Published on Web 10/04/2010

Engineered Biocompatible Nanoparticles for in Vivo Imaging

Applications
Shu Chen,†,‡ Lijun Wang,§ Suzanne L. Duce,| Stuart Brown,§ Stephen Lee,†
Andreas Melzer,§ Sir Alfred Cuschieri,*,§ and Pascal André*,†
School of Physics and Astronomy (SUPA), UniVersity of St Andrews,
St Andrews KY16 9SS, U.K., School of Chemistry (EaSt CHEM), UniVersity of St Andrews,
St Andrews KY16 9ST, U.K., Institute for Medical Science and Technology, UniVersity of
Dundee, Dundee DD2 1FD, U.K., and DiVision of Biological Chemistry and Drug DiscoVery,
College of Life Sciences, UniVersity of Dundee, Dundee DD1 5EH, U.K.
Received July 23, 2010; E-mail: Pascal.Andre@st-andrews.ac.uk; a.cuschieri@dundee.ac.uk

Abstract: Iron-platinum alloy nanoparticles (FePt NPs) are extremely promising candidates for the next
generation of contrast agents for magnetic resonance (MR) diagnostic imaging and MR-guided interventions,
including hyperthermic ablation of solid cancers. FePt has high Curie temperature, saturation magnetic
moment, magneto-crystalline anisotropy, and chemical stability. We describe the synthesis and characteriza-
tion of a family of biocompatible FePt NPs suitable for biomedical applications, showing and discussing
that FePt NPs can exhibit low cytotoxicity. The importance of engineering the interface of strongly magnetic
NPs using a coating allowing free aqueous permeation is demonstrated to be an essential parameter in
the design of new generations of diagnostic and therapeutic MRI contrast agents. We report effective cell
internalization of FePt NPs and demonstrate that they can be used for cellular imaging and in vivo MRI
applications. This opens the way for several future applications of FePt NPs, including regenerative medicine
and stem cell therapy in addition to enhanced MR diagnostic imaging.

1. Introduction development of exogenous MRI contrast agents.12 Common T2-

Nanoparticles (NPs) have attracted considerable interest as
diagnostic and therapeutic tools for biomedicine,1-3 with
magnetic NPs offering unique opportunities for magnetic
separation, targeted drug delivery, and hyperthermic ablation
of cancer and as contrast agents in magnetic resonance imaging
(MRI).4-11 MRI is a powerful, noninvasive medical imaging
technique where the signal originates predominately from water
and lipids. Medical diagnosis requires enhanced contrast
between normal and pathological tissues, resulting in the
†
SUPA, University of St Andrews.
‡
EaSt CHEM, University of St Andrews.
§
Institute for Medical Science and Technology, University of Dundee.
|
Division of Biological Chemistry and Drug Discovery, University of
Dundee.
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contrast agents with prolonged intravascular retention, improved
tissue delineation, high chemical stability, and improved
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system and therefore should enable long blood circulation half-
life, thereby improving their ability to reach specific targets.4,16
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engineered NPs, forming an inert and biocompatible outer shell.
Not only does it create a protective shell against chemical
degradation, but it also allows surface functionalization with
functional alkoxysilanes.7 An outer coating with functional
organic molecules has many advantages, including steric and/
or electrostatic repulsion between the NPs, which can increase
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Engineered Biocompatible Nanoparticles
water solubility and stability in biological environment.17
Positively charged NPs demonstrate increased cell internalization
efficiency and biomedical molecules binding capability for
multimodal imaging applications and drug delivery.4,18,19
Characterized by high Curie temperature and magneto-
crystalline anisotropy, iron-platinum metallic alloy NPs have
emerged as promising candidates for a new generation of
magnetic nanomaterials.20 Over the past decade, several chemi-
cal pathways have been developed to synthesize FePt NPs with
controlled stoichiometry, size, and shape.5 Such synthetic routes
include co-reduction of Fe and Pt precursors through both low-
temperature emulsion protocols and the high-temperature polyol
pathway,21-25 along with the thermal decomposition route.26-31
The latter approach often relies on Pt(acac)2, Fe(CO)5, or
Collman’s regeant precursors mixed with alkyl amines and acids
solubilized in high-boiling-point solvents heated at temperatures
of ∼300 °C and above to control the growth, composition, and
morphology of the NPs.
Studies have demonstrated the biomedical potential of FePt
NPs,6 such as in magnetic separation32,33 and hyperthermic
ablation34,35 and as T2 MRI contrast agents.36-38 However,
toxicity remains a major concern, preventing their use as a
bioplatform for diagnosis and therapy.37,39,40
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ARTICLES
In this article, we describe the fabrication of FePt NPs
specifically designed to enhance their magnetic properties for
biomedical applications. We demonstrate that, in contrast with
previous reports, FePt NPs can be made nontoxic, and we
provide the first data on their cellular uptake mechanisms. We
report a 6-fold increase in the FePt-based T2 contrast properties
compared to those of clinical iron oxide NPs. The relationship
between the MRI contrast properties and the NPs’ architecture
is explored and rationalized as the basis for the design of NPs
as enhanced MRI contrast agents. Finally, we report the first
observations of cellular and in ViVo MR imaging with FePt NPs.
Our results opens the way for several applications of FePt NPs,
such as regenerative medicine and stem cell therapy, in addition
to enhanced MR diagnostic imaging, thus providing a platform
to develop novel diagnostic and therapeutic agents.
2. Results
Figure 1 displays the schematic structure of a clinical iron
oxide NP coated with dextran (Feridex, Figure 1A1) and two
FePt NPs: the face-centered-cubic (fcc) FePt NP surface
functionalized with cysteamine (fcc-FePt-A, Figure 1B1) and
the fcc FePt NP coated with silica and surface-functionalized
with (3-aminopropyl)triethoxysilane (fcc-FePt-silica-A, Figure
1C1). The preparation of the family of FePt NPs is described
in the Supporting Information.27,30,17,41
In agreement with published literature,42 transmission electron
microscopy (TEM) revealed that the commercial iron oxide NPs
samples (Figure 1A2) are composed of aggregated crystals
approximately 5 nm in diameter, embedded in an organic
coating. These aggregates form large particles of a size
distribution ranging from 20 to >50 nm. In contrast, the fcc-
FePt-A NPs are well dispersed, with diameters of ∼5 nm (Figure
1B2). The fcc-FePt-silica-A NPs are coated with a homogeneous
silica shell of ∼17 nm in thickness, leading to a total diameter
of ∼40 nm (Figure 1C2).
The powder X-ray diffraction (XRD) pattern of the Feridex
iron oxide confirms the presence of magnetite (Fe3O4), the broad
peaks at 38°, 45°, and 55° corresponding to the (220), (311),
and (400) peaks, respectively (Figure 1A3). Figure 1B3,C3
shows peaks around 51° and 60°, characteristic of fcc-FePt (111)
and (200) peaks, respectively. The (111) peak position suggests
that the NPs’ composition includes between 40 and 45% of Fe
(Table 1). For the silica-coated NPs, the SiO2 XRD characteristic
peak is observed at ∼28° (Figure 1C3). The crystal size is ∼3.6
nm for both fcc-FePt-A and fcc-FePt-silica-A NPs (Table 1).
Zero-field-cooling and field-cooling (ZFC-FC) of Feridex
samples highlight a 45 K blocking temperature (Tb, Figure 1A4).
At 2 K, i.e., lower than Tb, Feridex hysteresis loop measurements
display ferrimagnetic behavior with a small coercivity value of
300 Oe and a saturation moment (Ms) of 101 emu/g of Fe. At
300 K, Feridex is superparamagnetic with no coercivity, as
illustrated in Figure 1A5. The Ms value is 82 emu/g of Fe, in
agreement with the literature.16,42 In comparison, the fcc-FePt-A
NPs have a higher Tb of ∼90 K (Figure 1B4). More importantly,
when compared to Feridex, the Ms of fcc-FePt-A NPs is ∼2
times larger at 2 K and 35% stronger at 300 K (Figure 1B5,
Table 1). After silica coating, the blocking temperature is
reduced to 50 K, and the ZFC slope below Tb is steeper (Figure
(41) Koole, R.; van Schooneveld, M. M.; Hilhorst, J.; Donega, C. D.; Hart,
D. C.; van Blaaderen, A.; Vanmaekelbergh, D.; Meijerink, A. Chem.
Mater. 2008, 20, 2503–2512.
(42) Jung, C. W.; Jacobs, P. Magn. Reson. Imaging 1995, 13, 661–674.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 15023
ARTICLES Chen et al.

