Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Citrate coated iron oxide nanoparticles with enhanced relaxivity for

in vivo magnetic resonance imaging of liver fibrosis
Ariya Saraswathy a , Shaiju S. Nazeer a , Madhumol Jeevan a , Nirmala Nimi a ,
Sabareeswaran Arumugam b , Vijayakumar S. Harikrishnan c , P.R. Harikrishna Varma d ,
Ramapurath S. Jayasree a,∗
a
Biophotonics and Imaging Lab, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura,
Thiruvananthapuram 695012, Kerala, India
b
Division of Implant Biology, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura,
Thiruvananthapuram 695012, Kerala, India
c
Division of Laboratory Animal Science, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura,
Thiruvananthapuram 695012, Kerala, India
d
Bio Ceramics Laboratory, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura, Thiruvananthapuram
695012, Kerala, India

a r t i c l e i n f o a b s t r a c t

Article history: Superparamagnetic iron oxide nanoparticles are widely used for the magnetic resonance imaging (MRI)
Received 20 September 2013 applications. The surface characteristics, magnetic properties, size and targeting efficiency of the material
Received in revised form 1 February 2014 are crucial factors for using the same as contrast agents. We report a simple synthesis method of citrate
Accepted 20 February 2014
coated iron oxide nanoparticles and its systematic characterization. The developed system is highly water
Available online 2 March 2014
dispersible with an average particle size of 12 nm. The particles in water are monodisperse and are
found to be stable over long periods. The efficiency of the material to de-phase water proton has been
Keywords:
studied for various concentrations of iron using longitudinal (T1 ) and transverse (T2 ) weighted MRI.
Contrast agents
Superparamagnetism
The coating thickness of the nanoparticle was optimized so that they exhibited a high transverse to
Citrate longitudinal relaxivity (r2 /r1 ) ratio of 37.92. A clear dose-dependent contrast enhancement was observed
Magnetic relaxivity in T2 weighted in vivo MR imaging of liver fibrosis model in rodents. The labelling efficacy of the particle
Stability and the intracellular magnetic relaxivity were also investigated and presented. The particles were also
MRI tested for blood and cellular compatibility studies. Development of fibrosis and presence of iron in the liver
was confirmed by histopathological analysis. From this study, we conclude that the citrate coated ultra
small superparamagnetic iron oxide nanoparticles (C-USPION) with optimized parameters like particle
size and magnetic property are capable of producing good MR contrast in imaging of liver diseases.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction or in the marginal delineation of premalignant lesions. Iron oxide

In medical diagnostics, MRI has gained wide acceptance due to
the high contrast difference exhibited among different structures
and non-invasive nature of the technique. Based on the relaxation
of water protons within the tissue, this technique requires addi-
tional contrast agents for accurate diagnosis, in certain pathological
conditions like soft tissue malignancy, early stage of malignancy
∗ Corresponding author. Tel.: +91 4712520273/9495948221;
fax: +91 471 2341814.
E-mail addresses: jayashreemenon@gmail.com, jayasree@sctimst.ac.in
(R.S. Jayasree).
nanoparticles (IONs) can reduce transverse relaxation rate r2 of the
water protons and hence can act as T2 contrast agents. The possibil-
ity of modifying IONs with various functional moieties to enable a
single entity with multiple functions has also increased its applica-
tion in fields like hyperthermia, drug delivery, cell separation and
magnetofection [1,2]. IONs based MR contrast agents are found to
be useful for the diagnosis of diseases affecting liver, lymph nodes
and arteries [3].
Chronic liver disease or liver cirrhosis remains as one of the
leading top ten cause of death and a major public health prob-
lem worldwide [4]. Liver fibrosis, the initial stage of cirrhosis is
reversible with proper and timely medical intervention whereas
cirrhosis is not. This highlights the need for early diagnosis of this

http://dx.doi.org/10.1016/j.colsurfb.2014.02.034
0927-7765/© 2014 Elsevier B.V. All rights reserved.
