+ APPENDIX_SUPPLEMENTAL MATERIAL

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APPENDIX: SUPPLEMENTAL MATERIAL
1. Methods
1.1. Synthesis of MgFe2O4 magnetic nanoparticles.
In order to realize a narrow size distribution, magnesium ferrite nanoparticles were prepared by
an ultrasonic assisted co-precipitation of ferric chloride (FeCl3 .6H2O, Ajax Finechem), ferrous
chloride (FeCl4 .4H2O, Sigma Aldrich) and magnesium chloride (MgCl2.6H2O, Sigma Aldrich) in an
aqueous solution, using trisodium citrate (C6H5Na3O7.2H2O, Sigma Aldrich) as an electrostatic
stabilizer and sodium hydroxide (NaOH) as an precipitant agent. Co-precipitation process was
carried out by placing the reactor chamber in an ultrasonic bath where the sample is exposed to
ultrasonic irradiation at a fixed output power of 25 W. The relevant amounts of ferric chloride
and magnesium chloride were mixed in 50 ml of DI water previously degassed by N 2 gas at 80
°C and continuously stirred for 10 min to form a homogenous and transparent salt solution. The
other solution was prepared by 20 ml of 6 M sodium hydroxide mixed with 0.0075 mol sodiumcitrate as precipitant solution. The salt solution was added drop-wise into the precipitant solution
with strong magnetic stirring under N2 blanket. The co-precipitated particles were aged for one
hour at 80 °C in order to complete the precipitation process. The resulting precipitates were
collected from the solution using a permanent magnet, which were then washed four times, i.e.
first with DI water, twice with ethanol, and finally with DI water to remove any excess ions and
salts from the suspension. The washed precipitates were centrifuged at 3000 rpm for 10 min, and
dried in an oven under vacuum at 50 °C for 1 day. Particles with sizes of 7 ± 0.5 nm, 10 ± 0.5 nm
and 13 ± 0.5 nm were produced by controlling the concentration of sodium citrate. The post
annealing treatment at successive temperatures of 973 K, 1073 K and 1173 K for 2 h was used to
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produce larger particles with sizes of 16 ± 1 nm, 21 ± 1 nm and 29 ± 1 nm, respectively. Samples
were collected and dried for structural and magnetic characterization.
1.2. Characterization techniques
X-ray diffraction (XRD) patterns were obtained on a Philips PW 1140/90 diffractometer using
Cu-Kα radiation. The mean particle size was calculated from the XRD pattern by using
Scherrer’s equation, and was confirmed by a transmission electron microscopy (TEM, Philips
CM20) for D < 13 ± 0.5 nm. IMAGE-J software was used to estimate the median size ±
(standard deviation) and size distribution of nanoparticles based on the TEM images. A
ZetaSizer Nano ZS (Malvern Instrument Ltd., UK) was used to measure the hydrodynamic size
(DH). Before hydrodynamic size measurements, the aqueous magnetic suspension was prepared
in DI water by re-dispersing magnetic nanoparticles in the presence of sodium citrate as a
dispersant, using ultrasonic disruption to break up any aggregates.
Fourier transform infrared (FTIR) spectra of the Mg-ferrite, dispersed in KBr and pelletized,
were obtained using a PerkinElmer Spectrum 100 series FTIR spectrometer in a wave number
ranging from 400 cm-1 to 4000 cm-1 with a resolution accuracy of 4 cm-1 under ambient
condition.
Thermo-magneto gravimetric analysis (TGMA) was utilized to measure the Curie temperature at
a heating rate of 40 °C/min in an argon atmosphere. The concentration of magnetic nanoparticles
in aqueous suspension was then adjusted to 10 mg/ml. 1 ml of the final suspension was used for
heating induction measurements. Specific values of the saturation magnetization (MS) and
coercivity (HC) were obtained using a vibrating sample magnetometer (VSM, RIKEN DENSHI)
operated at 77 K and 300 K with a maximum magnetic field of 10 kOe. A LakeShore 7000 series
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AC susceptometer was used to determine the blocking temperatures of samples in an AC
magnetic field of 100 A/m at a frequency of 137 Hz with a constant heating rate of 3.3×10-2 K/s.
The sample powders used for these magnetic measurements were sealed in a small amount of
epoxy resin.
The heat generation ability of magnetic nanoparticles dispersed in water was investigated using
an induction heating system (EASYHEAT 0224–Ambrell) with a heating coil consisting of 8
turns (N) with 2.5 cm diameter (D) and 4 cm length (L). The amplitude of the AC magnetic field
was fixed at H = 8 kA/m at frequency of 279 kHz. The changes in temperature of the magnetic
suspensions were monitored as a function of time using an IR Thermacams (FLIR Thermacam
SC2000) with accuracy of ±1 °C. In order to avoid as much as possible the heat exchange
between the magnetic suspension and the external environment, magnetic suspensions was
placed into a Dewar flask. The same ultrasonic dispersion procedure for hydrodynamic size
measurements was utilized before heat generation measurements in order to stabilize the
nanoparticles in their suspensions. The concentration of magnetic nanoparticles aqueous
suspension was then adjusted to 10 mg/ml. After concentration adjustment, 1 ml of the final
suspension was pipetted into a Dewar flask. In order to establish the background heating effect,
DI water without magnetic nanoparticles was exposed to the same external AC magnetic field
conditions for 30 min and then changes in the temperature was recorded.