Figure 1. Schematic representation (1), TEM images (2), XRD patterns (3), ZFC-FC (4), and hysteresis (5) curves of Feridex (A1-A5), fcc-FePt-A NPs
(B1-B5), and fcc-FePt-silica-A NPs (C1-C5).

Table 1. XRD, SQUID, and MRI Dataa

Ms (emu/g Fe)
sample DXRD (nm) FexPt1-x (%) Tb (K) at 2 K at 300 K Hc at 2 K (kOe) r1b (s-1 mM-1) r2b (s-1 mM-1)
fcc-FePt-A 3.6 ( 0.1 42.7 ( 0.7 90 201 112 0.7 2.5 ( 1.0 887 ( 32
fcc-FePt-silica-A 3.6 ( 0.1 43.8 ( 1.4 50 191 83 1.3 0.3 ( 0.1 210 ( 3
Feridex I.V.c (Endorem) 5.3 ( 0.1 - 45 101 82 0.3 0.9 ( 0.1 148 ( 2
a
DXRD is the crystalline grain size of the NPs; in FexPt1-x, x is the composition based on the FePt lattice constant (SI-Figure 1);43 Tb is the blocking
temperature; Ms is the saturation moment; Hc is the magnetic coercivity of the NPs; and r1 and r2 are the 1H relaxivity values for 1% agarose gel
containing Feridex, fcc-FePt-A, and fcc-FePt-silica-A NPs. b A range of other commercial MRI contrast agent relaxivity values are presented in the
Supporting Information (SI-Table 2). c Trademark of AMAG Pharmaceuticals, Lexington, MA.

15024 J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010

Engineered Biocompatible Nanoparticles ARTICLES

Figure 2. Viability of A375M cells incubated with fcc-FePt-A NPs (A). Fe released from fcc-FePt-A NPs (orange [) in pH 4.8 PBS (B1). Fe released from
fcc-FePt-A NPs (orange [) and from Feridex (4)49 in solutions at pH ∼4.8 of RPMI-1640 cell media containing 20 mM sodium citrate. The incubation
concentrations for FePt and Feridex49 were 120 and 25 µg/mL, respectively (B2). Photomicrographs (magnification ×40) of Prussian blue-stained A375M
cells cultured on coverslips in cell media (C1), media containing 30 µg/mL of fcc-FePt-A NPs (C2), and media containing 30 µg/mL of Feridex (C3) after
overnight incubation. TEM images of A375M cells after 16 h incubation in media containing 30 µg/mL of fcc-FePt-A NPs (D1-D3).

1C4). At 2 K and 5 T, the magnetization of fcc-FePt-silica-A
NPs is ∼5% lower than that of fcc-FePt-A and 2 times larger
than that of Feridex. At 300 K and 5 T, the magnetization of
fcc-FePt-silica-A NPs is ∼36% lower than that of fcc-FePt-A
NPs and similar to that of Feridex (Figure 1C5, Table 1).
Cytotoxicity of the NPs was evaluated in an A375M cell line
using fcc-FePt-A NPs and MTS assay with various concentra-
tions and incubation times.43 Both dose-response and time
course studies were performed (Figure 2A). As expected,44,45
and in contrast with bare iron oxide,46,47 Feridex’s dextran
organic coating does not lead to any detectable cytotoxicity.
At 120 µg/mL, fcc-FePt-A NPs exhibited cytotoxicity that
increases with incubation time. At a concentration of 60 µg/
mL, fcc-FePt-A NPs incubated with cells for 7 days showed a
∼15% loss of cell viability. Significantly, there is no loss of
cell viability at concentrations of 30 µg/mL and below, even
after prolonged incubation periods of up to 7 days. Similar
(43) See Supporting Information for details.
(44) Wang, L. J.; Wang, Z. G.; Frank, T. G.; Brown, S. I.; Chudek, S. A.;
Cuschieri, A. Nanomedicine-UK 2009, 4, 305–315.
(45) Dobrovolskaia, M. A.; McNeil, S. E. Nat. Nanotechnol. 2007, 2, 469–
478.
(46) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26–49.
(47) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26, 1565–1573.
(48) Skotland, T.; Sontum, P. C.; Oulie, I. J. Pharmaceut. Biomed. 2002,
28, 323–329.
results were obtained with fcc-FePt-A NPs and both MCF7 and
U2OS tumor cell lines (SI-Figure 5A,B).43
To confirm these results, the chemical stability of fcc-FePt-A
NPs in an acidic environment was investigated by incubation
of fcc-FePt-A in pH 4.8 buffer solutions. The concentration of
FePt NPs incubated in buffer solution is ∼120 µg/mL, to match
the highest NPs concentration used in the cytotoxicity study
(Figure 2A). In a pH 4.8 phosphate buffered saline (PBS),
cysteamine-coated FePt NPs show excellent chemical stability,
with only ∼0.6% Fe, i.e. <0.1 µg/mL, of iron released after 7
days of incubation (Figure 2B1). As suggested by the recent
literature,48,49 an in Vitro lysosomal model, i.e. RPMI-1640 cell
media containing 20 mM sodium citrate (pH 4.8), was also used
to match the metabolic conditions. In these more extreme
conditions (Figure 2B2), more Fe was releaseds0.3 µg/mL after
6 h and up to ∼3.3 µg/mL after 7 dayssbut importantly it
remained at a level comparable to the release observed with
Feridex.
The high chemical stability of fcc-FePt-A was investigated
further. Prussian blue staining experiments were carried out and
showed that FePt NPs have greater stability compared to Feridex
(Figure 2C1-C3).43 Indeed, photomicrographs obtained after
overnight incubation show no stain for both blank control sample
(Figure 2C1) and cells labeled with fcc-FePt-A NPs (Figure
(49) Arbab, A. S.; Wilson, L. B.; Ashari, P.; Jordan, E. K.; Lewis, B. K.;
Frank, J. A. NMR Biomed. 2005, 18, 383–389.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 15025
ARTICLES Chen et al.

Figure 3. T2-1 (s-1) vs [Fe] (mM) of the water in 1% w/v agarose gel containing (blue b) fcc-FePt-A, (green 2) fcc-FePt-silica-A, and (orange [) Feridex
(A1). 1H longitudinal relaxation time T2-weighted MRI images of 1% agarose gel/water solution containing fcc-FePt-A NPs with [Fe] ) 0.00 mM (a), 0.02
mM (CFePt ) 6 µg/mL) (b), and 0.04 mM (CFePt ) 12 µg/mL) (c) (A2). T2-weighted cellular imaging by MRI (3D spin echo, TE ) 30 ms, TR ) 1 s) of
A375M cells loaded with fcc-FePt-A NPs with a cell density of 10 × 103 cells/mL (a), 100 × 103 cells/mL (b), and 1000 × 103 cells/mL (c) (B). Embryo
injected in oVo with cell culture media (1 µL) containing no NPs (C) and 20 µg/mL fcc-FePt-A (D). 3D surface reconstruction of embryo eyes (blue) and
blood vessels (red) showing the position in yellow of the transverse and coronal images (C1,D1); dorsal view of transverse image of the embryo’s head
aligned through the eyes (C2,D2); and anterior view of coronal image of the embryo’s head aligned through the eyes (C3,D3). 2D slices from 128 × 128
× 128 3D Rare-8 MRI data set of day 4 quail embryo egg (TR/TE ) 250/25 ms), field of view of 30 mm and pixel dimensions of 0.234 mm/pixel. Labels:
y, yolk; a, albumen; b, brain; in-e, injected eye; un-e, un-injected eye; h, heart. Scale bar indicates 1 mm. All the MRI measurements were completed at 7.1
T.