 A. Saraswathy et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224
disease. Normal phagocytic function and the high iron affinity of the
liver macrophages, the Kupffer’s cells provide an easy pathway for
the passive targeting of iron oxide nanoparticles to the liver. This
property can be exploited for the early diagnosis of liver fibrosis
with MR imaging using IONs without additional targeting agents.
Intravenously administered IONs will reach liver as part of nor-
mal opsonization process [5]. Decrease in the Kupffer’s cell density
during the progression of liver disease may result in the differential
uptake of IONs. This enables clear visualization on T2 images of MRI
for the accurate and early diagnosis of fibrosis [6].
IONs need to be tuned for its properties like size, magnetization,
surface chemistry, stability, coating thickness and biocompatibil-
ity, to suit for an effective T2 contrast agent [7–10]. Mode of
synthesis also needs to chosen to facilitate the optimum results.
Among the various synthesis techniques, co-precipitation is pre-
ferred over other methods when considering the high yield, high
magnetic property and biocompatibility [11]. A major parameter
which contributes significantly to the proton relaxation efficacy
and cell–particle interaction of the particle is its outer shell [12,13].
This shell interacts with the outer layer of the magnetic core and
initiates the formation of a disordered layer, which in turn reduces
the magnetization value and increases the size of the whole unit.
Ultra small super paramagnetic iron oxide nanoparticles (USPI-
ONs) of hydrodynamic size less than 50 nm is often preferred as MR
contrast agents due to its long circulation and blood half-life time
[11]. Hence it is highly challenging to tune the magnetic nanopar-
ticle with optimum size and magnetic properties to suit for MR
imaging. Parameters like aqueous dispersibility and stability over
a long period of time also need to be taken care while choosing
the outer stabilizing shell and synthesis mode. The coating agents
with small ligands are reported to give high relaxation rates with
less particle size [14–16]. With an intention to optimize the parti-
cle size and magnetic properties, especially high T2 relaxivity, we
chose citrate to stabilize the USPIONs. Special consideration has
been given to make the final material biocompatible and water
dispersible while retaining its stability over a long period. A sys-
tematic characterization of the material and its efficacy in the early
diagnosis of liver fibrosis in rodent model has also been included in
this study. The developed C-USPIONs showed enhanced relaxivity
ratio than other contrast agents of similar sizes, reported so far. A
comparative evaluation of the magnetic relaxivity of various iron
oxide based MRI contrast agents reported previously has also been
made.
2. Experimental details
2.1. Materials
FeCl3 anhydrous, FeCl2 ·4H2 O, NaOH, tri-sodium citrate (TSC)
and 35% HCl (All from Merck, Germany/India) were used for
the preparation of citrate stabilized iron oxide nanoparticles.
The chemicals used for the cell culture studies were 3-[4,5-
dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT),
F12K medium, sodium bicarbonate, Gentamicin (Himedia,
India), Amphotericin B solution and Foetal Bovine Serum (FBS)
(Sigma–Aldrich, Germany). Carbon tetrachloride (CCl4 ) and olive
oil were used for the development of animal model for liver
fibrosis.
2.2. Synthesis of magnetic nanoparticles
Alkaline co-precipitation method of ferrous salts was followed
for the preparation of USPIONs with minor modifications from the
earlier reported method [17]. Briefly, an aqueous solution of 0.1 M
FeCl2 ·4H2 O and 0.2 M FeCl3 were mixed and magnetically stirred
217
at a temperature of 80 ◦ C and 1 M NaOH was added drop wise to
this mixture. At a pH of 12, black precipitate was observed indicat-
ing the formation of iron oxide nanoparticles. The precipitate was
washed initially with distilled water followed by washing with 1 N
hydrochloric acid. To stabilize the resultant iron oxide nanoparti-
cles with citrate through hydroxyl interactions, the nanoparticles
were dispersed in TSC. The final colloid was then centrifuged and
washed several times with deionized water to remove un-reacted
TSC, maintaining neutral pH. Combinations of different concentra-
tions of TSC, stirring time and temperature were tried to get a final
system with optimum coating thickness that demonstrated high
magnetic properties. Accordingly, C-USPIONs prepared with 0.1 M
TSC solution stirred for 6 h at 80 ◦ C were used for further studies.
