Download Nanotechnology in Medical Imaging - Arteriosclerosis, Thrombosis

ATVB In Focus
Molecular Imaging of Cardiovascular Disease
Series Editor: Zahi Fayad
Nanotechnology in Medical Imaging
Probe Design and Applications
David P. Cormode, Torjus Skajaa, Zahi A. Fayad, Willem J.M. Mulder
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Abstract—Nanoparticles have become more and more prevalent in reports of novel contrast agents, especially for
molecular imaging, the detection of cellular processes. The advantages of nanoparticles include their potency to generate
contrast, the ease of integrating multiple properties, lengthy circulation times, and the possibility to include high
payloads. As the chemistry of nanoparticles has improved over the past years, more sophisticated examples of
nano-sized contrast agents have been reported, such as paramagnetic, macrophage targeted quantum dots or
␣v␤3-targeted, MRI visible microemulsions that also carry a drug to suppress angiogenesis. The use of these particles
is producing greater knowledge of disease processes and the effects of therapy. Along with their excellent properties,
nanoparticles may produce significant toxicity, which must be minimized for (clinical) application. In this review we
discuss the different factors that are considered when designing a nanoparticle probe and highlight some of the most
advanced examples. (Arterioscler Thromb Vasc Biol. 2009;29:992-1000.)
Key Words: nanotechnology 䡲 molecular imaging 䡲 magnetic resonance imaging 䡲 drug delivery 䡲 gene therapy
O
ne of the main current focuses of research in medical
diagnostics is molecular imaging, as described in an
accompanying article in this issue by Choudhury et al.
Molecular imaging can facilitate early diagnosis, identify the
stage of disease, provide fundamental information on pathological processes, and can be applied to follow the efficacy of
therapy. Molecular imaging heavily relies on the development of sophisticated probes needed to detect biological
processes on the cellular and molecular level.1–5 Nanoparticulate probes have shown noteworthy advantages over single
molecule-based contrast agents. These advantages include
producing excellent contrast (eg, quantum dots6), integrating
multiple properties such as several types of contrast generating materials,7 lengthy circulation time,8,9 and the possibility
to include high payloads.10 Importantly, nanoparticles allow
the components of a molecular imaging contrast agent to be
easily assembled in an efficient ratio. As a result, very
exciting molecular imaging results are being reported, such as
agents that can be detected by multiple imaging techniques,7
agents that also deliver therapeutics,11 agents that detect
particular cell types,12 or agents that allow new imaging
systems to be developed.3,13 Other articles in this issue will
expound on the progress that has been made by applying
nanoparticles for molecular imaging in cardiovascular disease; we will confine our discussion to nanoparticle design.
The design of effective nanoparticle contrast agents for
molecular imaging requires careful consideration of the
properties required for the application in question. Once the
required properties have been established, candidate nanoparticle platforms may be identified. The particle synthesis can
then be optimized to generate an assembly with the appropriate contrast/therapeutics included, optimized surface coating, targeting properties, defined size, and a high degree of
biocompatibility. In this review we will first discuss the
current state of the art in nanoparticle contrast agent design
and give some examples of agents whose design is very
advanced. This is followed by a discussion about the available options and factors influencing nanoparticle design.
State of the Art Nanoparticle Contrast
Agent Design
The latest designs of nanoparticulate molecular imaging
contrast agents incorporate the appropriate contrast generating materials (eg, fluorescent, radioactive, paramagnetic,
superparamagnetic, or electron dense), targeting groups, a
biocompatible coating and the possibility for other functionalities such as a therapeutic.2 A schematic of a generalized
nanoparticle is shown in Figure 1A. Sources of contrast
enhancing materials or therapeutics can be included in the
particle core, within the coating, or attached to the surface.
This flexibility allows the inclusion of multiple types of
contrast agents or a combination of contrast and therapeutic
agents. This is an especially important feature for preclinical
Received October 9, 2008; revision accepted November 24, 2008.
From the Translational and Molecular Imaging Institute (D.P.C., T.S., Z.A.F., W.J.M.M.), Mount Sinai School of Medicine, New York; and the Faculty
of Health Sciences (T.S.), Århus University, Denmark.