2C2). In contrast, Feridex-labeled cells show significantly
staining (Figure 2C3) characteristic of the release of Fe in the
cells. These results confirm that the cysteamine coating provides
fcc-FePt-A NPs with high chemical stability.
The cysteamine coating is positively charged, resulting in
higher cellular uptake by human non-phagocytic A375M tumor
cells (Figure 2D1-D3) compared to Feridex. FePt NPs appear
to be internalized by an endocytotic pathway. Indeed, TEM
studies suggest a macropinocytosis cellular uptake mechanism
by the large protrusions of the plasma membrane around NP
clusters (Figure 2D2).50,51 The intracellular localization route
is endosome, early lysosome, and late lysosome, which are basic
cell biology phenomena. Both endosomes and lysosomes are
confined subcellular organelles, and, as both expected and
(50) Conner, S. D.; Schmid, S. L. Nature 2003, 422, 37–44.
(51) Kirkham, M.; Parton, R. G. Biochim. Biophys. Acta 2005, 1745, 273–
286.
15026 J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010
commonly observed in other NPs systems, the FePt NPs appear
typically “entrapped” inside these subcellular vesicles (Figure
2D3).
The effectiveness of fcc-FePt-A and fcc-FePt-silica-A as MRI
contrast agents was investigated by measuring the dependence
of the longitudinal relaxation rate (T1-1) and transverse relax-
ation rate (T2-1) of 1% agarose gels.43 Relaxivity (ri) quantifies
the enhancement of either the longitudinal or the transverse
relaxation rates of the protons in water. As expected at high
magnetic field, the FePt NPs and Feridex have a weak influence
on the water 1H longitudinal relaxation rate (Table 1 and
SI-Figure 7A).16,43 In contrast, FePt NPs have a significant effect
upon the aqueous transverse relaxation rate (Figure 3A1). The
relaxivity r2 of the fcc-FePt-silica-A NPs is 40% larger than
that of Feridex, which is attributed to the stronger magnetic
moment of the fcc-FePt-silica-A NPs (SI-Figure 7B) at the MRI
magnetic field (7 T).43 Importantly, the relaxivity r2 of the fcc-
FePt-A NPs is more than 6 times larger than that of Feridex.
Engineered Biocompatible Nanoparticles ARTICLES

Table 2. Summary of FePt Nanoparticles Cytotoxicity Studies Reported in the Literaturea

cytotoxicity
b
material Fe precursor coating cells [NPs] (µg/mL) [Fe] (µg/mL) tinc (h) cell damage (%) ref
FePt-CoS2 Fe(CO)5 CoS2 shell HeLa 1.5 ∼0.007 24 50 39
FePt-Cys Fe(CO)5 cysteine HeLa 5 ∼0.7 24 ∼10 39
15.5 ∼2.5 24 50
FePt-Fe2O3 Fe(CO)5 Fe2O3 shell, L-DOPA coating HeLa ∼8.3 ∼0.045c 24 50 37
∼3.8d
FePt-Fe3O4 Fe(CO)5 Fe3O4 shell HeLa 40 e 24 ∼10 37
10 e 72 ∼10
20 e 72 ∼30
FePt-COOH Fe(CO)5 bilayer of phospholipid HeLa ∼10.7 1.72 24 ∼40 52
(DSPE-PEG(2000) carboxylic acid lipid) A431 ∼10.7 1.72 24 ∼40
and oleic acid/oleylamine SK-BR3 ∼10.7 1.72 24 ∼20
HEK-293 ∼10.7 1.72 24 ∼30
A2780 ∼7.8 1.25 24 50
FePt-SiO2-A Na2Fe(CO)4 SiO2 shell A375M, MCF7, U2OS 200 ∼2 168 0 f
FePt-A Na2Fe(CO)4 cysteamine A375M, MCF7, U2OS 30 ∼5.3 168 0 f
60 ∼10.5 72 ∼10
a
Listed are NPs’ structure, precursors, and coating; “cells” stands for the cell line; [NPs] is the nanoparticle concentration; tinc is the incubation time;
cell damage is that after incubation. b Pt precursor always Pt(acac)2. c Fe contribution from Fe2O3 shell not included. d Fe contribution from Fe2O3 shell
included. e [Fe] cannot be calculated. f Data presented in this work.

