force as compared to blood flow,
resulting in aggregation at the right side wall of the capillary,
adjacent to the permanent magnet as shown in Fig. 7(A). The
Fig. 3. (a) Zeta potential of CUR-OA-SPIONs. (b) TEM image of CUR-OA-SPIONs.
Fig. 4. (a) XRD of SPIONs. (b) VSM curve of CUR-OA-SPIONs.
Fig. 5. FT-IR spectra of SPIONs, OA-SPIONs and CUR-OA-SPIONs.162 M. Anwar et al. / International Journal of Pharmaceutics 468 (2014) 158–164

complete aggregation of nanoparticles at the capillary wall was
visualized under bright field mode through a 5/C2 objective dry lens
as shown in Fig. 7(A), while the growth of the aggregate of
nanoparticles around (x = 0.0 mm) was observed under fluores-
cence mode through a 10/C2 objective dry lens as shown in Fig. 7(B),
respectively. This is because the fluorescence signal from the
nanoparticles was very weak for visualization under 5/C2 objective
dry lens as compared to the 10/C2 objective dry lens. The profile of
aggregation of CUR-OA-SPIONs depends upon the nature of
magnetic field generated inside the glass capillary. Due to the
effect of magnetic field over a region of x = /C64 mm along the length
of the capillary, the aggregation of CUR-OA-SPIONs spreads over
the right side wall of the capillary. For minimizing spreading of
aggregate of CUR-OA-SPIONs, the distance between permanentmagnet and the glass capillary can be optimized for a given set of
blood flow rate and the strength of the permanent magnet. The
CUR-OA-SPIONs form chain-like structures within the aggregate
due to dipole –dipole interaction between the nanoparticles as
shown in Fig. 7(B). The size of the aggregate of CUR-OA-SPIONs
increased gradually with time (t = 0+s to t = 500 s), while no
significant change in the size of the aggregate was observed after
time t = 500 s). As the aggregate size was increased, its outer
boundary moved away from the location of permanent magnet, as
a result of which the CUR-OA-SPIONs experienced a weak magnetic
force compared to blood flow and allowed SPIONs escaping from
the deposition at the right side wall of the capillary. The blockage
ratio (ratio of volume of aggregate to total volume of capillary)
resulting after a given time with respect to a particular blood flow
Fig. 6. DSC thermogram of CUR and SPIONs.
Fig. 7. (A) Bright field image showing the arrangement of magnet and capillary. (B) In vitro localization study of CUR-OA-SPIONs in blood after (a) t = 0 s, (b) t = 100 s, (c)
t = 200 s, (d) t = 300 s, (e) t = 400 s, (f) t = 500 s.M. Anwar et al. / International Journal of Pharmaceutics 468 (2014) 158–164 163

rate should be monitored in future, So that the capillary would not
be fully blocked/choked to the flow of blood through it, otherwise
arterial obstruction or venous obstruction will interrupt the blood
flow to an organ or body part.
5. Conclusions
We have synthesized spherical and monodispersed curcumin-
loaded SPIONs by using oleic acid as a stabilizer and Myrj 52 as a
surfactant. Apart from the anticancer activity of curcumin, oleic acid
and Myrj 52 will also promote its anticancer activity by suppressing
the over expression of HER2 receptor and by P-glycoprotein
inhibition, respectively. During in vitro localization study of CUR-
OA-SPIONs, the nanoparticles have been successfully localized or
aggregated at the side wall of the glass capillary with the use of a
permanent magnet located close to the glass capillary. Further
studies should emphasize on the design and development of
micro/nano-magnets so that the shape and size of the aggregation
can be optimized as per the shape and size of various tumors. Also,
the anti-cancer activity of CUR-OA-SPIONs should be evaluated to
reveal their promising therapeutic as well as theranostic applica-
tions as efficient targeted drug delivery systems.
Conflict of interest
The authors state no conflict of interest and have received no
payment in preparation of this manuscript.
Acknowledgements
This work is supported by the University Grants Commission
(UGC), India. We would like to thank Rama Scienti fic, New Delhi,
India, for designing the apparatus for the synthesis of SPIONs and
to Dr. P.K. Panigrahi for providing the lab facility in IIT Kanpur.
References
Aggarwal, B.B., Kumar, A., Bharti, A.C., 2003. Anticancer potential of curcumin:
preclinical and clinical studies. Anticancer Research 23, 363 –398.
Aggarwal, B.B., Sung, B., 2009. Pharmacological basis for the role of curcumin in
chronic diseases: an age-old spice with modern targets. Trends in Pharmaco-
logical Sciences 30, 85–94.