Correspondence to Willem J.M. Mulder, Translational and Molecular Imaging Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place,
Box 1234, New York, NY 10029. E-mail [email protected]
© 2009 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
992
DOI: 10.1161/ATVBAHA.108.165506
Cormode et al
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Figure 1. A, Generalized schematic of a nanoparticle-based
molecular imaging contrast agent. B, Schematic of a quantum
dot-based paramagnetic contrast agent targeted to macrophages via CD204 antibodies.
contrast agents so that they can be detected with a clinical
imaging modality (eg, MRI) and fluorescence techniques to
confirm the localization of the agent in the target tissues or
cell types. The combination of contrast and therapeutic agents
is known as “theranostics”14 and is attractive as it allows
imaging of drug, protein, or gene delivery. An important
Nanotechnology and Probe Design
993
aspect of nanoparticles is that multiple properties can be
easily integrated at the desired ratios of contrast and/or
therapeutic agents, fluorescence, and targeting groups.
Mulder et al recently reported a multifunctional nanoparticle for the imaging of macrophages in atherosclerosis.15
This agent comprises a micellular coating that encloses a
quantum dot core, as depicted in Figure 1B. The micelle is
formed around the quantum dot by hydrating a mixed film
that contains quantum dots, polyethylene glycol (PEG) modified phospholipids, gadolinium labeled lipids (for MRI
contrast), and a PEG-phospholipid that has a reactive group to
enable functionalization. The relative proportions of gadolinium to fluorophore to targeting moiety can be controlled by
the lipid/amphiphile inputs; these properties are combined in
the particle in only 2 steps, which is followed by a single
purification step. This methodology is much simpler and
more efficient than a multi-step synthesis of 1 molecule that
contains the targeting group, contrast agent, and fluorophore.
A team from Washington University has extensively investigated perfluorocarbon-based microemulsions as contrast
agents targeted to atherosclerotic plaque components such as
␣v␤3-integrin,11,16 fibrin,17 or collagen III.18 The perfluorocarbon core allows “hotspot” MR imaging using systems
tuned to the 19F nucleus19,20 and the inclusion of hydrophobic
drugs. In this highlighted example, the antiangiogenesis drug
fumagillin was included in the particle, along with gadolinium chelates in the phospholipid coating to provide contrast
for conventional MRI (Figure 2A).11 The particle was targeted using a peptidomimetic specific for the ␣v␤3-integrin,
which is overexpressed in angiogenesis. In a theranostic
approach, the authors applied the agent twice to a rabbit
model of atherosclerosis in which angiogenesis is pronounced
Figure 2. A, Schematic depiction of a microemulsion-based contrast agent/drug delivery nanoparticle. B, MR images of the aortas of
hyperlipidemic rabbits injected with the microemulsion contrast agent with or without the antiangiogenesis drug fumagillin. The superimposed color maps denote the percent signal enhancement in the aorta. C, Quantification of enhancement on agent injection pre- and
1 week posttreatment showing reduction of angiogenesis when the drug was included in the particle. Parts of this figure reproduced,
with permission, from Winter et al.11
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in the adventitia of the arteries. When MR images were
acquired 4 hours after the first injection there was a significant increase in the intensity of the aorta attributable to
accumulation of the contrast agent, whereas after the second
injection, 1 week later, the increase in intensity of the aorta
was scant (Figure 2B and 2C). This effect was ascribed to the
antiangiogenic effect of the fumagillin delivered in the first
injection, hence reducing microvessel density and therefore
accumulation of the agent after the second microemulsion
application.
Iron oxide nanoparticles are sensitive contrast agents for MRI.
A highly sophisticated iron oxide particle has recently been
reported to knock out gene expression for therapeutic intervention.21 The nanoparticle has a cross-linked aminated dextran
shell, to which was first attached the near-infrared dye Cy5.5,
then the membrane translocation myristoylated polyarginine
peptide (MPAP), and lastly siRNA for gene silencing (Figure
3A). The particle design allows its detection by both MR and
fluorescence techniques, enables efficient uptake by cells,
and can silence specific genes. In initial experiments, the
siRNA used was antisense to green fluorescence protein
(GFP). Mice were inoculated with 2 tumors, one expressing
GFP, the other red flurorescent protein (RFP). Uptake of the
particle in these tumors was established by MR and fluorescence methods both in vitro and in vivo. Fluorescence
imaging revealed that expression of GFP was knocked down
by the siRNA-iron oxide particle by as much as 85%, whereas
red fluorescent protein expression remained unaffected. Furthermore, a therapeutic version of the particle was created
where the siRNA attached was antisense to survivin, an
inhibitor of apoptosis proteins. In this case also, nanoparticle
uptake was established using MR and fluorescence imaging
performed 24 hours after administration of the agent (Figure
3B and 3C). After 4 injections of the agent over 2 weeks,
survivin levels in the tumors of these mice were knocked
down to 3% of the levels in control animals (Figure 3D). In
addition, apoptosis and necrosis was increased in the tumors
of treated mice, as found via immunofluorescence and histology, hence this iron oxide–siRNA agent is an effective
therapeutic for cancer.