Figure 3A2 displays T2-weighted MRI images of various
concentrations of fcc-FePt-A NPs in agarose gels. The signal
intensity reduces and the image appears darker as the NPs
concentration increases. The FePt NPs produce an observable
change in image contrast at concentrations as low as 6 µg/mL,
which is well within the safety range indicated by the cytotox-
icity data (Figure 2A). Similarly, signal intensity loss was
observed in cells labeled with fcc-FePt-A NPs as the cell density
increases from 10 × 103 to 1000 × 103 cells/mL (Figure 3B).
The in ViVo potential of FePt NPs as MRI contrast agents
was demonstrated by injecting NPs in cell culture media into
the eyes of day 4 quail embryos in oVo. Figure 3C,D displays
3D MRI image data sets after injection. The transverse and
coronal images through the embryo’s head are presented. The
uninjected eye (un-e) acts as an internal control. There is no
difference in the contrast between the uninjected and the
uppermost eye (in-e) which had been injected with a 1 µL of
culture medium only (Figure 3C2,C3). However, when the
injection medium contained 20 µg/mL of fcc-FePt-A NPs, a
very distinct hypointensity appears in the injected eye (Figure
3D2-D3). This was consistently observed when varying the
NPs concentration.
This thus demonstrates the capability of nontoxic FePt NPs
as potent MRI probes for cellular imaging, even at very low
NP concentrations. Labeling the cells and these magnetic NPs
will allow, for instance, cell migration to be studied, with their
biodistribution and behavior monitored in ViVo, especially as
we have demonstrated that the effect of contrast agents can be
observed at non-cytotoxic concentrations lower than 30 µg/mL.
3. Discussion
The major hurdle preventing the wider use of FePt NPs in
biomedicine is their potential toxicity. In the literature, there
are many inconsistencies in FePt cytotoxicity, leaving the subject
rather confused. A summary of the cytotoxicity data of FePt
NPs is presented in Table 2. While it was reported that 24 h
incubation of FePt-cysteine NPs had no significant toxicity at
concentrations below 5 µg/mL, these compounds resulted in
50% cell damage at 15.5 µg/mL;39 FePt-Fe3O4 NPs showed no
significant cell viability damage up to 3 days of incubation at
10 µg/mL.37 In contrast, FePt-Fe2O3 and FePt-CoS2 were
reported to induce significant cell viability damage of over 50%
even at concentrations of NPs as low as 1.5 and ∼8.3 µg/mL,
respectively.37,39 A recent toxicity study with phospholipids-
coated fcc-FePt NPs showed 50% cell viability damage above
1.25 µg/mL Fe concentration (∼7.8 µg/mL Fe40Pt60 NPs).52 In
this latter study, the high toxicity was attributed to fast Fe release
from phospholipids-coated fcc-FePt NPs. It was reported that
∼6% Fe was already released after 6 h of incubation, and the
percentage of Fe released increased to ∼20% after 24 h of
incubation in pH 4.8 PBS.52 While iron is essential for almost
all living organisms, it is toxic. It can act as a catalyst in the
Fenton reaction, which generates a wide range of free radical
species, including hydroxyl radicals. These are among the most
reactive free radical species known and have the ability to react
with a wide range of cellular constituents, including pyrimidine
bases of DNA, and also to trigger lipid peroxidation in cellular
membranes, leading to cell death.53
In this study, the fcc-FePt-silica-A NPs have good biocom-
patibility and, as expected, the silica coating forms an inert shell
(SI-Figure 5C).43 In contrast to previously published results,
our study demonstrates that cysteamine-coated fcc-FePt NPs
also have very high chemical stability in pH 4.8 PBS. Fe release
after 24 h of incubation is only ∼0.3%, with only a slight
increased to 0.6% after 7 days of incubation (Figure 2B1). In
an in Vitro model (i.e., cell medium containing sodium
citrate),48,49 fcc-FePt-A NPs were found to release as little Fe
as Feridex (Figure 2B2), with ∼5% Fe found to be released
after 24 h and 20% after 7 days. This confirms that the low pH
in the endosome/lysosome environment is not sufficient to
dissolve the NPs and that the presence of endogenous citrate
or similar metallic chelates is instrumental to the NPs degrada-
tion by binding to solubilized Fe atoms.48,49 The results
presented herein are significant as they suggest that, when
considering the Fe release, the safety of fcc-FePt-A NPs is
comparable to that of Feridex under similar experimental
conditions. The chemical stability of the fcc-FePt-A NPs is also
supported by the Prussian blue staining experiments (Figure
2C1-C3).
(52) Xu, C.; Yuan, Z.; Kohler, N.; Kim, J.; Chung, M. A.; Sun, S. J. Am.
Chem. Soc. 2009, 131, 15346–15351.
(53) Crichton, R. R.; Wilmet, S.; Legssyer, R.; Ward, R. J. J. Inorg.
Biochem. 2002, 91, 9–18.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 15027
ARTICLES
The high chemical stability of cysteamine-coated FePt NPs
certainly contributes to the NPs’ noteworthy low cytotoxicity.
Of course, iron release is only one parameter that contributes
to toxicity. It is quite possible that differences in the NPs
preparation, surface coating, ligand exchange, and extraction
protocols could account for the spread of the cytotoxicity data
reported in the literature. The toxicity may not necessarily be
due to the material itself through the release of its constituent.
In the present study, we used Collman’s reagent, Na2Fe(CO)4,
to control the FePt NPs stoichiometry and to ensure the
simultaneous presence of Fe and Pt species during the formation
of FePt alloy NPs; the use of Fe(CO)5 has been shown to form
NPs with a core and a shell rich in platinum and iron,
respectively.31 After ligand exchange, the extraction process was
repeated up to six times to ensure removal of hazardous
chemicals such as chloroform, oleic acid, and oleylamine, while
the thiol group provides a strong bond between the cysteamine
and the NPs’ surface, preventing the ligands from becoming
lethal. During the review of this publication, cysteamine-coated
FePt NPs were reported to induce, after 24 h, very low cell
damage on Veron cells;38 however, it is noticeable that the
strong bonding to the surface of the NPs, protecting them from
the cells’ environment, can be contrasted with, for instance, a
coating made with a bilayer structure of phospholipids and oleic
acid/oleylamine.52 Indeed, even though this phospholipid ap-
proach provides aqueous solubility, it is also characterized by
a weaker bonding strength and a more dynamic behavior,
allowing the coating to slowly disperse in the cell. By doing
so, the “free” ligands can alter the cell viability as well as the
release of the NP constituents as the surface of the particles is
exposed to the cell, and the NPs’ chemical integrity can be
compromised further. Such outcomes have been reported by
Xu et al., although they elegantly focused on the opportunity
this provided to use the NPs as metal reservoirs to kill cancer
cells.52
While the very high chemical stability of fcc-FePt-A NPs
was demonstrated in this report, cytotoxicity is observed at high
concentrations above 30 µg/mL FePt and long incubation times.
This could be explained by contributions from (i) the very small
release of Fe, which has been found to be <1% after 168 h and
(ii) the high concentration of positive surface charges, which
could also interact with the more negatively charged cell
membranes and induce membrane disruption.54 In addition,
ligands with strong anchoring groups can reduce the toxicity,
as illustrated in this publication, and it is noticeable that the
present results were obtained with commercially available
ligands (i.e. cysteamine), so one should expect enhanced
viability from, for instance, custom-made dithiolate ligands, such
as those developed recently for quantum dots.55,56 These could
provide cell viability at NP concentrations higher than 30 µg/
mL.
In this context, the little consistency observed when consider-
ing FePt NPs’ cytotoxicity data available in the literature stresses
that the measured toxicity should always be associated with the
nanocolloids, i.e. inorganic NPs + organic stabilizer. The
interplay between the components will indeed contribute to