2.3. Physico-chemical characterization
The core size of USPIONs and C-USPIONs was examined using
transmission electron microscopy at 100 kV (TEM, JEM-2010,
Hitachi-JEOL, Tokyo, Japan). Hydrodynamic sizes and the surface
charge of the prepared nanoparticles over a period of 6 months
were measured using a dynamic light scattering method (DLS, Zeta-
sizer NanoZS90, Malvern Instruments, UK). Using X’Pert PRO X-ray
diffraction (XRD), the phase and crystalline properties were inves-
tigated. Crystal structure was determined from the position and
intensities of diffraction peaks observed in the diffraction angle
range, 2 = 10–80◦ using Cu K˛ radiation of 1.54 Å at 40 kV and
20 mA current.
Thermo gravimetric analysis (TGA) and differential scanning
calorimetric (DSC) analysis were used to evaluate the thermal prop-
erties of the prepared materials. TGA was also used to evaluate the
amount of citrate bound to the IONs. TGA and DSC of lyophilized
C-USPIONs and TSC were performed using SDT 2960 V2.2B (Simul-
taneous TGA-DTA, TA Instruments, Delaware, USA) and DSC – Q20
V24.4 instrument. TGA was run within a temperature range of
25–1200 ◦ C applying a constant heating rate of 10 ◦ C/min. DSC was
performed at a heat flow rate of 5 ◦ C/min within the temperature
range of 25 ◦ C to 500 ◦ C.
Fourier transform infrared (FTIR) spectra of USPIONs, C-USPIONs
and TSC were also recorded using Thermo Nicolet 5700 FTIR spec-
trometer (USA) in the diffuse reflectance mode. To enable high
signal to noise ratio, 64 scans were acquired at a resolution of
4 cm−1 .
2.4. Magnetic property characterization
Magnetic property and the saturation magnetization of the
developed nanoparticles were evaluated using a vibration sample
magnetometer (VSM), EG&G PAR 4500. Powder samples of USPI-
ONs and C-USPIONs were placed in a uniform magnetic field and
the magnetic hysteresis at room temperature was studied.
Magnetic relaxivity measurements were performed on a 1.5 T
whole body MRI scanner (MAGNETOM Avento Tim, Siemens,
Munich, Germany) using a 12 channel head coil. Aqueous phantoms
with concentration of iron ranging from 0 to 0.45 mM were pre-
pared in de-ionized water and used for relaxivity studies. Scanning
parameters used were temperature, T = 22 ◦ C, FOV = 20 cm × 40 cm,
slice thickness = 10 mm.
An inversion recovery sequence was used for the measurement
of longitudinal relaxation time T1 . The repetition time (TR) and echo
time (TE) were set at 4000 and 11 ms, respectively and the MR sig-
nal was measured by changing the inversion time (TI) from 50 to
3000 ms. A modified T2 relaxometry spin echo sequence was run
at three different planes of the phantoms for T2 relaxometry mea-
surements. For a fixed TR of 2000 ms, MR signal was measured for
varying TE values of 15–120 ms. T1 and T2 relaxation times were cal-
culated from the resulting MRI pixel intensity maps with respect
218 A. Saraswathy et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224
Fig. 1. (a) TEM images of USPION and C-USPION showing average particle size of 12 nm and non-aggregation on citrate coating. (b) Hydrodynamic size distribution of USPION
and C-USPION.
to each concentration. r1 and r2 values were calculated as per ear-
lier reports and were plotted against the iron concentration and
relaxivity value was determined by the linear fit [18,19].
2.5. Cellular interaction studies
Human lung cancer cell line A549 was used for checking cell via-
bility, uptake and in vitro MRI studies. This particular cell line was
chosen because it contains high level of de-saturated fatty acids as
observed in fibrosed liver cells [20,21]. MTT assay was performed in
triplicate for the in vitro toxicity assessment of developed materials
at different concentrations (25, 50, 100, 300, 600, and 900 ␮g/ml)
using standard protocols [22].
Prussian blue staining of A549 cells incubated with different
concentrations of C-USPIONs (0–0.9 mM) confirmed the uptake and
localization of particles in the cells.
