Magnetic Resonance in Medicine 62:1497–1509 (2009)
High-Contrast In Vivo Visualization of Microvessels
Using Novel FeCo/GC Magnetic Nanocrystals
Jin Hyung Lee,1† Sarah P. Sherlock,2† Masahiro Terashima,3 Hisanori Kosuge,3
Yoriyasu Suzuki,3 Andrew Goodwin,2 Joshua Robinson,2 Won Seok Seo,2,4 Zhuang Liu,2
Richard Luong,5 Michael V. McConnell,1,3 Dwight G. Nishimura,1 and Hongjie Dai2*
FeCo-graphitic carbon shell nanocrystals are a novel MRI
contrast agent with unprecedented high per-metal-atom-basis relaxivity (r1 ⴝ 97 mM-1 sec-1, r2 ⴝ 400 mM-1 sec-1) and
multifunctional capabilities. While the conventional gadolinium-based contrast-enhanced angiographic magnetic MRI
has proven useful for diagnosis of vascular diseases, its
short circulation time and relatively low sensitivity render
high-resolution MRI of morphologically small vascular structures such as those involved in collateral, arteriogenic, and
angiogenic vessel formation challenging. Here, by combining
FeCo-graphitic carbon shell nanocrystals with high-resolution MRI technique, we demonstrate that such microvessels
down to ⬃100 ␮m can be monitored in high contrast and
noninvasively using a conventional 1.5-T clinical MRI system,
achieving a diagnostic imaging standard approximating that
of the more invasive X-ray angiography. Preliminary in vitro
and in vivo toxicity study results also show no sign of
toxicity. Magn Reson Med 62:1497–1509, 2009. © 2009
Wiley-Liss, Inc.
Key words: FeCo/GC; nanocrystal; contrast agent; high-resolution angiography; PAD
Noninvasive in vivo visualization of morphologically
small vascular structures is of pivotal importance for the
diagnosis of cardiovascular (1) and cancerous (2) diseases
in which collateral, arteriogenic, and angiogenic vessel
formation plays a critical role in their etiopathology. Moreover, monitoring the therapeutic effects in patients with
vascular diseases necessitates the availability of a tool that
is noninvasive and provides high-contrast and high-spatial-resolution angiographic images up to the scale of microvessels (i.e., ⬃100 ␮m). However, noninvasive imaging
of microvessels continues to pose technical challenges for
present angiographic approaches. For example, X-ray angiography necessitates an invasive insertion of a catheter,
and the use of ionizing radiation and iodine contrast adds
1Department of Electrical Engineering, Stanford University, Stanford, California, USA
2Department of Chemistry, Stanford University, Stanford, California, USA
3Division of Cardiovascular Medicine, Stanford University, Stanford, California, USA
4Department of Chemistry, Inorganic and Bio-Materials Center of BK21,
Sogang University, Seoul, Korea
5Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California, USA
†These authors contributed equally.
*Correspondence to: Hongjie Dai, PhD, William Keck Science Building, Rm
125, Stanford University, 380 Roth Way, Stanford, CA 94305-5080, USA.
E-mail: [email protected]
Received 11 March 2009; revised 6 May 2009; accepted 16 June 2009.
DOI 10.1002/mrm.22132
Published online 26 October 2009 in Wiley InterScience (www.interscience.
wiley.com).
© 2009 Wiley-Liss, Inc.
potential danger and discomfort for the patient. In addition, the contrast level of the intravenously (i.v.) injected
iodine contrast agent drops rapidly and can be difficult to
time for small vessels where the flow is slow. Likewise,
while conventional MR vascular imaging is routinely performed using gadopentetate dimeglumine contrast agents,
similar to iodine, there is high contrast during the first
pass, but the rapid drop in contrast leaves insufficient time
for high-resolution small vessel imaging. Furthermore,
with clinical MRI scanners, the imaging resolution is typically in the millimeter scale, which is not suitable to
visualize micron-scale collateral and/or angiogenic vessels.
These challenges posed for current vascular imaging
techniques provide a high impetus to seek the development of blood pool agents with high sensitivity and low
toxicity capable of labeling vessels below millimeter
scales. Some recent examples include Fenestra VC威 (Advanced Research Technologies Inc., Montreal, Canada) for
CT, macromolecular agents (3-5), protein-binding agents
(6,7), and ultrasmall iron-oxide nanoparticles (8) for MRI.
In this study, we report a significant progress we were
able to achieve in visualizing microvessels through the use
of novel FeCo-graphitic carbon shell (FeCo/GC) nanocrystals coupled with high-resolution in vivo MR vascular
imaging technique. A rabbit hind limb peripheral ischemia
model (9) was used for demonstration. To this end, our
attempts were made possible by the unique properties of
FeCo/GC nanocrystals (10,11): (1) the highest magnetization among all magnetic materials (allowing high relaxivities and thus sensitivity), (2) biocompatibility due to
graphite encapsulation and chemical functionalization/
pegylation, and (3) positive intravascular T1 contrast capability (12). By utilizing the ultrahigh sensitivity and long
blood circulation characteristics of these nanocrystals, we
aimed to demonstrate that low-dose (approximately 20fold reduction metal dose compared to conventional Gdbased contrast agent) FeCo/GC-based MRI (13) of microvessels (⬃100 ␮m) can be achieved using a ubiquitous
conventional 1.5-T clinical scanner. Finally, the results of
our studies suggest that FeCo/GC nanocrystals are likely to
be safe and nontoxic, without causing adverse side effects
to the health of animals monitored over several months
post-i.v. administration.
MATERIALS AND METHODS
FeCo/GC Nanocrystal Synthesis
FeCo/GC Synthesis and Functionalization
FeCo/GC nanocrystal synthesis and concentration determination by calcination and ultraviolet-visible spectros-
1497
1498
copy were performed as previously described (10).
FeCo/GC nanocrystals were functionalized using a phospholipid (PL)-branched-polyethylene glycol (PEG) surfactant (14). A nanocrystal suspension was prepared by sonicating FeCo/GC nanocrystals in water with 1 mg/mL of
surfactant for 1 h. To remove aggregates, the suspension
was centrifuged at 15,000 revolutions per minute for 6 h.
Excess PL-PEG was removed by filtration and put in phosphate-buffered saline prior to injection. All samples were
made fresh and the concentration was determined before
use. The difference with the previous sample described in
Seo et al. (10) is that PL-branched PEG was used as surfactant instead of the PL-2k-PEG. The centrifugation time
was also increased to 6 h instead of 5 min at a higher speed
of 15,000 revolutions per minute instead of 10,000 g.
Lee et al.
mM for a kg rabbit, the following formula (Eq. 1) was used
to calculate the required injection volume ( mL).
␯⫽
共60 ⫻ m ⫹ 1兲x
5⫺x
[1]
Blood Circulation Time Measurement
To estimate the blood circulation time, normal rabbits
were injected with FeCo/GC nanocrystals while the aorta
signal was monitored using T1-weighted pulse sequences.
The time-dependent aorta signal gave an estimate of the
blood circulation time.