Aspects of Nanoparticle Contrast
Agent Design
Nanoparticle Selection
A wide range of nanoparticles has been proposed for use as
contrast agents with each imaging modality requiring nanoparticles of different properties for contrast production. In this
section we will briefly describe the nanoparticles that have
been applied for each modality.
Gadolinium-labeled nanoparticles such as liposomes,22 micelles,12 microemulsions,23 lipoproteins,24 viruses,25 and carbon nanotubes26 are suitable for providing contrast for T1weighted MRI via the paramagnetism of this element.
Superparamagnetic iron oxides have been widely exploited to
induce contrast for T2(*)-weighted MRI.1 As both types of
MRI can be carried out using any MRI system, one can
choose between iron or gadolinium as the basis of contrast,
depending on the application. Although the sensitivity of
detection of gadolinium chelate enriched nanoparticles is
generally lower than that of iron oxide nanoparticles,27
gadolinium produces positive contrast that is easy to assign to
agent accumulation in that tissue. Iron oxides induce negative
contrast or signal loss, and because there are many sources of
signal loss in MR images other than iron oxide accumulation,
it may be hard to ascribe signal loss to the iron contrast agent
with certainty. New image acquisition sequences that produce
positive contrast from iron oxides may mitigate this difficulty.28,29
There are several factors to weigh when deciding whether to
use iron oxides or gadolinium based nanoparticles. For
example, if the target to be imaged is expressed in very low
concentrations, the sensitivity of iron oxide is very useful.
Iron oxides are also well suited for imaging in tissues that
produce homogenous MRI signals and have predictable
structures, such as the brain.30 In regions of the body where
the structure is less predictable and signal voids commonly
occur, such as the abdomen, gadolinium-based agents have an
advantage as they produce positive contrast.
Quantum dots provide excellent contrast for fluorescent
imaging. They have wide excitation windows,31 narrow
emission windows,32 they fluoresce with high efficiency,6 and
are not prone to photobleaching.33 When applying fluorescent
labels to other kinds of nanoparticles, using fluorophores that
both absorb and emit light in the near-infrared window (650
to 900 nm), where the absorbance of tissue is low, is
advocated.34 This is important for fluorescence tomography,
where light has to pass through substantial tissue thickness,
but not important if the application is confocal microscopy,
for example, as light only has to penetrate tissue sections
around 10 ␮m thick.
The nanoparticles that have been proposed as CT contrast
agents tend to be based on electron dense and high atomic
number elements such as iodine,35 bismuth,36 or gold.37
Because contrast in computed tomography requires high
payloads of such elements to be delivered, most researchers
have based their agents on solid nanoparticles, but some have
proposed liposomes in whose aqueous interior iodinated
molecules are trapped.38 Gold nanoparticles are becoming a
popular choice for CT,39 although it is unclear as yet whether
this is the optimal material to use; other elements have
superior X-ray attenuation at clinical energies.40 Recently
Hyafil et al reported a solid, iodine-based nanoparticle that
was specifically taken up by macrophages in a rabbit model
of atherosclerosis.35 This report demonstrated the possibility
of molecular imaging in vivo using CT, an application for
which CT had been thought to be too insensitive.41
Ultrasound requires contrast agents that provide backscatter of sound waves to the scanner head. This is normally
achieved using micron-sized bubbles (and hence not technically nanoparticles) of a water insoluble gas such as decafluorobutane.42 The bubbles require coatings to make them
stable, but coatings that produce excellent stability reduce the
particle flexibility needed to yield good contrast. As a
consequence, the coatings currently used often compromise
between stability and flexibility.43 Multi-layer vesicles have
also been used as ultrasound contrast agents, although it has
been shown that the echogenic response they generate may be
attributable to entrapped gas pockets.44 In addition, micro-
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Nanotechnology and Probe Design
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Figure 3. A, Schematic depiction of an iron oxide– based MR contrast agent that can deliver siRNA. B, MR image of a mouse pre- and
24 hours postadministration of the agent. Change in pixel color in the tumor (arrow) from red-yellow to blue indicates accumulation of
the agent. C, Fluorescence image of the mouse with emission seen from the tumor (arrow), also indicating agent accumulation. D,
Silencing of the Birc5 gene by the siRNA-labeled nanoparticle (MN-NIRF-siSurvivin) reduces Survivin expression in the tumor and leads