MRI of the C-USPION labelled cells was performed using a
method similar to that of the particle phantom. For this, the cells
were incubated with different concentrations of iron in the range of
0–0.9 mM. For the T2 measurement, a spin–echo sequence with TE
ranging from 50 to 800 ms and TR of 6500 ms was run. The images
from three different planes were acquired and the pixel intensity
with respect to each concentration was extracted.
2.6. Blood compatibility studies
Aggregation studies and hemolysis assay of C-USPIONs were
evaluated on red blood cells (RBC), white blood cells (WBC) and
platelets, as per the reported protocols [23,24]. Saline and water
were used as negative and positive controls respectively. Aggrega-
tion was assessed by checking the morphology of the cells using
phase contrast microscopy (Leica DM IRB, Germany). Absorbance
at 541 nm was considered for hemolysis assay.
2.7. Animal model development and in vivo MRI
A CCl4 induced progressive hepatic fibrosis, which is charac-
terized by a progressive increase in the deposition of extracellular
matrix proteins in the liver, was developed in 8 weeks old male
Wistar rats with an average body weight of 220 g, as per reported
protocol [25]. Study was approved by the Animal Ethics Commit-
tee of the Sree Chitra Tirunal Institute for Medical Sciences and
Technology (Order no: B 2982011 IX, dated: 19-10-2011) in accor-
dance with the regulations of Committee for the Purpose of Control
and Supervision of Experiments on Animals, India. Liver function
tests (LFT) for liver enzymes, serum glutamic oxaloacetic transam-
inase (SGOT) and serum glutamic pyruvic transaminase (SGPT)
were assessed to confirm the developed fibrosis. The animals with
elevated SGPT (1446 vs 59 iu/l of control) and SGOT (1809 vs 194 iu/l
of control) were selected for in vivo MRI.
In vivo MRI was performed with multisection T2 -weighted TSE
sequence (TR 5780 ms; TE 125 ms; FOV 98 mm × 140 mm; slice
thickness 3 mm; flip angle 90). All animals were anaesthetized
using ketamine and xylazine mixture before the MRI scan. After
acquiring the pre-contrast images, a dose of 2.17 mg/ml (0.04 mM)
of Fe/kg body weight of C-USPIONs was injected through tail
vein and post-contrast images were acquired within 10–15 min.
Pre- and post-contrast images were quantified for pixel intensity
using the software Syngo (Siemens, Germany) and the variation of
fibrosed area was specifically analyzed using Image J software.
After 2 h of post-contrast MRI, the animals were sacrificed and
liver extracted for histological analysis, to confirm the development
of fibrosis model and also to establish the presence of iron in liver
using special staining. The formalin fixed tissues were processed
using standard histopathological techniques. Masson’s Trichrome
(MT) staining for the detection of collagen fibres and the Pearls’
Prussian blue (PB) stain for assessing the presence of iron were
also carried out.
3. Results and discussion
A simple co-precipitation method was adopted to synthesize
USPIONs with improved magnetic properties, maintaining the ultra
small particle size. Selection of TSC as the coating agent and the
optimized coating thickness reduced the particle aggregation and
preserved the magnetic properties to a great extent. Ambient con-
ditions were maintained for the covalent attachment of the TSC
with USPIONs to generate enhanced magnetic relaxivity and highly
dispersed colloidal suspension which was stable over a period of
six months. The physico-chemical and magnetic characterizations,
in vitro cytotoxicity, cellular uptake, blood compatibility assess-
ments and animal imaging of C-USPIONs were performed and
presented.
3.1. Physico-chemical characterization
The particle size and crystalline phase of USPIONs and C-
USPIONs were determined using three established techniques like
TEM, DLS (Fig. 1) and XRD (Fig. 2a). TEM micrograph indicates
that both USPIONs and C-USPIONs have narrow size distribution of
10–15 nm with an average diameter of 12 nm. Well dispersed pat-
tern of particles is observed in the TEM of C-USPIONs compared to
an aggregated pattern in USPIONs. DLS showed the hydrodynamic
diameter of USPIONs and C-USPIONs as 24 and 30 nm respectively.
