Supporting information
1. Materials and Methods
1.1 Synthesis of Iron Oxide (IO) and Oleic acid capped Iron Oxide (IOL)
The iron oxide nanoparticles (IO) were synthesized by co–precipitation of iron (III) and iron (II)
salts. Oleic acid was used as a capping agent to prevent the aggregation of iron oxide
nanoparticles (IOL). Different volumes of oleic acid (0.1, 0.2, 0.4, 0.6, 0.8 & 1.0 mL) are
introduced into the reaction mixture and heated to 80oC for 20 min. The lyophilized IOL
nanoparticles (nps) were dispersed in n–hexane (20 % w/v).
1.2 Characterization of FIO: The synthesized iron oxide nanoparticles (IO), oleic acid capped
iron oxide nanoparticles (IOL) and fluorescent iron oxide nanoparticles (FIO) were characterized
for their physico–chemical properties with various techniques. The particle size and zeta
potential was obtained from the Zetasizer (Nano ZS, Malvern Instruments, UK). The colloidal
stability was studied using Particle size analyzer (Bluewave, Microtrac, USA). The surface
morphology was observed under field emission scanning electron microscope (JSM 6701F,
JEOL, Japan). The presence of oleic acid, BSA and sulphorhodamine B in the iron oxide samples
were studied using FTIR spectroscopy (Spectrum 100, Perkin Elmer, USA). The ultrafine
structure and lattice arrangement was analyzed using field emission transmission electron
microscopy (JEM 2100F, JEOL, Japan). The crystallinity of the iron oxide nanoparticles was
studied using the x–ray diffractometer (D8 Focus, Bruker, Germany). The magnetic properties of
various iron oxide samples (IO, IOL, FIO) were analyzed using SQUID (MPMS–XL, Quantum
Design, USA). The thermogravimetic analysis was performed using simultaneous TG–DTA
(SDT–Q600, TA Instruments, USA) from 28 °C to 1000 °C at a rate of 10 °C/minute. The nature
of the iron oxide nanoparticles was monitored using UV–Vis–NIR spectrometer (Lambda 750,
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Perkin Elmer, USA) between 250 and 2000 nm. The fluorescence property of FIO was studied
by dispersing the sample in millipore water using a spectrofluorimeter (LS–45, Perkin Elmer,
USA) between wavelengths 500–650 nm with a bandwidth of 2.5 nm. The excitation wavelength
was found to be 565 nm and emission occurred at 582 nm.
1.3 Magnetic Resonance Imaging: The magnetic resonance imaging experiments were carried
out using a 1.5 T scanner (Sonata, Siemens, Germany) and the phantom containing agar gels
were positioned at the iso-centre of the magnet. T1 and T2 weighted images were obtained in the
coronal section. For T1-calculation,images were acquired with a spin-echo pulse sequence using
the following parameters: a slice thickness of 5 mm with a TE of 15 ms and with variable TR
(repetition time) of 100,200,300, 500, 600, 800, 1000, 1500, 2000, 3000, 4000, and 5000 ms.
Similarly, using the spin-echo sequence the T2 relaxation was calculated using a fixed TR of
5000 ms and with variable TE of 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260,
280, 300, and 320 ms. The slice thickness was kept at 5mm. The FOV (field of view) used was
240x240 with a matrix size of 512x512.
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2. Results and Discussion
The fluorescence spectrum of FIO nanoparticles.
Intensity (units)
1000
Without magnetic field
With magnetic field
800
600
400
200
0
570
580
590
600
Wavelength (nm)
Figure S1: The emission spectrum of FIO showing the intensity differences in the
presence and absence of magnetic field.