Synthesis of PL-Branched-PEG
Rabbit Aortic Stenosis
This surfactant was prepared by mixing one equivalent of
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG5000NH2 (Sunbright DSPE-050-PA, NOF Corp., Tokyo, Japan)
with 1.5 equivalents of (methyl-poly(ethylene oxide)12)3poly(ethylene oxide)4-N-hydroxysuccinimide ester (Pierce
Biotechnology, Rockford, IL) in methylene dichloride
overnight, followed by the addition of two equivalents of
N,N-dicyclohexylcarbodiimide (Sigma Aldrich, St.
Louis, MO). Dicyclohexylcarbodiimide was used to reactivate any potentially hydrolyzed NHS groups on PEG
during storage. The solvent was evaporated after another
24-h reaction and replaced with water. After a 10-min
centrifugation at 10,000 g, to remove the insoluble solid
(untreated dicyclohexylcarbodiimide), the supernatant
was collected, filtered through a 0.2-␮m membrane, and
lyophilized for storage at –20°C.
Stenosis of the aorta was generated by high-cholesterol
feeding, in combination with an amaroid constrictor. The
aortic procedure was performed approximately 1 week
after initiation of a high-fat diet (1% cholesterol and 4%
coconuts oil). All surgical procedures were conducted under general anesthesia and sterile conditions, as previously described (15). The animal was given ketamine
(35 mg/kg) and xylazine (5 mg/kg) intramuscularly 30 min
before the operation. After endotracheal intubation, anesthesia was maintained with 1.5% halothane and oxygen
administered at a rate of 65-80 mL/min through an automatic ventilator (Harvard Respiration Pump). A midline
abdominal incision approximately 5 cm long was made
and then the abdominal aorta was dissected free. An MRcompatible amaroid constrictor (Research Instruments
SW, Escondido, CA) (16) was placed around the abdominal aorta at 1-2 cm below the renal arteries. The constrictor
consisted of a casein bar with central lumen housed in an
acrylic covering, rather than the standard metallic covering. In the body, the dehydrated casein absorbs fluid and
swell, thus inducing a progressive stenosis of the vessel
over several months. Finally, the chest was closed and
followed by air suctioning to resume physiologic chest
pressure. MR images were obtained ⬎6 months after the
surgery. After the scan, the rabbit was euthanized by an i.v.
injection of pentobarbital (40 mg/kg).
Particle Size Measurement
The hydrodynamic size after functionalization with PLbranched-PEG was measured through dynamic light scattering with a Brookhaven 90 Plus Particle Size Analyzer.
Light was collected at a scattering angle of 90°.
In Vitro/In Vivo Experiment Setup
All animal experiments were performed according to the
protocol approved by the Stanford University Administrative Panel on Laboratory Animal Care.
General Rabbit Protocol
New Zealand white rabbits weighing ⬃3.5-5.5 kg were
used for all in vivo experiments. Rabbits were first anesthetized with an injection of ketamine/xylazine cocktail
with an intramuscular injection (ketamine 35 mg/kg ⫹
xylazine 5 mg/kg). Then, contrast agents were injected
through the catheterized ear vein. Immediately after the
contrast agent injection, 1 mL of saline was injected. The
contrast agents were injected in 5 mM concentration solution. The contrast agent injection amount was determined
assuming the rabbit blood volume ratio of 60 mL/kg. Given
the contrast agent concentration of 5 mM, rabbit blood
volume ratio assumption of 60 mL/kg, and 1-mL saline
push, to achieve a target blood volume concentration of
Rabbit Hind Limb Ligation
Unilateral hind limb ischemia (9) was induced in New
Zealand white rabbits (3- to 4-kg body weight). Animals
were subcutaneously premedicated with ketamine (40 mg/
kg) and xylazine (4 mg/kg) and intubated. The endotracheal tube was connected to a ventilator, which maintained ventilation, using 2 L oxygen mixed with 1-3%
isoflurane. A longitudinal incision was then performed,
extending from a point 4-5 cm proximal to a point 1-2 cm
proximal to the patella. The superficial femoral artery was
dissected free and ligated at 2 places by 4-0 silk (Ethicon,
Sommerville, NJ). Subsequently, the animals were weaned
from ventilation and transferred to the animal care facility.
MRI images and X-ray angiograms were obtained approximately 6 weeks after the surgery. The rabbits were euthanized after the X-ray imaging.
FeCo/GC Magnetic Nanocrystals
X-ray Angiography
After the MRI scan, an X-ray angiogram of the rabbit hind
limb was acquired using OEC 9600 (40k Vp, 0.2 mA, 30
frames/sec) cardiac C-arm (OEC Medical systems, Inc., Salt
Lake City, UT). After induction of anesthesia, as previously described, vascular access was obtained using standard 4-Fr vascular sheath, placed in the right or left carotid
artery. A 4-Fr diagnostic catheter (Judkins right type) was
advanced into the descending aorta and the aortoangiography was performed. After X-ray angiography, animals
were euthanized by KCl infusion. The vessels were excised and were perfusion fixed with phosphate-buffered
saline followed by 10% formalin and then placed in 10%
formalin for fixation.
Rabbit Femoral Artery Histology
The excised occluded femoral artery was sectioned and
stained with hematoxylin-eosin, elastic van Gieson stains.
The stained histology confirmed the formation of total
occlusion with the rabbit hind limb surgical ligation
model.
FeCo/GC Nanocrystal Excretion
Normal rabbits were repeatedly injected (5 mL of 3-mM
solution injected five times on day 1 and 9 mL of 3-mM
solution injection on day 6) with FeCo/GC nanocrystals.
Images were obtained with the same T1-weighted sequence
as in the blood circulation time measurement experiment.
In Vitro FeCo/GC Toxicity Study
1499
months post-injection, blood was drawn from the submandibular vein or via cardiac puncture into heparinized
tubes for analysis.
Histologic evaluations were done on three treated mice
and two control mice 3 months after the injection of the
nanocrystal solution. All soft tissues were collected and
examined, preserved with formalin for 48 h, and then
routinely processed for light microscopic examination of
hematoxylin-eosin-stained sections.
MRI Data Acquisition
All MRI data were acquired using a 1.5-T GE Excite wholebody MRI system (maximum gradient amplitude: 40
mTm-1; maximum slew rate: 150 mTm-1 ms-1).
Relaxivity (r1 and r2) Measurements
T1 and T2 were measured at six different metal concentrations of FeCo/GC nanocrystal suspensions (0.05, 0.1, 0.2,
0.4, 0.8 mM: Fe and Co concentration combined). The
corresponding particle concentration can be obtained by
multiplying 6.1854 ⫻ 10⫺8 to the mM concentration (e.g.,
0.2 mM metal concentration ⫽ 12.38 nM particle concentration). For the T1 measurement, inversion recovery sequences were used (17) with a pulse repetition time (TR) of
6000 ms and inversion time of 50, 100, 150, 200, 250, 300,
350, and 2000 ms. For the measurement of T2, spin-echo
sequences were used (17), with a TR of 3000 ms and echo
time (TE) of 8, 12, 20, 30, 50, 80, 160, 320 ms. The field of
view (FOV) was 22 ⫻ 22 cm2 and the image matrix size was
128 ⫻ 128.