Here also, aggregation of the particles is visualized only in USPIONs
and was absent after the citrate modification. The high dispersivity
observed in C-USPIONs compared to USPIONs is attributed to the
 A. Saraswathy et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224
Fig. 2. (a) XRD pattern of USPION and C-USPION. (b) TGA curves of C-USPION and
tri sodium citrate (inset) showing thermal decomposition of citrate.
hydroxyl interaction of citrate molecules to the surface of iron oxide
nanoparticles, which reduces the inter particle interaction consid-
erably, compared to that of USPIONs. Powder XRD analysis showed
an inverse spinal structure of magnetite with the indices (3 1 1),
(4 4 0), (2 2 0), (5 1 1), (4 0 0), (6 2 0) and (5 3 3) [16,26]. The sharp-
ness of the XRD peaks is an indication of higher order of crystallinity
in the case of C-USPIONs compared to USPIONs.
Zeta potential, TGA, DSC and FTIR spectral analysis were also
used for the complete characterization of nanoparticles. Zeta
potential increased to −19.6 mV after the citrate coating compared
to −9.29 mV of USPIONs. TGA plot of TSC shows four distinct stages
of thermal decompositions around 171, 321, 557 and 1120 ◦ C corre-
sponding to the total weight loss of 13.23, 26.52, 45.51, and 98.29%
due to elimination of bound water, exothermic phase transition,
endothermic phase transition and linear degradation respectively
(Fig. 2b). TGA curve of C-USPIONs also show four similar stages of
decomposition around 147, 315, 597 and 1188 ◦ C corresponding
to the total weight loss of 2.3, 4.6, 5.7 and 7.1%. These phase tran-
sitions indicates the step-by-step degradation of the citrate due
to the dissociation of citrate–Fe bonds. The shift observed in the
decomposition temperature of C-USPIONs from that of TSC is due
to the effect of catalytic behaviour of iron oxide nanoparticles in the
system [27,28]. The high temperature of around 1200 ◦ C required
to eliminate the total weight of 7.1% of citrate shows the strong
binding of the same to the material.
219
Fig. 3. Coating confirmation and stability studies. (a) FTIR spectra of C-USPION,
USPION and TSC. (b) Zeta potential measurements of C-USPION and USPION showing
high stability over 6 months.
The DSC curve of TSC shows characteristic endotherms at 162.2
and 318.2 ◦ C (Supplementary Fig. S1). The change in crystallinity of
TSC is very well illustrated by the sharp endothermic peaks. In the
case of C-USPIONs, sharp endotherms are absent, but the broadness
along with a minor shift in the peaks that appear around 156.8 ◦ C
and 328 ◦ C confirms the presence of citrate over USPIONs [29].
The FTIR spectra of USPION, TSC and C-USPION (Fig. 3a) shows
the vibrational modes associated with C O and Fe O groups and
the characteristic peaks of the citrate. USPION shows prominent
peaks of Fe O vibrations at 572 and 450 cm−1 corresponding to the
tetrahedral and octahedral sites of magnetite respectively [16,30].
The peaks at 1618 and 1397 cm−1 corresponding to the asymmetric
and symmetric stretching of carboxyl group confirms the pres-
ence of citrate in C-USPIONs. The shift observed in the asymmetric
stretching mode to lower wave number region is an indication of
the presence of strong hydrogen bonded system [16,31].
3.2. Stability
Zeta potential and hydrodynamic diameter of C-USPIONs and
USPIONs were monitored over a period of 6 months. During this
period, only 20% change in zeta potential (−19.6 to −15.7 mV) was
observed for C-USPIONs which proves the stability of the system
even after 6 months (Fig. 3b). Hydrodynamic size also remained
220 A. Saraswathy et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224

Fig. 4. Magnetic property evaluation. (a) Magnetic hysteresis curve of USPION and C-USPION. (b) T2 weighted MR phantom images of USPION and C-USPION for varying
concentrations (0–0.45 mM) and transverse (r2 ) relaxation rates of C-USPION and USPION showing enhanced relaxation rate of 102 mM−1 s−1 on citrate coating.