to apoptosis in this tissue. Parts of this figure reproduced, with permission, from Medarova et al.21
emulsions have been reported to act as ultrasound contrast
agents.45
Conceptually, it is possible to include drugs in any nanoparticle, as drugs may be attached to surfaces, incorporated in
coatings, or stowed in cores. It is worth noting the hydrophobic/hydrophilic nature of the drug, however, when making the
choice of nanoparticle for drug delivery. Hydrophobic drugs
are more easily and more stably incorporated into nanoparticles with a hydrophobic compartment than those without,11
and the corresponding concept holds true for hydrophilic
drugs.46
Coating Types
A lot of functionally active materials used to generate
contrast for molecular imaging have a very low biocompatibility, which leads to fast excretion, low circulating half-life,
decreased stability, and potential toxicity. Therefore, great
efforts have been undertaken to render these materials bio-
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Figure 4. A, Schematic depictions of lipid-coated and bare
silica particles that have a
quantum dot in their cores. B,
MRI of the vasculature of a
mouse before and after administration of the paramagnetic,
PEG-lipid– coated silica particles. C, Cell viability assays
indicating lowered cytotoxicity
of the lipid-coated particles. D,
Blood clearance profiles of the
silica particles showing the
enhanced circulation half-life of
the coated particles. E, Transmission electron micrographs of
mouse lungs after injection with
silica particles. Large aggregates of the particles were
found in the lungs of mice
injected with bare particles. The
images in this figure are reproduced, with permission, from
van Schooneveld et al.55
applicable. A variety of different approaches have been
reported, using materials such as phospholipids,27 dextran,47
polyvinylpyrrolidone,48 PEG,49 or silica as coatings.50 Furthermore, it is now common practice to incorporate additional
functional labels in the coating corona, rendering the nanoparticles multimodal.2 These coatings increase the hydrophilicity of nanoparticles, thereby decreasing aggregation, but as
well as this improvement in physical properties there are
beneficial biological effects. By coating nanoparticles that are
otherwise foreign to the body, considerably enhanced blood
half-lives are produced, by avoiding opsonization and “fooling” the defense mechanism of the body to some extent.
PEGylation has been very widely used as a coating strategy
for nanoparticles to enhance their circulation half-life by
avoiding fast removal by the reticuloendothelial system.9,51
Such prolonged circulation half-lives can be advantageous as
they allow an increased amount of the injected material to
reach and bind the desired sites. Silica has been used as a
nanoparticle delivery platform for a variety of cargoes such as
drugs,52 genes,53 proteins,54 or contrast-generating materials.50 Recently it has been shown by van Schooneveld et al
how quantum dot-containing silica particles can be coated
with PEGylated lipids to improve their biocompatibility, as
compared to silica particles with unmodified surfaces (Figure
4A).55 The lipid-coated particles had paramagnetic lipids
included in this coating as well and hence could be used to
visualize the vasculature (Figure 4B). These lipid-coated
particles displayed lower cytotoxicity, exhibited a 10-fold
increase in circulation half-life, and did not form aggregates
in the lungs (Figure 4C through 4E). In summary, the
lipid-coating caused a spectacular improvement in short-term
biocompatibility, however it remains to be investigated how
silica is metabolized and excreted from the body over longer
periods.
An alternative strategy to synthetic coatings is the use of
natural nanoparticles such as viruses or lipoproteins, thereby
evading recognition by the body’s defense systems. To this
aim, 2 routes have been pursued. Firstly, the exterior/surface
of the natural nanoparticle can be modified to contain contrast
generating ions and dyes.25,56 The second approach forms
novel blends of inorganic and natural materials by including
inorganic nanoparticles in the core of the virus or lipoprotein.57–59 The latter approach was recently exemplified by a
study by Cormode et al, where iron oxides, quantum dots, or
gold nanoparticles were incorporated into high-density lipoprotein (HDL) (Figure 5A), as shown by negative stain
transmission electron microscopy (TEM) in Figure 5B.59 The
iron oxide, quantum dot, and gold HDL nanoparticles were
termed FeO-HDL, QD-HDL, and Au-HDL, respectively.
Paramagnetic or fluorescent phospholipids were incorporated
into the particles so that each particle provided contrast for
MRI and fluorescence techniques, as well as for CT in the
case of Au-HDL. These particles were shown to be taken up
by macrophage cells in vitro and in a mouse model of
atherosclerosis with MRI, fluorescence imaging, and computed tomography (Figure 5C through 5G).