To test in vitro toxicity of FeCo/GC nanocrystals, 30 ␮L of
a highly concentrated solution was added to macrophage
cells in 100 ␮L of cell medium. Cells were cultured with
Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum and 1% penicillin-streptomycin
in a 5% CO2 atmosphere at 37°C. Macrophage cells were
incubated in a 96-well plate for 48 h, with varying concentrations of FeCo/GC nanocrystals functionalized with
PL-branched-PEG. Prior to measuring cell viability, all medium and nanocrystals were removed from the wells, followed by the addition of 100 ␮L of fresh medium and 20
␮L of CellTiter 96威 AQueous One Solution Reagent (Promega Co., Madison, WI). Following incubation for 1.5 h,
the absorbance was recorded at 490 nm and the cell viability was determined.
A standard GE head coil was used. Fat was suppressed
with a spectral-spatial excitation pulse. Three-dimensional (3D) stack-of-spirals acquisition was designed to
have 40 interleaves and 40 kz-phase encodes. The FOV was
designed to be 30 ⫻ 30 ⫻8 cm3, with a resolution of 1 ⫻
1 ⫻ 2 mm3. TR was 20 ms, with a flip angle of 60°. The
resulting volume scan time is 32 sec.
For all the in vivo studies, TR and TE were chosen to be
as short as possible under the constraint of obtaining
proper FOV and resolution to maximize contrast. High flip
angles were used since the Ernst and Anderson (18) angle
for the FeCo/Gc nanocrystals at 0.2 mM concentration is
around 60°.
In Vivo FeCo/GC Toxicity Study
In Vivo Demonstration of High Sensitivity
To evaluate the toxicity of FeCo/GC nanocrystals in vivo,
16 female Balb/c mice weighing 17-21 g were injected with
4 nm FeCo/GC PL-branched-PEG-carboxylate. The PLbranched-PEG-carboxylate was prepared by mixing fourarm PEG-N-hydroxysuccinimide (Mn ⬃10,000; Laysan Bio,
Inc., Arab, AL) with 1,2-distearoyl-3-sn-glycero-3-phosphoethanolamine (Lipoid LLC, Ludwigshafen, Germany)
in a 1:1 molar ratio overnight and then purifying by dialysis. The injected nanocrystal dose was ⬃210 ␮L of a
solution with a metal concentration of 15.5 mM. This dose
yields a ⬃2.2- to 2.7-mM blood pool concentration, which
corresponds to ⬃11-14 times the rabbit dose. After 1 or 3
For the comparison of the enhancement effects in rabbit
aorta, 3D spoiled-gradient recalled echo (SPGR) images
were acquired with a TR of 33.3 ms, TE of 4 ms, flip angle
of 60° and fat saturation. The FOV was 18 ⫻ 18 ⫻ 5.6 cm3,
with a matrix size of 192 ⫻ 192 ⫻ 28.
Blood Circulation Time Measurement
High Spatial Resolution MRI of Blood Vessels
The high-resolution images comparing ferumoxtran-10
and the FeCo/GC 7-nm particles’ performance were obtained using a custom-designed pulse sequence with a
spectral-spatial excitation and stack-of-spirals acquisition
1500
incorporated into an SPGR sequence. The spectral-spatial
excitation pulse (19) was designed to selectively excite
water (excluding fat) while simultaneously limiting the
slab volume. The pulse duration was 8.536 ms. The FOV
was designed to be 4 ⫻ 4 ⫻ 1 cm3, with a resolution of 78 ⫻
78 ⫻ 500 ␮ m3. TR was 40 ms and TE was 5.128 ms, with
a readout duration of 16.224 ms and flip angle of 60°. 3D
stack-of-spirals acquisition was designed to have 200 interleaves and 20 kz-phase encodes. The resulting volume
scan time is 2 min 40 sec. To improve signal-to-noise ratio
(SNR), nine images were averaged.
Imaging Angiogenesis in Rabbit Aorta
The rabbit aorta was imaged with a 3D SPGR sequence
with a TR of 35 ms, TE of 4 ms, flip angle of 60°, and fat
saturation. The FOV was 18 ⫻ 18 ⫻ 2.8 cm3, with a matrix
size of 256 ⫻ 256 ⫻ 28.
Lee et al.
Signal Intensity vs Concentration Calculation
To calculate the expected signal enhancement vs concentration of various contrast agents in Fig. 3b, T1, T2 values
expected for each concentration level (C) were calculated
from T1, T2 values of blood without any contrast (T10 ⫽
1200 ms, T20 ⫽ 327 ms at 1.5 T (20)) and r1, r2 of each
contrast agents as follows (Eq. 2).
1
1
1
1
⫽
⫹ r 1 ⫻ C,
⫽
⫹ r2 ⫻ C
T 1 T 10
T 2 T 20
[2]
Then, the resulting signal intensity from the T1, T2 was
calculated for the case of SPGR sequence used for the
imaging of the aorta. (TR: 33.3 ms, TE: 4 ms, flip angle: 60°)
(Eq. 3).
S共C兲 ⫽ M̈ 0 ⫻ sin␪ ⫻
1 ⫺ e⫺TR/T 1共C兲
*
⫻ e⫺T E/T2共C兲
1 ⫺ cos␪ ⫻ e⫺TR/T 1共C兲
[3]
Monitoring Angiogenic Vessel Growth in Rabbit Hind Limb
Two different protocols were used to image the rabbit hind
limb. In both cases, the aforementioned spectral-spatial
excitation was used. The large FOV covering both hind
limbs was used to show the difference between the two
limbs in terms of vessel distribution. The hind limb ischemia model is expected to result in angiogenic vessel
growth in a large area of the limb, unlike in the case of
local angiogenesis observed in the aortic stenosis. To image a large FOV covering both hind limbs, a 5-inch surface
coil was placed on top of a rabbit in supine position. A
coronal plane was scanned with an imaging FOV of 20 ⫻
20 ⫻ 3 cm3 and resolution of 300 ⫻ 300 ⫻ 500 ␮ m3. TR
was 40 ms and TE was 5.128 ms, with a spiral readout
duration of 7.36 ms and flip angle of 45°. 3D stack-ofspirals acquisition was designed to have 200 interleaves
and 60 kz-phase encodes. The resulting volume scan time
is 8 min.
Once the region of the stenosis was identified, a small
FOV image was obtained using a 1-inch surface coil (Fig.
4c) in higher resolution. The FOV was 6 ⫻ 6 ⫻ 3 cm3,
resolution was 117 ⫻ 117 ⫻ 500 ␮ m3 TR was 40 ms and TE
was 5.128 ms, with a spiral readout duration of 16.224 ms
and flip angle of 45°. 3D stack-of-spirals acquisition for
this protocol was also designed to have 200 interleaves
and 60 kz-phase encodes. The resulting volume scan time
is 8 min. To improve SNR, two images were averaged.
MRI Data Analysis
S共C兲
was then calculated and plotted to show the enhanceS共0兲
ment ratio.
Signal Intensity Measurement
For the comparison of the signal intensities, the crosssectional images were selected along the aorta. Signal intensity was measured by averaging the signal across the
ROI selected within the aorta.
Angiogram
For the aortic, circumflex iliac, and femoral artery angiography images, maximum intensity projection (MIP) was
calculated from the whole volume or selected slices from
the volume (25 slices out of 60 total selected for Fig. 6c).