the cytotoxicity results, with, for instance, a weak bonding to
the NPs’ surface increasing the probability of the nanocolloids
(54) Bilensoy, E. Exp. Opin. Drug DeliV. 2010, 7, 795–809.
(55) Han, G.; Mokari, T.; Ajo-Franklin, C.; Cohen, B. E. J. Am. Chem.
Soc. 2008, 130, 15811–15813.
(56) Mei, B. C.; Susumu, K.; Medintz, I. L.; Mattoussi, H. Nat. Protoc.
2009, 4, 412–423.
15028 J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010
Chen et al.
being toxic. It then appears that, to be able to compare
cytotoxicity data in a meaningful way, one must to develop and
use standardized approaches including the correlation between
(i) the evolution of the nanocolloids’ integrity, relying on both
the inorganic NPs and the organic stabilizers, and (ii) the cell
viability. It should be noticed that, in the context of apparently
toxic NPs, the nanocolloids’ solution stability could easily be
measured by, for instance, time-resolved transmission monitored
sedimentation experiments, as while the ligands come off the
NPs’ surface, the NPs tend to aggregate and fall out of solution.
The cellular internalization of Feridex is poor due to the weak
binding of the neutral dextran coating to the plasma membrane,
which limits the capability of cell internalization by the fluid-
phase endocytosis pathway.57,58 Enhancing cellular uptake of
dextran-coated NPs is possible with transfection agents and
further surface functionalization but was not relevant to the
present study.44 In contrast, the amine-coated FePt NPs, fcc-
FePt-A and fcc-FePt-Silica-A, are efficiently internalized directly
without requiring the use of any external mediation such as
transfection agents or electroporation, illustrated with non-
phagocytic human cells in Figure 2D1. The uptake appears to
be mediated via macropinocytosis, a nonspecific endocytic
process, which involves the internalization of large areas of
plasma membrane together with significant amounts of fluid
(Figure 2D2). Irregular macropinosome vesicles larger than 1
µm are usually generated when membrane protrusions fuse back
to the plasma membrane (SI-Figure 6A).43,50,51 Other pathways
could be involved, e.g., lipid raft-dependent mechanisms or the
clathrin-dependent pathway;50,51,59,60 however, we did not
observe any evidence supporting their involvement. Further
studies are needed to clarify the exact cellular internalization
mechanism. However, and as expected once inside the cells,
the FePt NPs that are stabilized by Coulombic interactions
appear to be confined in specific subcellular organelles, most
likely endosomes and lysosomes (Figure 2D3). This is of
significant importance when developing FePt NPs-based bio-
medical platforms and can be attributed to the interplay between
the cationic surface of the NPs and the negatively charged cell
membrane.
By considering a family of FePt NPs, we established that,
despite the attractiveness of the silica coating, it weakens the
NPs’ magnetic moment and their applicability in clinical MRI.
This may be the result of a reduction of the magnetic effective
volume and/or an alteration of the material. In support of this
interpretation, the strong base environment associated with the
silica coating can partially oxidize the FePt surface, resulting
in a thin layer of softer magnetic material like iron oxide or
iron silicide, which because of its thinness is not visible by XRD
or TEM.61,62
The fcc-FePt-A NPs demonstrated a very high MRI relaxivity
r2, which can be attributed to the combination of (i) an
intrinsically high magnetic moment, (ii) a thin organic coating,
(57) Arbab, A. S.; Bashaw, L. A.; Miller, B. R.; Jordan, E. K.; Lewis,
B. K.; Kalish, H.; Frank, J. A. Radiology 2003, 229, 838–846.
(58) Arbab, A. S.; Yocum, G. T.; Wilson, L. B.; Parwana, A.; Jordan, E. K.;
Kalish, H.; Frank, J. A. Mol. Imaging 2004, 3, 24–32.
(59) Dausend, J.; Musyanovych, A.; Dass, M.; Walther, P.; Schrezenmeier,
H.; Landfester, K.; Mailander, V. Macromol. Biosci. 2008, 8, 1135–
1143.
(60) Raffa, V.; Ciofani, G.; Vittorio, O.; Riggio, C.; Cuschieri, A.
Nanomedicine 2010, 5, 89–97.
(61) Lee, D. C.; Mikulec, F. V.; Pelaez, J. M.; Koo, B.; Korgel, B. A. J.
Phys. Chem. B 2006, 110, 11160–11166.
(62) Thomson, T.; Terris, B. D.; Toney, M. F.; Raoux, S.; Baglin, J. E. E.;
Lee, S. L.; Sun, S. J. Appl. Phys. 2004, 95, 6738–6740.
Engineered Biocompatible Nanoparticles
and (iii) their small size, resulting in effective dispersion across
a sample. While this combination created very effective T2 MRI
contrast agents even at µg/mL concentrations, these results also
underline the importance of engineering the interface of NPs
and demonstrate that a strong magnetic moment such as that of
FePt, combined with a thin coating layer having an open
molecular structure allowing very close aqueous proximity, are
essential parameters for the design of new generations of
diagnostic and therapeutic MRI contrast agents.
Finally, we demonstrate that FePt NPs can be successfully
used as a potent cellular and in ViVo MRI probes. This is
illustrated by both cell MRI (Figure 3B) and distinct hypointen-
sity of ∼1 mm in size observed in the in ViVo environment into
which the FePt NPs were injected (Figure 3D and SI-Figure 8).43
Their more than 6 times higher relaxivity r2 allows using less
NPs than with iron oxide contrast agents such as Feridex, which
makes FePt NPs very promising contrast agents to identify small
lesions such as lymph node metastases.
4. Conclusion
In conclusion, we have presented the synthesis and charac-
terization of a family of FePt NPs: fcc-FePt-A NPs and fcc-
FePt-silica-A NPs. The reduction of magnetic properties ob-
served with both SQUID magnetometry and MRI relaxivity of
silica-coated NPs is attributed to the formation of very thin layers
of oxides and silicides. In sharp contrast with some previous
reports, our biocompatibility studies clearly demonstrate and
explain the absence of cytotoxicity at concentrations below 30
µg/mL, even after 7 days of incubation. The biocompatibility
is mainly attributed to the strong surface coating and the
extraction protocols. Another striking feature for cellular imag-
ing is the ability of the FePt NPs to efficiently enter tumor cells
without requiring any external mediation. The cellular uptake
by non-phagocytic tumor cells occurs via an apparent macro-
ARTICLES
pinocytosis mechanism. The 7.1 T MRI studies, in Vitro and in
ViVo, have confirmed that both fcc-FePt-A and fcc-FePt-silica-A
NPs are stronger T2 contrast agents than commercial Feridex,
with fcc-FePt-A T2 relaxivity (r2) being more than 6 times larger.
The superior capability of MRI contrast enhancement associated
with the FePt NPs makes them an ideal platform for the design
of diagnostic and therapeutic agents for image-guided magnetic
drug delivery and hyperthermic tumor ablation. The findings
of this study underline the importance of engineering magnetic
NP interfaces combining intrinsically strong magnetic moment
with coating layers. Finally, our studies have also demonstrated
that FePt NPs can be used successfully for cellular and in ViVo
imaging, thus confirming the potential of FePt NPs in regenera-
tive medicine and stem cell therapy.
Acknowledgment. The authors gratefully acknowledge the
Scottish Universities Physics Alliance for funding of a SUPA
Advanced Research Fellowship (P.A.) and the James and Enid Nicol
Trust for funding a studentship (S.C.). The authors thank Dr. Lorna
Eades (EaSt CHEM, University of Edinburg) for the ICP-OES
measurements, Prof. Kate Storey (University of Dundee) for use
of the microinjection equipment, Dr. Steve Francis (University of
St Andrews) for the FTIR measurements, and Profs. David Cole-
Hamilton and Peter Bruce (University of St Andrews) for discussion
and access to instruments. S.L.D. acknowledges the Wellcome Trust
(WT081039) for funding.
Supporting Information Available: Experimental section
including the NPs synthesis and characterization, protocols,
cytotoxicity, cellular uptake, and MRI application data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA106543J
J. AM. CHEM. SOC. 9 VOL. 132, NO. 42, 2010 15029
 Engineered Biocompatible nanoParticles for in vivo Imaging Applications