in the acceptable range (30–50 nm) after 6 months which rules out
any aggregation. For USPIONs, the initial zeta potential of −9.29 mV
gradually increased and reached a positive value after 1 week and
attained a constant value of +20 mV after 6 months. Hydrodynamic
size also varied from 24 nm to 1 ␮m, indicating aggregation of the
particles, in the absence of the stabilizing agent. Zeta potential and
hydrodynamic size of C-USPIONs remained almost constant for the
first two months and was stable without significant changes after 6
months. Stability of C-USPIONs is important and can be correlated
with the shelf life of the developed system for its use as an MRI
contrast material. The stability of C-USPIONs also implies absence
of dissociation of citrate from iron oxide and subsequent leakage
of citrate, and the aggregation of iron oxide particles. Till date, no
report is available on the time dependent stability study of IONs
suspensions.
3.3. Magnetic property characterization
Magnetic properties like proton relaxation efficiency, magnetic
phase and saturation magnetization which are crucial factors of a T2
contrast agent were studied using VSM and MR imaging techniques.
The hysteresis loop at room temperature showed zero coerciv-
ity (Fig. 4a) for both materials which is a characteristic property
of superparamagnetic materials. USPIONs and C-USPIONs showed
high saturation magnetization of 62.7 and 57.5 emu/g respectively.
Coating effect of citrate over the magnetic particles would have
caused the minor reduction in the magnetization value. Superpara-
magnetic property exhibited by the particles indicates the single
domain existence and the absence of aggregation, which is an
advantage over ferromagnetic particles [32].
Based on the pixel intensity expressions of the MRI images,
longitudinal and transverse relaxivity (r1 and r2 ) of USPIONs and
C-USPIONs were calculated. Increase in the concentrations of the
particles resulted in a loss of signal intensity in the T2 weighted
MR images. Transverse relaxivity was calculated from the slope
of linear plots of 1/T2 versus iron concentration. An enhanced r2
value of 102 mM−1 s−1 is achieved for C-USPIONs compared to
57 mM−1 s−1 of USPION (Fig. 4b). This increase in r2 will contribute
to the desired delay in relaxation of the protons bound to the citrate
coated USPION system. The high magnetic moment induced by the
 A. Saraswathy et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224 221

Fig. 5. Cell uptake of C-USPION. Microscopic images of Prussian blue stained A549 cells labelled with C-USPIONs at different concentrations.

strong magnetic inhomogeneity around the particles causes high
r2 compared to USPION [14]. The observed maximum relaxation
was shown by the C-USPIONs which had citrate coating thick-
ness optimized to about 7%, whereas all other batches showed less
enhancement. This finding correlates the effect of coating thickness
and relaxivity of the particles.
In T1 weighed MR images also showed increase in pixel
intensity of the phantom images with the increase in iron con-
centration. T1 weighed MR images and r1 values calculated by
the linear fit are given in Supplementary Fig. S2. r1 of 1.53
and 2.69 mM−1 s−1 are observed respectively for USPIONs and
C-USPIONs. It has been reported that SPIONs of particle size
less than 50 nm exhibit T1 contrast in addition to T2 contrast
when used in moderate concentrations and also improve the
long blood half life period and reduce the chance of accumu-
lation in reticulo-endothelial system [11,15,16]. The T1 contrast
exhibited by C-USPIONs was not appreciable compared to that
of the T2 contrast exhibited on T2 weighted imaging. Hence, we
highlight only the T2 contrast property of the material, in this
study.
Relaxivity ratio (r2 /r1 ) is an important parameter to ensure the
efficiency of a contrast agent [12,14]. A comparative study of the
relaxivity ratio of contrast agent developed in this study and that
of commercially available and earlier reported iron oxide based sys-
tems are given in Supplementary Table S1. Compared to other iron
oxide based contrast agents of similar sizes, r2 of 102 mM−1 s−1
and relaxivity ratio of 37.91 obtained in this study for C-USPIONs
are found to be very high which is a clear indication for devis-
ing the same as an efficient T2 contrast agent. There are reports
which state that iron oxide particles exhibit much higher relaxivity
when coatings like alginate or poly ethylene glycol are employed.