Due to the high contrast provided by FeCo/GC, small vessels are observed in high quality, despite the partial volume effect that can be expected from the lower resolution
in the through-plane direction. The smallest visible vessel
sizes are down to the in-plane image resolution of 703 ␮m
for the aortic, 78 ␮m for the circumflex iliac, and 117 ␮m
for the femoral artery angiography.
Signal Intensity Plot
To evaluate the spatial resolution of small vasculature,
cross-sectional signal intensity was plotted across small
vasculature. A single dot on the plot represents each voxel.
Relaxivity (r1 and r2) Measurements
The data were analyzed to estimate T1 and T2 values
through nonlinear least-squares fits to the inversion recovery and spin-echo decay curves sampled at various TE.
The slope of T1-1 and T2-1 vs concentration (r1, r2) was then
calculated through linear fitting. The slope, which is the
rate of T1-1 and T2-1 change with the contrast concentration,
indicates the efficacy of the contrast agent as a T1 or T2
contrast agent (17).
SNR Measurement
Blood pool signal SNR was measured by selecting an ROI
within the blood pool. The average signal intensity (Sb)
was then divided by the SD (␴b) to obtain SNR (Eq. 4).
SNR ⫽
Sb
␴b
[4]
FeCo/GC Magnetic Nanocrystals
1501
FIG. 1. FeCo/GC nanocrystals with branched-PEG functionalization for in vivo applications. a: Transmit electron microscope (TEM) image
showing the FeCo/GC nanocrystals. b: A schematic diagram showing a FeCo/GC nanocrystal with PL-branched-PEG attached. c: Picture
of a FeCo/GC solution at 0.2-mM metal molar concentration. d: Hydrodynamic size distribution of 7-nm nanocrystals with PL-branchedPEG measured by dynamic light scattering. The graph shows the number of counts at each diameter out of 313 total counts. The results
show that 99% of the sample is between 19.2 and 28.4 nm, but most of it (93%) is at or below 25.8 nm.
Contrast Resolution Measurement
Contrast resolution of the blood pool vs the surrounding
tissue was measured by selecting ROIs in the blood pool
and the adjacent tissue (usually muscle tissue). Average
signal intensity in blood pool ROI (Sb) and the surrounding
tissue ROI (Sm) was then measured to calculate contrast
resolution as shown in Eq. 5.
contrast resolution ⫽
Sb ⫺ Sm
Sb ⫹ Sm
[5]
RESULTS
FeCo/GC Nanocrystals
The FeCo-Graphitic shell nanocrystals (Fig. 1a) were synthesized as previously reported (10), with the particle core
size distribution of 7 ⫾ 1.2 nm (Fig. 1a). We made an
important advancement in the current work by chemical
functionalization of the graphitic shells of the nanocrystals
using PL with 10 kDa branched-PEG chains (Fig. 1b) (14).
Clusters of nanocrystals were removed by increasing the
centrifugation time (10). The hydrodynamic size was
23.1 ⫾ 2.6 nm (Fig. 1d). The branched PEG and cluster
removal resulted in longer blood circulation time and de-
layed uptake by the reticuloendothelial system for the
improved nanocrystal particles due to branched pegylation (14,21). A blood circulation half-life of approximately
4 h was measured for nanocrystals i.v. injected into rabbits
(Fig. 2). The better declustering reduced the r2/r1 ratio,
which is desirable for highly sensitive positive-contrast
blood pool imaging. As seen in Fig. 3a, compared to results
reported earlier (10), the improved FeCo/GC has higher r1
relaxivity (97 mM-1 s-1) and lower r2 (400 mM-1 s-1) relaxivity and thus lower r2/r1. These relaxivities were among
the highest on the per-metal-atom basis among all known
MRI contrast agent materials, owing to the superior magnetic properties of FeCo alloy crystals (10,22-25). Per-particle basis r1 relaxivity is 1.5682 ⫻ 1012 mM⫺1 s⫺1 and r2
relaxivity is 6.4668 ⫻ 1012 mM⫺1 s⫺1.
In Vivo Demonstration of High-Sensitivity MRI Using FeCo/
GC Contrast Agent
To test the comparative relaxivity and thus sensitivity
improvement of FeCo/GC contrast agent, we i.v. injected
equal amounts (blood pool target concentration of 0.2 mM
of metal) of commercially available ultrasmall particle iron
oxide agent ferumoxtran-10 (Combidex威; AMAG Pharmaceuticals, Inc., Cambridge, MA) and our FeCo/GC contrast
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FIG. 2. Seven-nanomolar FeCo/GC contrast agent was injected to
a normal rabbit and the blood circulation time was measured by
monitoring the signal intensity of the aorta. The signal enhancement
in the low concentration range (⬍0.2 mM) is approximately linear.
Therefore, the blood circulation half-life can be estimated by estimating the time point where the signal intensity enhancement is half
of that obtained immediately after the injection. The blood half-life is
estimated to be approximately 4 h.
agents into rabbits and carried out MRI of the rabbit aorta
(Fig. 3c,d). At the same targeted 0.2-mM blood pool metal
concentration, ferumoxtran-10 exhibited a positive signal
enhancement factor of approximately 3.8 (Fig. 3c) with a
blood pool SNR (determined by the blood pool signal
Lee et al.
intensity divided by the SD; see Materials and Methods) of
5.6, and contrast resolution (as defined in Materials and
Methods) of 0.60, while FeCo/GC nanocrystals displayed a
substantially higher positive enhancement factor of 9.5
(Fig. 3d) and blood pool SNR of 13.9, and contrast resolution of 0.80 consistent with the higher relaxivities of the
latter than the former (r1 ⫽ 15 mM-1 s-1 vs 97 mM-1 s-1).
Fig. 3b shows the calculated contrast enhancements for
various contrast agents (see also Materials and Methods)
based on the measured r1, r2 values (Fig. 3a) for a T1weighted SPGR sequence. Consistent with experimental
results, calculations find that FeCo/GC is expected to give
a high contrast enhancement factor of approximately 9
(Fig. 3b, red curve) at 0.2 mM per-metal-atom basis concentration. The increased positive enhancement (see the
steep upward slope with increasing concentration of the
contrast agent) in the low-concentration region is attributed to the high r1 relaxivity. The reversal of the contrast at
higher concentration is due to the T2 effects caused by the
large r2.
Ferumoxtran-10 has a long circulation time (blood halflife of approximately 24 h), but the r1, r2 relaxation effects
are such that even at 1-mM concentration (five times that
used for enhancement factor of 9 using FeCo/GC), an enhancement factor of only 7 can be achieved (Fig. 3b, green
curve). However, due to contrast reversal, further increase
in ferumoxtran-10 concentration will not result in additional contrast enhancement.
To achieve the same contrast enhancement as the
FeCo/GC nanocrystals at 0.2-mM concentration, the clinically prescribed Magnevist contrast agent would require a
minimum of 10-fold higher concentration in the blood
FIG. 3. Relaxivities of commercial
and our FeCo-graphitic carbon
shell nanocrystals measured at
1.5 T and highly sensitive MRI enabled by FeCo/GC nanocrystals.
a: The newly improved FeCo/GC
material with a more uniform size
through elimination of clustering
resulted in a smaller r2/r1 ratio.