Shu Chen,a,b Lijun Wang,c Suzanne Duce,d Stuart Brown,c Stephen Lee,a Andreas Melzer,c Sir Alfred Cuschieri,c Pascal André a*
a
School of Physics and Astronomy (SUPA), University of St Andrews, St Andrews KY16 9SS (UK)
b
School of Chemistry (EaStChem), University of St Andrews, St Andrews KY16 9ST (UK)
c
Institute for Medical Science and Technology, University of Dundee, Dundee DD2 1FT (UK)
d
College of Life Sciences, University of Dundee, Dundee DD1 5EH (UK)

* Pascal.Andre@st-andrews.ac.uk, a.cuschieri@dundee.ac.uk

Supplementary Information

SI-I. Experimental Section surface was further functionalized with (3-aminopropyl)

SI-I.1. FePt nPs preparation
Materials: All chemical reagents, unless otherwise stated,
were purchased from Sigma, used without further purification but
degassed before use: Disodium tetracarbonylferrate-dioxane
complex (Na2Fe(CO)4•1.5 C4H8O2), platinum(II) acetylacetonate
(Pt(acac)2, 97 %), oleylamine (70 %), oleic acid (90 %),
cysteamine (CA, HSCH2CH2NH2, BioChemika, ≥ 98.0 %),
tetraethylorthosilicate (TEOS, Si(OC2H5)4, GC, ≥ 99.0 %), (3-
Aminopropyl)triethoxysilane (APTES, H2N(CH2)3Si(OC2H5)3, ≥
98 %), IGEPAL® CO-520 (NP-5, (C2H4O)n•C15H24O, n ~ 5).
When available ACS grade was chosen to select the solvents:
dibenzyl ether (≥ 98.0 %), hexane (99.0 %), ethanol (≥ 99.5 %),
chloroform (≥ 99 %, anhydrous), methanol (≥ 99.8 %), cyclo-
hexane (GC, ≥ 98.0 %), ammonium hydroxide aqueous solution
(NH4OH, 28.0 - 30.0 %).
FePt nPs synthesis: all the syntheses were carried out inside a
glove box. A mixture of Pt(acac)2 (1 mmol), oleyl amine (8
mmol) and oleic acid (4 mmol) in 10 mL of dibenzyl ether was
placed in a 50 mL round bottom flask connected to a condenser.
Under stirring, the mixture was heated up to 100 °C for 1 h to
remove oxygen and moisture, then Na2Fe(CO)4 (1 mmol) in 10
mL dibenzyl ether mixture was added and heated up to 150 °C for
1h. The mixture was further heated up to reflux ~ 300 °C to for 3
h. The dark solution was cooled down to room temperature and
after washing with hexane and ethanol, the nPs were collected by
centrifugation.1-2
fcc-FePt-A: Ligand exchange experiments were carried out in
a glove box according to a modified protocol based on the
literature.3 For a typical experiment, 30 mL hexane was added
into 3 mL (13.5 mg/mL) of as-synthesized oleic acid/ oleylamine
coated fcc-FePt hexane solution. To remove the excess of ligands,
the nPs were precipitated by addition of 60 mL ethanol and
collected by centrifugation. The nPs were redispersed in 15 mL of
chloroform assisted with sonication. 15 mL of cysteamine in
methanol solution (0.5 M) was added dropwise to the FePt
NPs/CHCl3 mixture under vigorous stirring. The final solution
was left under stirring for 48 h to complete the ligand exchange.
To extract the nPs, they were redispersed in 10 mL ethanol and
sonicated, 20 mL hexane was added and the solution was
centrifuged. This process was repeated twice to remove CHCl3,
methanol and the excess of ligands. The nPs were then
redispersed in 10 mL ethanol and 20 mL deionized water mixture,
sonicated and collected by centrifugation. This step was repeated
6 times to obtain positively charged nPs stable in deionized water
for more than 8 months (SI-Figure 1).
triethoxysilane.4 Reverse microemulsions were prepared by
mixing under vigorous stirring 10 mL cyclohexane, 1.3 mL NP-5
and 50 µL DI H2O. 2 mg (~ 3-4 nmol) of FePt nPs were then
dispersed in 1 mL cyclohexane and added dropwise into the
reverse microemulsion. After 15 min, 80 µL TEOS was added
dropwise. After another 15 min, 150 µL NH4.H2O (28-30 %) was
added dropwise. The solution was kept under constant stirring at
for 72 h. To form amine functionalized FePt-silica nPs, 100 µL of
APTES was added after 48 h and kept stirring for another 24 h.
The nPs were precipitated by centrifugation after addition of 3
mL of ethanol and 2 mL of methanol. The nPs were redispersed
in 5 mL of ethanol and precipitated by centrifugation after
addition of 10 mL of hexane. This step was repeated up to 6 times
to completely remove the surfactant. FePt-silica nPs were stable
both in ethanol and DI water, while FePt-silica-A nPs were stable
in deionized water.
SI-I.2. Experimental Techniques
TEM: Transmission electron microscopy (TEM) images were
recorded using a Gatan CCD camera on a JEOL JEM-2011
electron microscope operating at 200 kV. The chemical
composition of FePt nPs was examined with energy-dispersive X-
ray spectroscopy (EDX) using an Oxford Link system installed in
the JEM-2011 microscope.
XRD: Wide-angle powder X-ray diffraction (XRD) data were
collected on a Stoe STADI/P powder diffractometer operating in
transmission mode and with a small angle position sensitive
detector. Incident radiation was generated using a FeKα1 source
(λ=1.936 Å). The strongest (111) peak was fitted with Lorentzian-
shaped peaks using STOEwinXpow and Kaleida-Graph softwares
to precisely determine the diffraction peak positions. The
crystalline grain size, DXRD, of the FePt nPs was calculated
according to Scherrer’s formula:5
0.9λ
D XRD = (eq. 1)
B cos θ
where DXRD is the “ average” dimension of the crystallites, λ is
the wavelength of the X-ray source (for Fe source is equal to
0.193604 nm), B is the full width at half maximum of the peak
intensity, θ is the glancing angle.
The atomic composition of FePt NPs was calculated based on
the linear relations between lattice constant, a, and Fe percentage,
Fe %, reported by Bonakdarkpour et al.6
From SI-Figure 1, the following equations can be obtained:
x%Fe < 40 % a = - 0.0014 x%Fe + 3.929 SI-eq. (1)
x%Fe > 40 % a = - 0.0041 x%Fe + 4.039 SI-eq. (2)