2.1 Particle size and zeta potential: The size and zeta potential of the samples were measured
through DLS technique and the results were tabulated in table S1. All experiments were carried
out in triplicates. The average hydrodynamic particle size of iron oxide was found to be in the
micron range, which is strongly indicative of a high level of agglomeration. Such a phenomenon
is common for magnetic nanoparticles that tend to attract each other forming an aggregate in the
absence of any stabilizer. Addition of oleic acid brings down the average particle size to about
210 nm indicating a stabilization of the nanoparticles by the oleic acid coating. Introduction of
sulphorhodamine B slightly increases the particle size to about 340 nm indicating that this
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modification did not accelerate the aggregation process and retained the stabilizing effect of oleic
acid. The zeta potential of iron oxide becomes more negative after incorporation of oleic acid,
which is not significantly altered when modified with sulphorhodamine B. The enhanced
negative zeta potential implies a greater colloidal stability of the nanoparticles as is evident from
the particle sizes obtained [1]. The stabilization of iron oxide nanoparticles by oleic acid might
be attributed to a steric repulsion offered by the long acyl chains of oleic acid. Another facet that
emerges from the observed particle sizes for these samples using laser diffraction and dynamic
light scattering is that these techniques measure the hydrodynamic radius of a particle and hence
do not represent its true size. Presence of oleic acid coating on the surface of iron oxide will tend
to reduce its hydrophilicity thereby contributing to a reduction in the hydrodynamic radius. Thus,
in order to obtain a true perspective on the effect of surface modifications on the size of a
particle, alternate techniques such as transmission electron microscopy (TEM) needs to be
employed.
Table S1. Particle size and zeta potential of iron oxide (IO), oleic acid capped iron oxide (IOL)
and fluorescent iron oxide (FIO).
Sample
Average size (nm)
PDI
Zeta potential (mV)
IO
1264.33 ± 99.30
0.60 ± 0.27
–22.53 ± 0.66
IOL
211.53 ± 13.52
0.45 ± 0.01
–33.13 ± 1.75
FIO
341.23 ± 38.42
0.40 ± 0.05
–32.06 ± 0.80
Further the stability of FIO nanoparticles was checked in water, PBS and FBS at different time
points using laser diffraction method (Figure S1). The results showed that FIO nanoparticles are
stable for up to 3h in water and PBS whereas it showed aggregation at 3h in FBS. The colloidal
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stability of the FIO in FBS is less compared to that of PBS and water. The particle size increases
because of the adsorption of the protein on the surface of the FIO leading to an increase in the
hydrodynamic radius of the nanoparticles. Thus the nanoparticles aggregate with the time and at
3 h it shows maximum aggregation. In case of the PBS and water it shows similar trend that the
increase in the particles size with respect to time where, after 6 h it shows maximum aggregation.
The FIO aggregation is higher in case of PBS compared to water, because the salt in the PBS
facilitates the aggregation according to the Oswald ripening where as in case of the water the
aggregation is limited.
Figure S2. Colloidal stability of FIO nanoparticles determined by laser diffraction method in
(A) Water (B) PBS and (C) FBS.
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Table S2. The d spacing values for all the samples from selected area electron diffraction
patterns and XRD compared with the theoretical values.
d spacing values
hkl
IO
IOL
FIO
XRD
Theoretical
2.86
2.82
2.89
2.91
2.97
220
2.45
2.42
2.49
2.50
2.53
311
2.04
2.01
2.08
2.07
2.1
400
1.65
1.65
1.67
1.69
1.71
422
1.55
1.53
1.60
1.59
1.61
511
Table S3. The magnetization (Ms) values of iron oxide (IO), oleic acid capped iron oxide (IOL)
and Fluorescent iron oxide nanoparticles
Sample
Saturation Magnetization (emu/g)
Core Magnetization
300 K
4K
(emu/g)
IO
53.62
60.83
54.71
IOL
52.16
60.48
71.45
FIO
42.53
49.61
72.08
Table S4. Relaxation rates (r2) calculated
Relaxivity (r2)
Relaxivity (r1)
(mM-1 s-1)
IO
(mM-1 s-1)
Regression
Coefficient (R2)
r2/r1
61.67
0.40
0.98
154.18
IOL
29.78
0.81
0.99
36.78
FIO
53.20
0.50
0.95
106.41
Sample
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3. References
[1] Qin C, Li C, Hu Y, Shen J, Ye M. Facile synthesis of magnetic iron oxide nanoparticles
using 1-methyl-2-pyrrolidone as a functional solvent. Coll Surf A  2009;336:130–134.
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