These are important properties
for in vivo applications where long
blood circulation with reduced
uptake from the reticuloendothelial system is of interest. b: Calculated signal enhancement vs concentration for commercial and
FeCo/GC agents. MRI showing
the rabbit aortic signal enhancement by (c) ferumoxtran-10 and
(d) FeCo/GC nanocrystals, respectively; 0.2-mM blood pool
concentration was targeted for
both agents. The difference in resulting signal enhancement factors of 3.8 for ferumoxtran-10 and
9.5 for FeCo/GC was observed,
consistent with calculations in (b).
FeCo/GC Magnetic Nanocrystals
1503
FIG. 4. In vivo high-resolution MRI.
(a) A vasculature MIP image enhanced by 1-mM blood pool concentration of ferumoxtran-10. (b) A vasculature MIP image enhanced by
0.2-mM blood pool concentration of
FeCo/GC. (c) Picture of 1-inch-diameter custom-designed surface coil
used for this experiment. (d) Crosssectional signal intensity plot across
a small vasculature confirms that it
has a sharp single voxel (78 ␮m) peak,
suggesting vessel size on the order of
100 ␮m. Even at a concentration five
times higher than that used for FeCo/
GC, ferumoxtran-10 does not provide
sufficient contrast to visualize small
vasculature (⬍100 ␮m).
pool (Fig. 3b, dotted blue curve). However, since Gd-based
contrast agents, such as Magnevist, have a short blood
half-life, it is not viable to equate a 10-fold increase in
injection with a 10-fold increase in the actual (and more
relevant) blood pool concentration. Therefore, even higher
dosage of Gd-based contrast agent would be necessary to
achieve the intended concentration within the blood pool.
High-Spatial-Resolution MRI of Blood Vessels
To investigate the potential of FeCo/GC nanocrystals for
high-resolution MRI of vessels such as those involved in
microvasculature growth, we developed an ultra-high-resolution MR angiography technique by utilizing surface
coils (26) (Fig. 4) and highly optimized pulse sequence for
high-spatial resolution imaging (27,28) (see Materials and
Methods). Blood vessels in the posterior side of the upper
hind limb of healthy rabbits were imaged by MRI following the injection of ferumoxtran-10 and FeCo/GC at a targeted 0.2-mM blood pool concentration. Fig. 4b demonstrates the in vivo visualization of rabbit superficial circumflex iliac artery and branches (29) (blood pool SNR:
11.9; contrast resolution: 0.77) with diameters less than
100 ␮m, using FeCo/GC. In contrast, ferumoxtran-10, even
at five times higher dosage (1-mM targeted blood concentration) than FeCo/GC failed (blood pool SNR: 5.8; contrast
resolution: 0.58) to label small vasculature (Fig. 4a).
Imaging Microvessels in Rabbit Aorta
The high sensitivity and long circulation time of FeCo/GC
nanocrystals make them ideally suited to label morpholog-
ically small vessels as typically displayed by collateral,
arteriogenic, and angiogenic vessels. Fig. 5 displays the
results of our studies investigating the progressive stenosis
of the rabbit aorta through FeCo/GC-labeled MR angiography (Fig. 5a). The results obtained using a 1.5-T clinical
MRI with a quadrature head coil and i.v. injection of the
FeCo/GC nanocrystals suggest that we were able to label
vessel emergence at a submillimeter scale around the aortic occlusion. Despite the limited spatial resolution of the
image acquisition, the high vessel contrast (blood pool
SNR: 14.0; contrast resolution: 0.81) provided by FeCo/GC
nanocrystals allows not only visualization of the larger
collateral vessels but also a putative ability to label finerscale structures such as arteriogenic and potentially angiogenic vasculature (Fig. 5b). This result further provides
initial proof of concept of highly sensitive MRI of small
microvessel growth using our FeCo/GC nanocrystal contrast enhancement.
Monitoring Morphologically Small Microvessels in
Angiogenic Rabbit Hind-Limb Model
Next, we explored high-resolution MRI of microvessels
using the rabbit hind-limb ischemia model, which has
been well established for studying microvessel formation
in peripheral arterial disease (9,30). A total occlusion (Fig.
6) was surgically induced in the right femoral artery, and
the spontaneous formation of microvessels was monitored
6 weeks postsurgery by both standard X-ray angiography
and our FeCo/GC MRI.
The presence and location of spontaneously formed microvessels were confirmed through standard X-ray angiog-
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Lee et al.
FIG. 5. FeCo/GC enhanced MRI at
1.5 T of rabbit microvessels around
aorta stenosis. (a) An FeCo/GC-enhanced 3D SPGR MIP image
shows small microvessel formation
around an aortic stenosis in a
rabbit. The spontaneously formed
tortuous vessels can be clearly
seen in (b).
raphy. Here, X-ray angiography revealed a stenosis in the
right femoral artery (Fig. 7a). Tortuous small spontaneously formed vessels connecting the proximal and distal
parts of the occluded femoral artery were observed (Fig.
7a). An angiogram obtained in a rabbit left hind limb
(normal artery without surgery) was also obtained for comparison (Fig. 7b). Note that the X-ray angiogram was obtained by invasively inserting a catheter from the carotid
artery down to the femoral artery where the iodine contrast
agent was injected during imaging.
The MIP of the FeCo/GC contrast-enhanced MRI image
(Fig. 7c) highlighted both the arteries and nearby veins
since the imaging was performed in steady state. In the
surgically operated right hind limb, narrowing and occlusion of the femoral artery were observed by MRI next to the
femoral vein (Fig. 7c, Fig. 8a,b). Interestingly, we also
observed a significant number of tortuous small vessels in
the right hind limb connecting the proximal and distal part
of the femoral artery (see Fig. 7c and Fig. 8), comparable to
those seen using the more conventional X-ray angiography
(Fig. 7a,b), suggesting that they may indeed represent collateral, arteriogenic, and/or angiogenic microvessels induced by the applied hind limb ischemia (blood pool SNR
and contrast resolution in Fig. 7c: 11.4 and 0.71; blood
pool SNR and contrast resolution in Fig. 8b,c: 14.2 and
0.70).
DISCUSSION
In the present study, we investigated the properties of a
novel FeCo/GC contrast-enhanced MR angiographic ap-
FIG. 6. Normal femoral artery shows large lumen area in the middle (a). The femoral artery taken from the portion identified as the occlusion
site (b,c) shows that the artery is completely occluded. The histology was obtained around the ligation (b) and in the proximal portion of
the complete total occlusion (CTO) (c). The left images in (b) and (c) show hematoxylin-eosin staining result, and the right images show the
elastic van Gieson staining result. These results show that the rabbit hind limb ligation model successfully created CTO.