fcc-FePt-silica-A: The fcc-FePt-silica-A nPs were prepared by

hydrolysis of tetraethylorthosilicate (TEOS) and the silica shell

S. Chen et al. March-July 2010 S 1/6

 3.95
3.90
a (Angstrom)
3.85
3.80
3.75
Fe Pt (%)
x 100-x
3.70
0 20 40 60 80 100
SI-Figure 1. FePt lattice constant (a) analysed by XRD vs. composition
curve reported by Bonakdarkpour et al. 6
SQUID: A 5.0 Tesla Superconducting Quantum Interference
Device (SQUID) from Quantum Design (MPMS XLTM) was used
to characterize the nPs magnetic properties. The nPs were
dispersed in polyvinylpyrrolidone matrix (VPolymer/VnPs = 20) to
prevent interaction between the nPs while the resulting sample
was loaded into a low background gelatin capsule. Zero-Field
Cooled and Field Cooled (ZFC/FC) measurements were
completed as follow: the sample was first cooled from room
temperature to 2 K without any external field, next a small field
100 Oe was applied and the nPs magnetization was recorded as
the temperature was increased up 275 K. The FC curve was
obtained by cooling the sample back to 2 K under a 100 Oe
magnetic field. The magnetization was then measured while the
temperature was increased up to 275 K. Hysteresis measurements
were completed at temperatures of 2 K and 300 K. The
magnetization of the gelatine capsules and the PVP matrix was
subsequently subtracted.
ICP-OES: FePt nPs were dissolved by addition of
hydrochloric acid (Trace SELECT®, ≥ 37 %) and nitric acid
(Trace SELECT®, ≥ 69.0 %) mixed in 3:1 as volume ratio and left
overnight at room temperature. After full dissolution of the nPs,
the solution was further diluted with deionised water to prepare
inductively coupled plasma-atomic emission spectrometry
measurement (ICP-OES, Perkin Elmer Optima 5300 DV).
The conversion of the weight concentration of FePt metal core
into Fe molar concentration was completed according to the
following equation:
C R
[Fe] = FePt - A Fe (eq. 2)
M w- Fe
where [Fe] (mM) is the molar concentration of iron, CFePt (µg/mL)
is the FePt core concentration, Mw-Fe the molecular weight of the
iron atom, RFe is the mass ratio of Fe in FePt metal core. The mass
of the ligand was neglected. The FexPt1-x nPs composition was
found to be x ~ 0.43 by XRD and ICP-OES. The mass ratio of Fe
in the nP core (RFe ~ 0.18) was calculated with the equation below:
xM w- Fe
RFe = (eq. 3)
xM w- Fe + (1 − x )M w - Pt
A similar approach as described by equation 4 was used for
fcc-FePt-silica-A nPs:
C R'
[Fe] = FePt -silica - A Fe (eq. 4)
M w - Fe
where [Fe] (mM) is the molar concentration of iron, CFePt-silica-A is
the concentration of FePt-silica-A in µg/mL, R’Fe is the mass ratio
of Fe in FePt-silica-A nPs which is ~ 0.01 according to eq. 5,
′ = yRFe
RFe (eq. 5)
where y is the mass ratio of FePt in FePt-silica-A nPs, i.e. [FexPt1-
x]y[silica-A]1-y, which was measured experimentally by ICP-OES
and found to be ~ 0.052. As an illustration, according to eq. 4 and
S. Chen et al.
5, the weight concentration of FePt-silica-A nPs at 100 µg/mL
corresponds then to ~ 0.02 mM of Fe in molar concentration.
SI-I.3. Cell culture
Materials: Human breast adenocarcinoma cell line MCF7
(ATCC; Cat# HTB-22), human osteosarcoma cell line U2OS
(ATCC; Cat# HTB-96) were grown in Dulbecco’s Modified
Eagle Medium (DMEM; GIBCO, Invitrogen, Paisley, UK).
Human malignant melanoma cell line A375M was kindly
provided by Dr Daniele Bergamaschi (Institute of Cell and
Molecular Science, Barts and The London, Queen Mary’s School
of Medicine and Dentistry) and was grown in RPMI-1640
medium (GIBCO, Invitrogen). All media were supplemented with
10 % fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin,
and 100 µg/mL streptomycin. Cells were grown under standard
cell culture conditions in 5 % CO2 at 37 °C to reach confluence of
60-70 % before subjected to any further treatments.
Cellular labelling/uptake of FePt nPs and TEM: Cell culture
media were removed and replaced with the media containing
FePt-A (30 µg/mL). For control experiments, media without FePt-
A was used. After 12-16 h of culture, cells were washed and fixed
with 4 % (w/v) paraformaldehyde/2.5 % (v/v) glutaraldehyde
buffered in 0.2 mol/L PIPES as a standard buffer solution. After
30 min in fixative the cells were scraped off the flasks in1 mL of
fixative, transferred to Eppendorf tubes and cetrifuged at 14000 g
for 20 min. Pellets were washed in 0.2M PIPES and post fixed in
1 % aqueous osmium tetraoxide for 1 h, washed in water, and
dehydrated in graded ethanol and propylene oxide before
embedding in Durcupan resin (Sigma) and polymerisation at 60
°C for 24 h. 70 nm sections were cut on a Leica UCT
ultramicrotome, mounted on Pioloform coated 100 mesh copper
grids, stained with uranyl acetate and lead citrate before being
examined in a Tecnai 12 electron microscope. Images were
recorded in Digital Imaging Plates and scanned in a Ditabis
Micron scanner (Pforzheim, Germany).
Cell viability study by MTS assay: The MTS CellTitre 96
AQueous One Solution Cell Proliferation Assay (Promega,
Southampton, UK) was used to determine cell viability in control
and FePt nPs treated cells. This colorimetric assay is based on the
conversion of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy
methoxy-phenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS)
compound to a colored, soluble formazan product in
metabolically active cells. Cells were seeded at densities 1,250-
10,000 cells/well in 96-well plates depending on the duration of
incubation time and cultured overnight. Cell culture media were
then removed and replaced with the media containing the FePt
nPs at various concentrations with the iron content being
determined by XRD and ICP-OES. For control experiments,
media without FePt nPs was used. MTS assay was performed at
specific time intervals by addition of CellTiter 96® AQueous One
Solution reagent to each well, in triplicate, and further incubation
of cells with the reagent for 3 h at 37 °C in 5 % CO2. The colour
development, i.e. absorbance, was read at 490 nm using a plate-
reader (E-Max; Molecular Devices, CA, USA) and absorbance
values of both labelled and control cells were subtracted by that
of blank wells. Cell viability in labelled cells was expressed as
percentage of that from the corresponding control cells.
Fe release experiment: 3.6 mg (~ 0.6 mg Fe) of FePt fcc-
FePt-A nPs in aqueous solution was dispersed in a dialysis tubing
(MWCO 1000, Spectrum Laboratories, Inc.) which was further
immersed into a 30 mL PBS (pH 4.8) or RPMI-1640 (20 mM
March-July 2010 S 2/6
sodium citrate, pH 4.8) bath held at a constant temperature of 37
°C.7-8 Aliquot buffer solution was then sampled at 6, 12, 24, 48,
72, 168 h, and it composition was subsequently analyzed with
ICP-OES.
Prussian blue staining and light microscopy analysis: To
gain insight in intracellular iron ions distribution, cells grown on
glass coverslips or cytospun on glass slides were washed with
PBS to remove the excess of ferumoxides and then fixed with 4 %
para-formaldehyde for 30 min. They were then washed with
double distilled water (ddH2O) and incubated for 30 min with 2.5
% potassium ferrocyanide in 2.5 % HCl to produce Fe(III)
ferrocyanide (Prussian blue). Samples were then counterstained
with nuclear fast red for cell nuclei. Cells were examined using a
Zeiss microscope Axiovert 200 (Zeiss, Oberkochen, Germany) at
x20, x40 and x63 magnification and Axiovision 4.6 software
(Zeiss). Cells were considered Prussian blue positive if
intracytoplasmic blue granules could be identified.
Preparation of Agarose Gel Phantoms for MRI of FePt nPs
and FePt nP-labeled Cells: In the gel MRI study, 1 % (w/v) low
melting point agarose was dissolved in deionized water at 60-70
°C. The desired amount of nPs, i.e. Fe concentration as
determined by XRD and ICP-OES was then added to the solution
to obtain the targeted concentrations (up to 0.25 mM) and mixed
gently. For cellular MRI, FePt labelled melanoma A375M cells
were washed three times with PBS and harvested, suspended in
low melting point agarose gel (1 % in PBS at densities of 104,
105, 106 cell/mL, respectively. Samples were then transferred into
200 µL thin wall tubes. In both cases, samples were rapidly
cooled on ice to form homogenous gel suspensions and kept at 4
°C until further experiments.
Quail Eggs Preparation: Fertilized Japanese quail (Coturnix
japonica) eggs were obtained from Rosedean, Huntingdon,
Cambs. The eggs were then placed vertically with air sac
uppermost in a humidified incubator with a temperature of 38 °C,
this was referred to as Day 0. On Day 4, the shell above the air
sac was pierced and cut away to remove the inner shell membrane
and expose the embryo. The Day 4 embryos typically lay on their
sides, the upper eye is visible allowing the solution containing the
nPs to be injected into it using syringe with thin glass capillary
needle. The top of the eggs were re-sealed with adhesive tape and
then imaged. fcc-FePt-A nPs coated with either cysteamine or
silica were added to cell culture media at concentrations ranging
from 1-100 µg/mL. A control medium without nPs was also used.
SI-I.4. Micro-MRI
Micro-MRI data were acquired on a Bruker Avance FT NMR
spectrometer with a wide bore 7.1 Tesla magnet resonating at
300.15 MHz for 1H, fitted with Bruker micro-imaging magnetic
field gradients. A birdcage radio frequency resonator with an
internal diameter of 30 mm was used. MRI pulse sequences were
taken from the Bruker Paravision® library. All acquisitions were
made at 19 ºC. Two acquisition sequences were collected and
averaged to improve the signal-to-noise ratio and reduce artefacts.
Relaxation measurements were determined from 128 x 128
axial planes across the samples with field of view of 30 mm, in-
plane resolution of 0.234 mm/pixel and image slice thickness of 1
mm. In a typical experiment, 9 tubes were studied
simultaneously, gels with no contrast agent acted as a standard. A
recycle time (TR) of 12 s was used to avoid saturation. T1
relaxation times were measured using an inversion recovery
(180º-TI-90º) imaging pulse sequence; 8 different inversion times
S. Chen et al. March-July 2010
(TI) that ranged from 100 to 15000 ms were applied and the echo
time (TE) was 4 ms.
T2 relaxation times were measured using a CPMG (Carr Purcell
Meiboom Gill) spin echo imaging pulse sequence; a train of 16
echoes was acquired and the delay (τ) between 180º pulses ranged
from 8 to 15 ms depending on the samples. Single exponential
relaxation times were calculated from experimental data. Each
sample was studied three times and average Longitudinal and
transverse relaxation rate calculated.
ri is defined as longitudinal relaxivity (i=1) or transverse
relaxivity (i=2), and calculated with the following equation:9
1 1
ri [CA ] = − (eq. 2)
Ti Tio
where [CA] is the concentration of contrast agent, 1/Ti (i=1 or 2)
is the longitudinal or transverse relaxation rate with the presence
of contrast agent, 1/Tio is the relaxation rate of the medium in the
absence of contrast agent.
The 128 x 128 x 128 three-dimensional RARE-8 (rapid
acquisition with relaxation enhancement) experiments were
acquired with TR of 250 ms and TE between 180º pulses of 25 ms.
The field of view was 30 mm and in-plane resolution was 0.234
mm/pixel. The scanning time was 34 minutes.
3D representation of the embryo eye and major blood vessels
were produced using Amira® software (Visage Imaging, Inc. San
Diego, CA USA).
SI-II. FePt nanoParticles Characterization
SI-II.1. Ligand exchange
Hexane and water are not miscible, and water which is denser
(1.0 g/mL vs 0.7 g/mL) lies beneath the hexane phase. This
provides a straightforward illustration of the effectiveness of the
ligand exchange protocol described in the Section SI-I.1.
The oleic acid and oleylamine capped FePt nPs are only
soluble in hexane giving a black color to the upper phase while
the DI water remains colorless (SI-Figure 2A). In contrast, SI-
Figure 2B presents the cysteamine capped FePt nPs which are
soluble in deionized water dispersion leading the lower phase to
B
A
O
H2N
Ligand
HO
exchange
NH3
FePt hexane
hexane HS
water water FePt
SI-Figure 2. Chemical structure of the ligands and photographs of FePt
nPs dispersions in hexane or water phase: oleic acid and oleylamine
capped FePt nPs (A), and cysteamine capped FePt (B).
be black whilst the upper hexane phase is remains transparent.
After having replaced oleic acid and oleylamine with cysteamine,
the nPs are soluble in water solution and stable for more than 8
months.
The efficiency of ligand exchange was then confirmed by
Fourier transform infrared spectroscopy (FTIR). The FTIR
S 3/6
spectra of nPs before and after ligand exchange are shown in SI- iron oxide or iron silicide, which because of its thinness is not
Figure 3, with all characteristic transmission peaks labelled and visible by XRD or TEM.13-14
matching the literature values.10 The upper spectra in SI-Figure 3
is characteristic of the nPs coated with oleic acid and oleylamine. SI-Table 1. SQUID data of the FePt nPs. Tb is blocking temperature, Ms
The peaks at 1552 and 1713 cm-1 can be assigned to the bidendate is the saturation moment and Hc is the magnetic coercivity of the nPs.
Sample Tb Ms Ms Hc
(-COO-) and monodendate -CO-) modes of oleic acid binding.
(2 K) (300 K) (2K)
The shoulder at 1595 cm-1 is due to the scissoring mode of the
(K) emu per g of Fe kOe
molecularly bonded oleylamine. The two peaks at 2854 and 2924 fcc-FePt-A 90 201 112 0.7
cm-1 correspond to the symmetric and asymmetric -CH2- fcc-FePt-silica-A 50 191 83 1.3
stretching modes of the oleyl group. The peak at 3005 cm-1 is
characteristic of the stretching of =C-H.
The original oleic acid and oleylamine coating of the FePt nPs SI-III. Cytotoxicity
surface was replaced by cysteamine through the ligand exchange SI-Figure 5A and B demonstrate that fcc-FePt-A nPs induce no
protocol described in Section SI-I.1. It efficiency is demonstrated cell viability damage at concentrations up to 30 µg/mL even after
by the bottom FTIR spectra in SI-Figure 3 characterised by i) the 7 days of incubation. Whilst, the same period of incubation with
FePt-oleic acid/oleylamine 60 µg/mL and 120 µg/mL nPs lead to a decrease of the cell
viability of only about 80 % and 70 % cell viability, respectively.
-CO- The results obtain with MCF7 and U2OS cells are consistent
Transmittance

=C-H
-NH
2 with A375M cells data presented in the core of this publication.
-COO- As expected the silica layer preserved the cell viability, with SI-
Figure 5C showing that fcc-FePt-silica-A nPs did not decrease
-CH -
human cell proliferative activity under culture conditions at
2
FePt-cysteamine concentration up to 200 µg/mL.