Anwar, M., Asfer, M., Akhter, S., Mohapatra, S., Warsi, M.H., Jain, N., Mallick, N., Jain,
G.K., Ali, A., Panigrahi, P.K., Ahmad, F.J., 2013. Aqueous phase transfer of oleic
acid coated iron oxide nanoparticles: influence of solvents and surfactants on
stability and pharmaceutical applications of ferrofluid. Magnetohydrodynamics
49, 191 –196.Boaz, M., Leibovitz, E., Dayan, Y.B., Wainstein, J., 2011. Functional foods in the
treatment of type 2 diabetes: olive leaf extract, turmeric and fenugreek, a
qualitative review. Journal of Functional Foods in Health and Disease 1, 472 –
481.
Bright, J.J., 2007. Curcumin and autoimmune disease. Advances in Experimental
Medicine and Biology 595, 425 –451.
Colomer, R., Menendez, J.A., 2006. Mediterranean diet, olive oil and cancer. Clinical
and Translational Oncology 8, 15–21.
Gregory, P.J., Sperry, M., Wilson, A.F., 2008. Dietary supplements for osteoarthritis.
American Family Physician 77, 177 –184.
Gupta, A.K., Gupta, M., 2005. Synthesis and surface engineering of iron oxide
nanoparticles for biomedical applications. Biomaterials 26, 3995 –4021 .
Henrotin, Y., Clutterbuck, A.L., Allaway, D., Lodwig, E.M., Harris, P., Mathy-Hartert,
M., Shakibaei, M., Mobasheri, A., 2010. Biological actions of curcumin on
articular chondrocytes. Osteoarthritis Cartilage 18, 141 –149.
Khopde, S.M., Priyadarsini, K.I., Palit, D.K., Mukherjee, T., 2000. Effect of solvent on
the excited-state photophysical properties of curcumin. Journal of Photochem-
istry and Photobiology 72, 625 –631.
Kurien, B.T., Dorri, Y., Scofield, R.H., 2012. Spicy SDS-PAGE gels: curcumin/turmeric
as an environment-friendly protein stain. Methods in Molecular Biology 869,
567 –578.
Lin, J.K., Chen, Y.C., Huang, Y.T., Lin-Shiau, S.Y., 1997. Suppression of protein kinase C
and nuclear oncogene expression as possible molecular mechanism of cancer
chemoprevention by apigenin and curcumin. Journal of Cellular Biochemistry
39–48.
Lin, L.I., Ke, Y.F., Ko, Y.C., Lin, J.K., 1998. Curcumin inhibits SK-Hep-1 hepatocellular
carcinoma cell invasion in vitro and suppresses matrix metalloproteinase-9
secretion. Oncology 55, 349 –353.
Mahady, G.B., Pendland, S.L., Yun, G., Lu, Z.Z., 2002. Turmeric (Curcuma longa) and
curcumin inhibit the growth of Helicobacter pylori , a group 1 carcinogen.
Anticancer Research 22, 4179 –4181 .
Mahmoudi, M., Sant, S., Wang, B., Laurent, S., Sen, T., 2011. Superparamagnetic iron
oxide nanoparticles (SPIONs): development, surface modi fication and appli-
cations in chemotherapy. Advanced Drug Delivery Reviews 63, 24–46.
Mishra, S., Palanivelu, K., 2008. The effect of curcumin (turmeric) on Alzheimer ’s
disease: an overview. Annals of Indian Academy of Neurology 11, 13–19.
Pan, Ch.J., Tang, J.J., Weng, Y.J., Wang, J., Huang, N., 2006. Preparation, characteriza-
tion and anticoagulation of curcumin-eluting controlled biodegradable coating
stents. Journal of Controlled Release 116, 42–49.
Sahoo, Y., Pizem, H.T., Fried, D., Golodnitsky, L., Burstein, C.N., Sukenik, G.M., 2001.
Alkyl phosphonate/phosphate coating on magnetite nanoparticles: a compari-
son with fatty acids. Langmuir 17, 7907 –7911 .
Simopoulos, A.P., 2001. The Mediterranean diets: what is so special about the diet of
Greece? The scienti fic evidence Journal of Nutrition 131, 3065S –3073S .
Tran, L.D., Hoang, N.M.T., Mai, T.T., Tran, H.V., Nguyen, N.T., Tran, T.D., Do, M.H.,
Nguyen, Q.T., Pham, D.G., Ha, T.P., Le, H.V., Nguyen, P.X., 2010. Nanosized
magneto fluorescent Fe3O4– curcumin conjugate for multimodal monitoring
and drug targeting. Colloids and Surfaces A 371, 104 –112.
Veiseh, O., Gunn, J.W., Zhang, M., 2010. Design and fabrication of magnetic
nanoparticles for targeted drug delivery and imaging. Advanced Drug Delivery
Reviews 62, 284 –304.
Wilken, R., Veena, M.S., Wang, M.B., Srivatsan, E.S., 2011. Curcumin: a review of anti-
cancer properties and therapeutic activity in head and neck squamous cell
carcinoma. Molecular Cancer 10, 1–19.
Xu, R., 2000. Particle Characterization: Light Scattering Methods. Kluwer Academic,
Dordrecht .164 M. Anwar et al. / International Journal of Pharmaceutics 468 (2014) 158–164