FeCo/GC Magnetic Nanocrystals
1505
FIG. 7. Imaging of microvessel in
rabbit hind limb model of peripheral arterial disease. a: X-ray angiogram obtained 6 weeks after
the surgery that induced stenosis
in the right femoral artery. Tortuous small vessels are seen
throughout the right hind limb. b:
X-ray angiogram obtained in a
normal left hind limb shows normal arterial vessels for comparison. c: MRI (1.5 T, 5-inch surface
coil) was performed 6 weeks after
the surgery with a FeCo/GC injection targeting a 0.2-mM blood
pool concentration. A coronal volume was scanned after the injection, with a 20 ⫻ 20 ⫻ 3 cm3 FOV
and 300 ⫻ 300 ⫻ 500 ␮ m3 resolution. MIP image shows the stenosis in the right femoral artery
and tortuous microvessel formation throughout the right hind limb
(red circle). Note the X-ray and
MR images are acquired in different projection angles. d: Highquality fat suppression and image
quality are demonstrated in selected cross-sectional images.
proach that displayed exceptional contrast resolution and
sensitivity for MRI using 1.5-T clinical scanners. While
significant prior advances in high-resolution imaging of
micro-vessels at 1.5 T exist (3-6,8,31-34), clear imaging of
microvessels on the order of 100 ␮m has continued to
present a challenge. Major challenges include insufficient
circulation time for high-resolution imaging, low sensitivity resulting in low contrast in microvessels, unpredictable
contrast, high dose, and toxicity (35). An ideal contrast
agent for angiography should have a sufficiently long
blood half-life (6,31-33) to allow for image acquisition and
should be efficient in generating predictable image contrast with low metal doses (3-5,31) and without the risk of
toxic side effects (33,34). FeCo/GC nanocrystals are long
circulating, are highly sensitive, generate contrast free of
complication from complex pharmacokinetics, and are stable and inert.
Here, we demonstrated a significant 20-fold reduction in
metal dosage (compared to the clinically used Gd-based
contrast dosage) attained by the use of FeCo/GC contrast
agent. The dosage of clinical Gd-based contrast agents
range from 0.1 to 0.4 mmol/kg of body weight, while
0.2 mmol/kg is a commonly used dose for MR angiography
at 1.5 T (36). Targeting a blood pool concentration of
0.2 mM is equivalent to approximately 0.01 mmol/kg dose,
which is 1/20 of the Gd-based contrast agent dosage for MR
angiography. The high sensitivity of our contrast agent is a
direct result of the ultra-high relaxivities of the FeCo/GC
nanocrystals originated from the high saturation magnetization of FeCo (approximately three times higher than iron
oxide). Although the exact relaxation mechanism is not
known, due to the graphitic shell fully surrounding the
metal core of the nanocrystal, relaxation must occur by an
outer-sphere mechanism. Any nonencapsulated metal that
1506
Lee et al.
FIG. 8. High-resolution MRI of microvessels. a: A zoom-in view of the right hind limb with the stenosis from Fig. 7c. b: A 6 ⫻ 6 ⫻ 3 cm3
FOV, 117 ⫻ 117 ⫻ 500 ␮m3 resolution MIP image (1.5 T, 1-inch surface coil) of the region near the occlusion. c: A MIP of 25 slices selected
from the full data set (60 slices). Small microvessels with diameters in the order of 100 ␮m are observed in the high-resolution images. d:
A further zoomed-in view of (c) and its cross-sectional signal intensity plot across a small vasculature confirms that it has a sharp single
voxel (117 ␮m) peak, suggesting vessel size on the order of approximately 100 ␮m.
could contribute to the relaxivity is removed by hydrofluoric acid (HF) etching prior to functionalization.
The high sensitivity and long circulation of the FeCo/GC
agent, combined with a high-resolution MRI technique,
allowed for exceptional SNR and contrast resolution. In
MRI, SNR is proportional to the voxel size. Reducing voxel
size leads to low SNR. Therefore, compensating for the
SNR loss of high-resolution imaging (down to 10 –100 ␮ m)
is of great importance. Some of the approaches include
using higher field strength scanners since SNR in MRI is
approximately proportional to the field strength. However,
high-field-strength systems (ⱖ 7 T) impose many problems
over clinical-field-strength systems (1.5 T), including patient safety and comfort, availability, and high cost. Therefore, we chose to utilize the standard clinical field strength
(1.5 T) with small surface coils (26,37) and 3D imaging that
allow SNR efficient acquisition. While the use of single
small surface coils reduces volume coverage, combining
with multiple surface coil phase arrays (38) can easily
recover volume coverage, making it possible to translate
the current technique into the clinical applications. Likewise, scan time reduction is important since imaging in
vivo involves motion. In high-resolution imaging, even a
small motion can lead to significant blurring. Therefore,
FIG. 9. MIP image of rabbit torso and in vitro toxicity data. a: Before any contrast injection, the liver typically shows bright signal and the
intestine shows darker signal than the liver. The rabbit was then injected with contrast agents at multiple time points. b: Six hours after the
latest injection, the rabbit liver shows dark signal and the intestine shows bright signal. This suggested that FeCo/GC contrast agent was
initially taken up by the liver and then excreted through the biliary system through the intestine and then feces, much like carbon nanotubes,
as reported recently. c: Percent viable macrophage cells after a 48-h incubation with varying concentrations of nanocrystals suspended
with PL-branched-PEG-(PL-3PEO). The vertical blue line represents the target blood pool concentration (0.2 mM) for imaging, and no toxic
effects are observed.
FeCo/GC Magnetic Nanocrystals
1507
FIG. 10. Body weight and histologic data for in vivo toxicity study. Body weight changes over time (a) in control mice and FeCo/GC injected
mice. Mice given the nanocrystal injection at day 0 were given a dose 11-14 times that needed for MRI. The lack of any significant deviations
in body weight, as compared to the control mice, supports the argument that there are no obvious toxic effects from the nanocrystals.
Hematoxylin-eosin-stained slices of a liver from a control mouse (b), nanocrystal-injected mouse (c), control spleen (d), and nanocrystalinjected spleen (e). The asterisks in (b) and (c) mark the portal triad. The arrows in (c) and (e) mark nanocrystals in macrophage cells. White
pulp and red pulp in images (d) and (e) are marked as w.p. and r.p., respectively. The scale bars in all images indicate 50 ␮m. No damage
is observed in any of the tissues collected from the nanocrystal-treated mice.
rapid imaging is of great importance. To further accelerate
image acquisition, we used 3D spiral encoding
(27,28,39,40) that reduced our encoding time by more than
a factor of 2. Furthermore, to ensure good background
suppression by avoiding fat signal and its blurring, water
frequency was selectively excited using spectral-spatial
excitation pulses (19).
Like any novel contrast agent, ensuring its biocompatibility and low toxicity is of crucial importance. Here,
several lines of evidences point to the biocompatibility of
FeCo/GC. First, the metal component is completely enclosed by a graphitic-carbon shell. The metal core remains
intact in harsh chemical processes such as soaking in a
solution of 10% HF in water (80%) and ethanol (10%). The
Table 1
Serum Chemistry and Hematology Data for balb/c Mice Injected With a High Dose of FeCo/GC Nanocrystals at 1 Month and 3 Months
Postinjectiona
Liver panel and blood
chemistry
Aspartate
aminotransferase
Alkaline phosphatase
Alanine aminotransferaseb
Total bilirubin
Albumin
Total protein
Phosphorus
Blood urea nitrogen
Hematology
White blood cell count
Red blood cell count
Hemoglobin
Hematocrit
Mean corpuscular
volume
Mean corpuscular
hemoglobin
Mean corpuscular
hemoglobin
concentration
aThe
1 mo
3 mo
Control
Unit
Reference range (literature)
Ave.
Std.
Ave.
Std.
Ave.
Std.