The cytotoxicity of nanoparticles was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide) assay in COS-7 cells (SV-40 transformed African Green Monkey
kidney fibroblast cells)1. COS-7 cell line (SV-40 transformed African Green Monkey kidney
fibroblast cells) were grown in Dulbecco’s modified Eagle’s medium (DMEM) complemented
with100 mg/ml streptomycin, 10% (v/v) FBS, and 100 mg/ml penicillin at 37°C in a gassed
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incubator with 5% CO2. The relative cell viabilities (%) to the control were calculated as
viability (%) = (mean absorbance of the sample/ mean absorbance of the control) × 100%.1
2. Supporting Results
2.1. Magnetization curves
FIG. S1. Magnetization curves at 300 K of magnetic nanoparticles with different sizes.
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2.2. Calorimetric measurement of different magnetic suspensions
FIG. S2. Change in temperature as a function of time at H = 8 kA/m and f = 279 kHz for
magnetic suspensions with different particle sizes.
2.3. In vitro cytotoxicity evolution
An interesting aspect is that the size of MgFe2O4 nanoparticles has an effect not only on the heat
generation rate, but also on the cytotoxicity. The interactions between nanoparticles and
biological organisms typically occur at the surface of the nanoparticles.2 It is widely accepted
that in endocytic mode, cellular uptake and efficiency of particle processing in the endocytic
pathway are dependent on the size of particles,2,3,4 and the size therefore has a dramatic effect on
the cell response to exposure. Hence, in order to assess the effect of particle size on the
cytotoxicity of MgFe2O4 nanoparticles, MTT assay were performed for magnetic suspension
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with concentration of 1 mg/ml for 72 h incubation. The quantitative MTT assay indicated that the
cell survival decreases slightly with the increase in the nanoparticles size. The cell survival rate
of MgFe2O4 nanoparticles were 100 ± 14%, 100 ± 11%, 98 ± 10%, 96 ± 7%, 90 ± 4% and 88 ±
3% for particle size of 7 ± 0.5 nm, 10 ± 0.5 nm, 13 ± 0.5 nm, 16 ± 1 nm, 21 ± 1 nm and 29 ± 1
nm, respectively (Figure S3).
FIG. S3. Cell viability of COS 7 cells incubated with medium containing bare MgFe2O4 ferrite
nanoparticles with different particle size. The relative cell viability results are the mean of three
experiments ± SD (standard deviation).
Notably, 100% of cells remained alive when they were incubated with a particle size of 10 ± 0.5
nm. The high protection against cell toxicity for particles with D < 13 ± 0.5 nm were attributed to
a strong negative surface charge due to the absorbed citrate groups on the surface of
nanoparticles. This strong negative charge density leads to strong repulsion force between
nanoparticles and an adjacent cell where the surface charges of COS-7 cells is negative.5 Such
charge-based protection prohibits the interaction of nanoparticles with the cell surface. The
nanoparticles with D > 16 ± 1 nm presented a slightly lower cell survival. A possible explanation
for this slight reduction in the cell viability could be due to the penetration rate of the particles. It
is known that the nanoparticles with larger sizes have a stronger tendency for particle penetration
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of cell membranes while the small particles stay on the surface. Thus, larger particle size render a
stronger stimulus on the cell surface and generated more pronounced damages to the cell
membrane structure over a period of time, causing cell lysis.2,3,4 Moreover, the protection effect
of citrate groups on the surface can be minimized for D > 16 ± 1 nm. It can be explained by
partial decomposition of citrate groups after post annealing at high temperature (Figure S4).
Additionally, increased toxicity for D > 16 ± 1 nm seems to be attributed to the higher
hydrodynamic size through similar mechanism for the particle size dependent toxicity.
2.4. FTIR spectra
FIG. S4. FTIR spectra of (a) 10 ± 0.5 nm and (b) 16 ± 1 nm nanoparticles.
The FTIR spectra of both the 10 ± 0.5 nm and 16 ± 1 nm particles are shown in Figs. S4(a) and
S4(b), respectively. It is clearly seen from the Fig. S4(a) that the 10 ± 0.5 nm particles show
several absorption bands at about 3425 cm-1 corresponding to hydroxyl groups and 1577 cm-1
and 1338 cm-1 related to the carboxyl groups (C=O) and (C-O), respectively. The appearance of
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the characteristic bands of carboxyl groups indicates that the carboxyl groups from trisodium
citrate exist in the structure of 10 ± 0.5 nm particles. In FTIR spectrum curve of the 16 ± 1 nm
particles in Fig. S4(b), the absorption bands at 1577 cm-1 and 1338 cm-1 decrease. On the other
hand, a significant spectroscopic band at 571 cm-1 appears which seems to be a characteristic
absorption band of magnesium ferrite. This increase indicates the more crystallization of Mgferrite after annealing at 700°C. The results of FTIR spectra can clearly show the decomposition
of citrate groups after post annealing at high temperature (700 °C for 16 ± 1 nm nanoparticles).
Hence, this result confirms slight reduction in the cell viability for nanoparticles with D > 16 ± 1
nm.
References:
1
M. R. Barati, K. Suzuki, C. Selomulya, and J. S. Garitaonandia, IEEE Trans. Magn. 49, 3460
(2013).
2
S. Sharifi, S. Behzadi, S. Laurent, M. L. Forrest, P. Stroeve and M. Mahmoudi, Chem. Soc.
Rev. 41, 2323 (2012).
3
H. Yin, H.P. Too, and G.M. Chow, Biomaterials 26, 5818 (2005).
4
N. D. Thorat, K. P. Shinde, S. H. Pawar, K. C. Barick, C. A. Betty and R. S. Ningthoujam, Dalton Trans.
41, 3060 (2012).
5
Y. Antov, A. Barbul, H. Mantsur, and R. Korenstein, Biophys. J 88, 2206 (2005).
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