-CH - -SH A B
2 15 (µg/mL) 60 (µg/mL) 15 (µg/mL) 60 (µg/mL)
-NH 30 (µg/mL) 120 (µg/mL) 30 (µg/mL) 120 (µg/mL)
2 120
MCF7 cell viability (%) 120

U2OS cell viability (%)

4000 3000 2000 1000 100 100
-1
Wavenumber (cm )
80 80
SI-Figure 3. FTIR spectra of as-synthesized nPs coated with oleic acid
60 60
and oleylamine (top) and cysteamine (bottom) after ligand exchange.
40 40
absence of -COO-M and -CO-M, =C-H peaks along with ii) the 20 20
dramatic decrease of the -CH2- stretching transmission mode. The 0 0
shoulder at 2560 cm-1 is associated with –SH,11 while the peaks at
1 3 5 7 1 3 5 7
Time (Day) Time (Day)
3350 and 3448 cm-1 are consistent with the –NH2 end group of the C 25 (µg/mL) 100 (µg/mL)
cysteamine.12 120
50 (µg/mL) 200 (µg/mL)
A375M cell viability (%)

100
SI-II.2. Silica Coating
80
The protocol associated with the silica coating was tuned to
provide homogeneous SiO2 shell as illustrated by the large scale 60

TEM image in SI-Figure 4A. In addition, careful control of the 40

experimental condition prevented the formation of silica nPs 20

without any FePt as illustrated in SI-Figure 4B. 0

1 3 5 7
Time (Day)
A B SI-Figure 5. Viability of MCF7 cells (A) and U2OS cells (B) incubated
with different fcc-FePt-A nPs concentrations (15, 30, 60, 120 µg/mL),
A375M cells (C) incubated with different fcc-FePt-silica-A nPs
concentrations (25, 50, 100, 200 µg/mL).

SI-IV. Cellular uptake

200 nm 50 nm
SI-Figure 4. TEM images of FePt coated with a silica shell: low
magnification (A), higher magnification (B).
The alteration of the magnetic properties of the nPs after the
silica coating (SI-Table 1) is attributed to the formation of thin
layers of iron oxide or iron silicide resulting in the reduction of
the magnetic effective volume and/or an alteration of the material.
In support of this interpretation, the strong base environment
associated with the silica coating can partially oxidize the FePt
surface resulting in a thin layer of softer magnetic material like
S. Chen et al.
SI-Figure 6A illustrates macropinocytosis, one of the main
non-specific endocytic pathways which involves the
internalization of large areas of plasma membrane together with
significant amounts of fluid. Large irregular vesicles called
macropinosomes with size larger than 1 µm are generated when
membrane protrusions fuse back with the plasma membrane as
indicated by the arrows on SI-Figure 6A.
SI-Figure 6B-C present TEM images of A375M cells incubated
with fcc-FePt-A nPs at FePt metal core concentration of 30
µg/mL and fcc-FePt-silica-A nPs at nPs concentration of 50
µg/mL after 16 h. SI-Figure 6B shows macropinocytosis uptake
mechanism. SI-Figure 6C shows intercellular presence of fcc-
FePt-silica-A nPs. Both fcc-FePt-A and fcc-FePt-silica-A nPs
demonstrated efficient non-phagocytic human cells internalisation
March-July 2010 S 4/6
 A SI-Table 2. 1H relaxivity values (r1 and r2) for agarose gel solution
containing Feridex I.V.® , fcc-FePt-A and fcc-FePt-silica-A nPs compared
to values of some commercial contrast agent base on literature (7.1 T, 300
MHz MRI, 37 ºC, in water).9
r1 r2
Sample
(s-1mM-1) (s-1mM-1)
fcc-FePt-A 2.5 ± 1.0 887 ± 32
fcc-FePt-silica-A 0.3 ± 0.1 210 ± 3
Feridex I.V.® (Ferumoxides) 0.9 ± 0.1 148 ± 2
Feridex I.V.® as reported 1.8* 132*
B C Ferucarbotran. A® 1.6* 205*
Ferumoxtran-10® 1.4* 71*
Ferumoxytol® 2* 95*
*
Experimental uncertainty was not reported.

published. The relaxivities values obtained with Feridex are

500 nm 50 nm
SI-Figure 6. Schematic of cellular uptake through macropinocytosis (A).
TEM image of A375M cells after 16 h incubation with DMEM cell media
solution containing 30 µg /mL of fcc-FePt-A nPs (B), 50 µg/mL fcc-FePt-
silica-A nPs (C).
by straightforward incubation, without any external allied force
such as transfection agents or electroporation.
SI-V. MRI
As expected for high magnetic fields such as 7.1 T used in this
work,9 all samples (i.e. Feridex, fcc-FePt-A, fcc-FePt-silica-A)
show weak effect on T1 relaxation time of 1H in 1 % agarose gel
water solution (SI-Figure 7A).
SI-Table 2 summarizes the relaxivity values r1 and r2 obtained
in this work and compare them with iron oxide data previously
consistent with literature’s values. In addition and as presented in
the core of the publication, it is noticeable that higher r2 values
were obtained with fcc-FePt based nPs compared with Feridex’.
SI-Figure 7B shows that at a magnetic field of 5 T and a
temperature of 300 K, fcc-FePt-A nPs have the highest
magnetization of 112 emu/g of Fe; however, fcc-FePt-silica-A
nPs and Feridex appear to have similar nPs magnetizations of
about 80 emu/g of Fe. By design, the commercial SQUID
magnetometer highest external magnetic field is 5 T, whilst MRI
experiments were performed at 7.1 T. However, a higher nPs
magnetization of fcc-FePt-silica-A nPs compared to Feridex can
be expected by extrapolating the magnetization curves on SI-
Figure 7 from 5 T to 7.1 T. It is indeed noticeable that Feridex has
already saturated at 5 T, while the silica coated FePt nPs had yet
to reach saturation magnetization.
SI-Figure 8 illustrates the effect of injecting embryo with fcc-
FePt-A nPs (1 mg/mL, SI-Figure 8, top row) and fcc-FePt-silica-
A nPs (27 µg/mL FePt core concentration, i.e. 560 µg/mL FePt-

A1 B1
A

0.8
1/T (s )
-1

0.7
1

0.6

0.5

0.4
[Fe] (mM)
0.3 A2 B2
0 0.05 0.1 0.15 0.2

150
B
100
M (emu per g Fe)

50

-50

-100

-150
-40 0 40 H (kOe)
SI-Figure 7. T1-1 (s-1) vs. [Fe] (mM) curve which illustrates 7.1 T MRI
studies of the effect of the presence of (blue ●) fcc-FePt-A, (green ▲) fcc-
FePt-silica-A and (orange ♦) Feridex I.V.® samples on 1H longitudinal
(T1) relaxation time of the water in 1 % w/v agarose gel (A). Hysteresis
curves of fcc-FePt-A nPs (blue ●), fcc-FePt-silica-A nPs (green ▲) and
Feridex (orange ♦) at 300 K. The empty symbols are magnetisation values
extrapolated at 7 T (B).
SI-Figure 8. Embryo injected in ovo with cell culture media (1 µL)
containing 1 mg/mL fcc-FePt-A nPs (top row) and fcc-FePt-silica-A nPs at
FePt core concentration 27 µg/mL (bottom row). 3D surface
reconstruction of embryo eyes (blue) and blood vessels (red) showing the
position in yellow of the transverse images (A); Dorsal view of transverse
image of the embryo’s head aligned through the eyes (B). 2D slices from
128x128x128 3D Rare-8 MRI data set of Day 4 quail embryo egg (TR ⁄ TE
= 250 ⁄ 25 ms), field of view of 30 mm and pixel dimensions of
0.234mm/pixel. Label: y = yolk, b = brain, in-e = injected eye, un-e = un-
injected eye; scale bar indicates 1mm.

S. Chen et al. March-July 2010 S 5/6

silica-A concentration, SI-Figure 8, bottom row). SI-Figure 8A
are 3D surface reconstruction which have been processed to
highlight embryo eyes in blue, the blood vessels in red, and the
nPs in green. In both cases, the presence of the nPs solution
produced a strong hypointensity in the images clearly visible
because of the darker contrast when comparing with the eye
which has not been injected (SI-Figure 8B).

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