IU/L 78
IU/L 137
IU/L 76.3
mg/dL
0.17
mg/dL
1.6
mg/dL
4.8
mg/dL
5.3
mg/dL 22.3
21.6
6.2
4.2
0.16
0.7
0
0.5
2.3
96.9
91.7
63.6
—c
1.2
4.7
6.1
22.1
35.3
29.4
23.3
—c
0.9
1.3
2.1
7.8
109.6
84
54.8
—c
—c
4.9
6.5
19
143.1
3.5
20.2
—c
—c
0.3
1.2
7.2
40–140
47–275
30–100
0.4–1.2
1.3–2.6
4.4–6.4
4.5–8.9
10–30
K/␮L
M/␮L
gm/dL
%
5.5
9.3
14.1
42.9
1.5
1.0
1.7
4.9
5.1
9.9
14.8
45.2
2.2
0.4
0.5
1.8
5.2
9.9
15.0
45.6
1.2
0.5
0.8
2.1
2–5.7
8.5–10.6
14.2–17.0
38.3–47.9
fL
46.2
0.4
45.9
0.2
46.1
0.5
40.9–50.3
pg
15.2
0.2
14.8
0.2
15
0.2
15.1–18.4
g/dL
32.9
0.1
32.7
0.4
32.6
0.2
34.2–40.6
results indicate that most values are either within reference range (41) or only slightly deviate from the given range and do not indicate
any toxic side effects due to nanocrystal injection. The blood data indicate that the liver, kidneys, spleen, and immune system were
functioning normally, with no signs of damage. Results are based on three to seven mice per group.
bReference range given is for CD-1 mice; all other ranges are for Balb/c mice (all mice less than 1 year of age).
c— ⫽ Data not obtained.
1508
graphitic-carbon shells are intact and fully encapsulating
the metal core in any biologically relevant environment
(10). Second, from our rabbit experiments, most of the
FeCo/GC material ends up in the reticuloendothelial system, including the liver, after several hours of blood circulation. This leads to dark signal in the liver due to high
concentration of the contrast material (Fig. 9a). Importantly, the liver then also starts to excrete the material
through the biliary system. This is evidenced by the fact
that following the dark liver signal, the intestine starts to
light up, suggesting that the FeCo/GC material is being
excreted into the intestine (Fig. 9b). The bright signal is
also indicative of the continued structural integrity and
functionality of the shell material since highly sensitive T1
contrast is only expected to be achieved if the particles
stayed intact without aggregation or oxidation. The liver
signal also starts to become lighter, which is an indication
of reduced contrast concentration in the liver (Fig. 9b).
Therefore, the material is believed to be excreted from the
body through the biliary system (14). In the future, we
expect to perform more complete biodistribution studies
to confirm the excretion.
For in vitro toxicity assessment, in addition to the initial
toxicity tests (e.g., cell proliferation assay) presented in
Seo et al. (10), we incubated macrophage cells in varying
concentrations of FeCo/GC solutions for 48 h. We then
measured the viability of cells and found no toxicity in
vitro, even at concentrations greater than 15 times the
blood pool target concentration (Fig. 9c).
In terms of in vivo toxicity, we injected a total of approximately 10 rabbits with the FeCo/GC material in high
concentrations (⬎5 ⫻ higher than blood pool target concentration for MRI) and monitored for over 6 months, and
no sign of the contrast agent–related adverse health effects
was observed. Notably, excretion by the biliary pathway
and lack of obvious toxic effects were similar to the observations of carbon nanotubes that have similar graphitic
surfaces and chemical functionalization as our nanocrystals (14). Furthermore, we conducted a pilot in vivo toxicity study by monitoring the body weight, behavior, blood
chemistry, and hematology (41) in mice for up to 3
months, followed by histologic analysis of soft tissues.
Compared to the untreated control group (five mice), there
were no abnormalities in body weight (Fig. 10a), behavior,
or blood test (Table 1). While accumulation of nanocrystals was present in the cytoplasm of hepatic resident macrophages and splenic macrophages, there was no evidence
of tissue damage or loss of organ function of the liver and
spleen (Fig. 10b-e) from the histologic evaluation or blood
biochemical testing. All other organs examined appeared
normal. The presence of nanocrystals in the liver and
spleen indicates that the macrophages play a role in the
metabolism of this agent and the liver may play an important role in nanocrystal excretion via the hepatobiliary
system. The retention of the nanocrystals in the liver and
spleen may be due to the long life span of the macrophages
in these organs. The lack of organ or tissue damage observed from histologic evaluation, combined with the normal blood biochemical data and lack of obvious behavioral
or body weight changes, suggests the overall nontoxic
nature of FeCo/GC nanocrystals.
Lee et al.
ACKNOWLEDGMENTS
This work was supported in part by the Stanford Bio-X
Interdisciplinary Initiative Grant, CCNT-TR Project 2 at
Stanford, NIH 1R01 HL075803, R01 HL078678, and R21
CA133492. The authors thank AMAG Pharmaceuticals,
Inc. for providing Ferumoxtran-10 (Combidex威) and GE
Healthcare for scanner support. The authors also thank
Jennifer Lyons and Juan M. Santos.
REFERENCES
1. Rivard A, Isner JM. Angiogenesis and vasculogenesis in treatment of
cardiovascular disease. Mol Med 1998;4:429 – 440.
2. Ross R. Angiogenesis: successful growth of tumours. Nature 1989;339:
16 –17.
3. Kobayashi H, Sato N, Hiraga A, Saga T, Nakamoto Y, Ueda H, Konishi
J, Togashi K, Brechbiel MW. 3D-micro-MR angiography of mice using
macromolecular MR contrast agents with polyamidoamine dendrimer
core with reference to their pharmacokinetic properties. Magn Reson
Med 2001;45:454 – 460.
4. Kobayashi H, Sato N, Kawamoto S, Saga T, Hiraga A, Ishimori T,
Konishi J, Togashi K, Brechbiel MW. 3D MR angiography of intratumoral vasculature using a novel macromolecular MR contrast agent.
Magn Reson Med 2001;46:579 –585.
5. Fink C, Kiessling F, Bock M, Lichy MP, Misselwitz B, Peschke P,
Fusenig NE, Grobholz R, Delorme S. High-resolution three-dimensional
MR angiography of rodent tumors: morphologic characterization of
intratumoral vasculature. J Magn Reson Imaging 2003;18:59 – 65.
6. Mahfouz AE. Ms-325 Epix. Curr Opin Investig Drugs 2000;1:476 – 480.
7. Gadofosveset: MS 325, MS 32520, Vasovist, ZK 236018. Drugs R D
2004;5:339 –342.
8. Weissleder R, Elizondo G, Wittenberg J, Rabito CA, Bengele HH, Josephson L. Ultrasmall superparamagnetic iron oxide: characterization
of a new class of contrast agents for MR imaging. Radiology 1990;175:
489 – 493.
9. Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara
N, Symes JF, Isner JM. Therapeutic angiogenesis: a single intraarterial
bolus of vascular endothelial growth factor augments revascularization
in a rabbit ischemic hind limb model. J Clin Invest 1994;93:662– 670.