But, such materials have a thick polymer coating resulting in par-
ticle size of around 200 nm or more [33]. In this study, we have
optimized the magnetic properties and relaxivity values without
compromising size of the particle.
3.4. Cellular interaction studies
The behaviour of the cells as expressed by its metabolic activ-
ity on interacting with different concentrations of the particles

Fig. 6. Blood aggregation study: RBC, WBC and platelet incubated with C-USPION, positive control water and negative control saline respectively.
222 A. Saraswathy et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224
Fig. 7. (a) Pre- and post-contrast in-vivo MR images after 10 min of injection, with typical ROIs drawn to quantify the pixel intensity from fibrosed areas (above). Pseudo
coloured images of the original MR images (below). (b) Pixel intensity of liver from pre- and post-contrast.
was evaluated. For particle concentration of 25 ␮g/ml, C-USPIONs
showed 98.5% cell viability which decreased to 70.3% at a concen-
tration of 900 ␮g/ml (Supplementary Fig. S3). Clinically approved
dose for MR imaging is 25 ␮g/ml for human of average body weight.
Previous reports show a maximum viability of 80% only at this con-
centration [34,35]. So it is clear that C-USPIONs prepared in this
study is more compatible even at higher concentrations compared
to many of the previously reported iron oxide based systems. Per-
haps, this property of the present system can address the issues of
previous iron oxide MRI contrast materials which had been with-
drawn from the market due to reasons related to safety concerns
[36].
The cellular uptake and labelling efficiency of C-USPIONs of var-
ious concentrations are shown in Fig. 5. Cell uptake of C-USPION is
clear on PB staining which gives an indication that the particles are
mostly concentrated in the cytoplasm. The normal morphology of
the cells remains undisturbed, even at higher concentrations. Inter-
nalization of the particle without any targeting moiety is achieved
due to the local neutralization effect of cell membrane to the repul-
sive interaction with the anionic particles. This property is an
added advantage of the system and is clear from the supremacy of
C-USPIONs over commercially available contrast agents like Reso-
vist and Endorem in the cell internalization [22,37]. The increase
in the cell internalization efficiency of the anionic surface modi-
fied SPIONs compared with bare SPIONs also supports the current
observations [38–40].
In vitro MR imaging of the cells with different concentrations
of particles showed gradual signal drop on T2 weighted image,
with increasing concentration of iron (Supplementary Fig. S4). This
demonstrates the significant signal dephasing efficacy of the par-
ticles taken up by the cells causing inhomogeneity in the nearby
water protons which is visualized as signal drop on T2 weighted
images.
3.5. Blood compatibility studies
For the safe administration of the particle through the blood
stream, absence of aggregation of hematocytes and hemolysis are
important. No aggregation was observed for any of the blood cells
on incubation with 100 ␮g/ml of C-USPIONs (Fig. 6).
Hemolysis assay performed with different concentrations of
C-USPIONs (25 ␮g/ml, 50 ␮g/ml, 100 ␮g/ml) also revealed similar
result (Supplementary Fig. S5). The percentage of lysis was less
than1% for different concentrations of C-USPIONs which is well
within the acceptable limits [24,41]. These results confirm the
blood compatible nature of C-USPIONs and hence could be adminis-
trated intravenously to the blood stream safely during MR imaging.
3.6. Animal model development and in vivo liver MR imaging
Liver uptake and imaging potential of C-USPIONs were evalu-
ated in vivo on rodent model of liver fibrosis. Development of liver
fibrosis was indicated by the LFT and the animals with elevated
level of liver specific enzymes, SGOT and SGPT corresponding to
the fibrosed state were selected for imaging (Supplementary Fig.
S6). Post-contrast MR images acquired within 10–15 min of the
administration of C-USPIONs showed hypointense liver compared
to pre-contrast images (Fig. 7). Estimated average signal inten-
sity was 48.09 and 78.89 respectively for pre- and post-contrast
liver images with a decrease in percentage signal intensity of 39%.
The hypointensity observed in the post-contrast T2 weighted MR
image is due to the heavy uptake of C-USPIONs by the liver cells.