10. Seo WS, Lee JH, Sun X, Suzuki Y, Mann D, Liu Z, Terashima M, Yang
PC, McConnell MV, Nishimura DG, Dai H. FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared
agents. Nat Mater 2006;5:971–976.
11. Kosuge H, Terashima M, Sherlock S, Lee JH, Dai H, McConnell MV.
Graphite/metal core-shell nanocrystals as MRI contrast agents to detect
vascular inflammation. Los Angeles, CA: 2008. 2105 p.
12. Lee JH, Seo WS, Terashima M, Suzuki Y, Yang P, McConnell MV,
Nishimura DG, Dai H. FeCo/graphitic carbon-shell nanocrystals as MRI
contrast agents for cellular and vascular imaging. Berlin, Germany:
2007. 858 p.
13. Lee JH, Sherlock S, Terashima M, Kosuge H, Seo WS, Suzuki Y, McConnell MV, Nishimura DG, Dai H. In-vivo ultra-high resolution imaging of small vessels using improved sensitivity and long circulation
time of FeCo-graphitic carbon shell nanocrystals. Los Angeles, CA:
2008. 2119 p.
14. Liu Z, Davis C, Cai W, He L, Chen X, Dai H. Circulation and long-term
fate of functionalized, biocompatible single-walled carbon nanotubes
in mice probed by Raman spectroscopy. Proc Natl Acad Sci U|S|A
2008;105:1410 –1415.
15. Xu C, Zarins CK, Pannaraj PS, Bassiouny HS, Glagov S. Hypercholesterolemia superimposed by experimental hypertension induces differential distribution of collagen and elastin. Arterioscler Thromb Vasc
Biol 2000;20:2566 –2572.
16. Omary RA, Frayne R, Unal O, Warner T, Korosec FR, Mistretta CA,
Strother CM, Grist TM. MR-guided angioplasty of renal artery stenosis
in a pig model: a feasibility study. J Vasc Interv Radiol 2000;11:373–
381.
17. Haacke EM, Brown RW, Thompson MR, Venkatesan R. Magnetic resonance imaging physical principles and sequence design. Wiley-Liss;
1999.
18. Ernst RR, Anderson WA. Application of Fourier transform spectroscopy to magnetic resonance. Rev Sci Instrum 1966;37.
FeCo/GC Magnetic Nanocrystals
19. Block W, Pauly J, Kerr A, Nishimura D. Consistent fat suppression with
compensated spectral-spatial pulses. Magn Reson Med 1997;38:198 –206.
20. Stanisz GJ, Odrobina EE, Pun J, Escaravage M, Graham SJ, Bronskill MJ,
Henkelman RM. T1, T2 relaxation and magnetization transfer in tissue
at 3T. Magn Reson Med 2005;54:507–512.
21. Mornet S, Vasseur S, Grasset F, Duguet E. Magnetic nanoparticle design
for medical diagnosis and therapy. J Mater Chem 2004;14:2161–2175.
22. Xu YH, Bai HM, Wang JP. High-magnetic-moment multifunctional
nanoparticles for nanomedicine applications. J Magn Magn Mater 2007;
311:131–134.
23. Lee JH, Huh YM, Jun Y, Seo J, Jang J, Song HT, Kim S, Cho EJ, Yoon HG,
Suh JS, Cheon J. Artificially engineered magnetic nanoparticles for
ultra-sensitive molecular imaging. Nat Med 2007;13:95–99.
24. Lee SW, Bae S, Takemura Y, Shim IB, Kim TM, Kim J, Lee HJ, Zurn S,
Kim CS. Self-heating characteristics of cobalt ferrite nanoparticles for
hyperthermia application. J Magn Magn Mater 2007;310:2868 –2870.
25. Xu YH, Wang JP. FeCo-Au core-shell nanocrystals. Appl Phys Lett
2007;91(233107).
26. Schenck JF, Hart HR Jr, Foster TH, Edelstein WA, Hussain MA. High
resolution magnetic resonance imaging using surface coils. Magn Reson Annu 1986;123–160.
27. Irarrazabal P, Nishimura DG. Fast three dimensional magnetic resonance imaging. Magn Reson Med 1995;33:656 – 662.
28. Lee JH, Hargreaves BA, Hu BS, Nishimura DG. Fast 3D imaging using
variable-density spiral trajectories with applications to limb perfusion.
Magn Reson Med 2003;50:1276 –1285.
29. McNally MA, Small JO, Mollan RA, Wilson DJ. Arteriographic study of
the rabbit lower limb. Anat Rec 1992;233:643– 650.
30. Greve JM, Chico TJ, Goldman H, Bunting S, Peale FV Jr, Daugherty A,
van Bruggen N, Williams SP. Magnetic resonance angiography reveals
therapeutic enlargement of collateral vessels induced by VEGF in a
murine model of peripheral arterial disease. J Magn Reson Imaging
2006;24:1124 –1132.
31. Kiessling F, Heilmann M, Lammers T, Ulbrich K, Subr V, Peschke P,
Waengler B, Mier W, Schrenk HH, Bock M, Schad L, Seminmler W.
1509
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Synthesis and characterization of HE-24.8: a polymeric contrast agent
for magnetic resonance angiography. Bioconjug Chem 2006;17:42–51.
Kiessling F, Morgenstern B, Zhang C. Contrast agents and applications
to assess tumor angiogenesis in vivo by magnetic resonance imaging.
Curr Med Chem 2007;14:77–91.
Barrett T, Kobayashi H, Brechbiel M, Choyke PL. Macromolecular MRI
contrast agents for imaging tumor angiogenesis. Eur J Radiol 2006;60:
353–366.
Port M, Corot C, Violas X, Robert P, Raynal I, Gagneur G. How to
compare the efficiency of albumin-bound and nonalbumin-bound contrast agents in vivo: the concept of dynamic relaxivity. Invest Radiol
2005;40:565–573.
Food and Drug Administration. Gadolinium-containing contrast
agents for magnetic resonance imaging (MRI): Omniscan, OptiMARK, Magnevist, ProHance, and MultiHance. Food and Drug
Administration; 2006.
Herborn CU, Runge VM, Watkins DM, Gendron JM, Naul LG. MR
angiography of the renal arteries: intraindividual comparison of double-dose contrast enhancement at 1.5 T with standard dose at 3 T. AJR
Am J Roentgenol 2008;190:173–177.
Wang J, Reykowski A, Dickas J. Calculation of the signal-to-noise ratio
for simple surface coils and arrays of coils. IEEE Trans Biomed Eng
1995;42:908 –917.
Wiggins GC, Triantafyllou C, Potthast A, Reykowski A, Nittka M, Wald
LL. 32-Channel 3 tesla receive-only phased-array head coil with soccerball element geometry. Magn Reson Med 2006;56:216 –223.
Thedens DR, Irarrazaval P, Sachs TS, Meyer CH, Nishimura DG. Fast
magnetic resonance coronary angiography with a three-dimensional
stack of spirals trajectory. Magn Reson Med 1999;41:1170 –1179.
Irarrazaval P, Santos JM, Guarini M, Nishimura D. Flow properties of
fast three-dimensional sequences for MR angiography. Magn Reson
Imaging 1999;17:1469 –1479.
Derelanko MJ, Hollinger MA. CRC handbook of toxicology. Boca Raton,
FL: CRC Press; 1995. 948 p.