Within the hypointense liver, hyperintense streaks are visualized,
which represents the Kupffer’s cell devoid fibrotic regions of the
liver. There is an excessive accumulation of extracellular matrix in
fibrosed liver and hence a decrease in the Kupffer’s cell density.
The amount of collagen increases and the ratio of fibro-connective
 A. Saraswathy et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 216–224
Fig. 8. Histopathological images of control (upper row) and fibrosed (lower row) liver section. H&E stained sections of normal liver with central vein and radiating hepatic
cords and fibrosed liver showing disruption of the tissue architecture, extension of collagen fibers and pseudo-lobulation. MT staining of normal liver shows the absence of
collagen strands where as fibrosed liver with abundant fibrous tissue (white arrows). PB stained sections of normal liver and C-USPION administered fibrosed liver showing
the presence of iron (black arrows).
tissue versus liver cells increases at the fibrosed sites [42,43]. Nor-
mally, C-USPIONs reach liver through normal opsonisation process
and will be taken up by the Kupffer’s cells. The negative surface
charge of C-USPIONs also favours the internalization through the
cationic sites of plasma membrane of the liver cells [44]. But in a
fibrosed condition, due to decrease in the density of Kupffer’s cells,
C-USPIONs uptake will be less, and hence appear as hyperintense
streaks, which are clearly visible in the MR images. A quantification
based on the pixel intensity from ROIs drawn on these hyperin-
tense streaks and corresponding ROIs from the pre-contrast images
showed significant increase in intensity which resulted in the clear
visibility of these streaks (Fig. 7 and Supplementary Fig. S7). A better
diagnosis through visualization of the extent of fibrosis is possible
from the pseudo coloured image (Fig. 7).
H&E stained sections of liver tissue showed increased inflam-
matory cell infiltration, ballooning of hepatocytes, fatty changes
and severe centrilobular necrosis. Perilobular fibrosis and por-
tal triad fibrosis was observed. MT staining revealed extensive
collagen deposition and pseudolobular formation in liver tissue.
These observations ascertained the development of liver fibrosis
in the rodent model. The presence of iron in the fibrosed liver
macrophages was also determined by PB staining of liver sections
(Fig. 8) [10,25,45].
4. Conclusions
In summary, we report the development of citrate coated USPI-
ONs based magnetic contrast agents for in vivo MR imaging of
liver fibrosis. The particles are highly water dispersible and also
stable over long period of time. High uptake of the material and
subsequent contrast difference were observed within 10–15 min
of intravenous administration of C-USPIONs. C-USPIONs exhib-
ited superparamagnetic nature with high magnetic saturation of
57.5 emu/g. The high transverse relaxation and transverse to lon-
gitudinal relaxation ratio exhibited by the particles indicates its
potential to use as T2 contrast agent for MR imaging. Long term
stability, cell viability, blood compatibility and the cell labelling
efficiency of the particles further recommend it as a safe and effi-
cient MRI contrast agent. The clear differentiation of the fibrosed
site within the MR image of whole liver suggests the applicability
of C-USPIONs as transverse MR contrast agent for liver associated
diseases at very early stage. The significantly high intensity of the
223
fibrosed area observed on post contrast images helps in the clear
visualization of the fibrosis and thus helps in the easy and accurate
diagnosis. In addition to the magnetic properties, the citrate coat-
ing of C-USPIONs can be utilized as a platform to modify them as
multifunctional nanoprobe in future.
Acknowledgements
The authors acknowledge the financial support received from
the Board of Research in Nuclear Sciences, Department of Atomic
Energy (DAE), Government of India. Ariya Saraswathy and Shaiju
S Nazeer acknowledge the Council of Scientific and Industrial
Research, New Delhi, India for senior research fellowship. Authors
also thank the Scientists in charge of BST, MIC, MOM and DTERT
divisions of BMT Wing and HOD, IS &IR for extending their support
in using some of their facilities. The support from Dr. P. A. Joy, NCL,
Pune in the VSM studies and Dr. M.R. Rekha, SCTIMST, in the cell
studies are also sincerely acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.colsurfb.2014.02.034.
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