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Nanotechnology and Probe Design
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are species specific and may generate immunologic responses. Smaller ligands can also be specific, are cheaper, but
may not be available for the target in question. Consequently,
phage display has been a boon to the field by identifying short
peptidic sequences for specific targeting.65 Furthermore, techniques such as that reported by Weissleder et al, where a large
library of small molecule-targeted nanoparticles were
screened against several cell types, are useful for identifying
cheap and simple targeting ligands.70 After identifying the
appropriate ligand, its linkage with the nanoparticle has to be
taken into consideration. The biotin-streptavidin linkage has
been frequently exploited,71 but gives immunogenic responses in patients.72 To avoid this immunogenic response,
ligands may be covalently attached to nanoparticles using a
variety of methods.73
Effect of Size
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Figure 5. A, Schematic depiction of nanocrystal-core high density lipoprotein (HDL). B, Negative stain TEM of FeO-HDL. C,
Photograph of pellets of cells incubated with QD-HDL, QD-PEG,
or without a contrast agent taken under UV irradiation. MR
images of the aorta of an apoE KO mouse taken (D) before and
(E) 24 hours after injection of FeO-HDL. F, Micro-CT image of
an excised aorta of a mouse injected 24 hours previously with
Au-HDL. G, Fluorescence image of an excised aorta of a mouse
injected 24 hours previously with QD-HDL. Parts of this figure
reproduced, with permission, from Cormode et al.59
Targeting Strategies
There are numerous methods of targeting nanoparticles. In
tissues where there is significant angiogenesis, such as in
cancer and inflammatory diseases like atherosclerosis,60 the
consequent leaky and chaotic vasculature results in an increased accumulation of particles with a long circulation
half-life attributable to the enhanced permeability and retention (EPR) effect.22,61 Therefore, in cases when angiogenic
tissues are the pathologies of interest no targeting ligands are
required, but it must be ensured that the nanoparticle coating
engenders a long blood half-life. Another passive form of
targeting uses dextran as the particle coating. Iron oxide
nanoparticles coated with dextran are avidly taken up by
macrophages.62 Several different types of molecules can be
attached to nanoparticles to yield active targeting, including
antibodies,12 antibody fragments,63 proteins,27,64 peptides,65
peptidomimetics,11 aptamers,66 sugars,67 and small molecules.68 It is important that the targeting ligand exhibits a high
degree of specificity, but there are other factors to consider as
well. For example, although antibodies are very specific, they
are expensive and their attachment may increase the size of
the particle by as much as 10 nm.69 Furthermore, antibodies
Nanoparticle size plays a critical role for a number of aspects,
including the cell types that can be targeted, payload, clearance/excretion, and the strength/quality of contrast produced.
The size of nanoparticles has a considerable impact on the
biodistribution of the particles and thus plays a prominent
role in the ability to target different cells. Inflamed pathological tissues that possess highly permeable vasculature, such as
tumors or atherosclerotic plaques, can be infiltrated by
nanoparticles. This infiltration is more likely if the nanoparticles are small.
The size range of nanoparticles is determined by the
components used for synthesis, and considerable efforts have
been devoted to developing procedures that produce particles
of a defined size. As a consequence the size and shape of
inorganic nanocrystals such as iron oxide, gold nanoparticles,
silica, or quantum dots as well as lipid-based nanoparticles
can now be precisely controlled.74 –77 Nevertheless, the total
diameter of a particle using the same core can widely differ
depending on the coating used.
Furthermore, the sizes of nanoparticles are important in the
excretion of the particles form the body. Choi et al showed
recently that there is a cut-off point in which nanoparticles are
cleared from the body via the renal system.78 With fluorescence techniques the authors showed that nanoparticles equal
or smaller than 5.5 nm are excreted through the renal system,
whereas particles larger than that are mainly taken up by the
reticuloendothelial system where they end up in the liver and
spleen. Here they are metabolized and excreted, or they
accumulate and potentially become toxic for the body. On the
other hand, if particles are small enough to be renally
excreted, their half-life is reduced.
Altering the size of nanoparticles may tune the contrast
they generate. For example, the fluorescent emission of
quantum dots (QDs) increases in wavelength with increasing
size.79 Therefore the emission of these particles can be tuned
to the desired wavelength by control of the size synthesized.
Iron oxide particles give excellent contrast for MRI in a very
wide size range (1 nm to 1 ␮m),27,80 but are only useful as
contrast agents in the nascent magnetic particle imaging
(MPI) technique when larger than 20 nm.81 Larger particles
can carry high payloads, which may be advantageous.
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As we have now discussed, size has an impact on biodistribution, clearance, and contrast produced. An important
question scientists face is, therefore, how to determine the
size of their particles.82 Many different techniques are currently available, but proper use of them is critical, because
each of the techniques return different information. For
example, transmission electron microscopy will give the
diameter of inorganic cores, but often not the coating,
scanning electron microscopy will return the diameter including the coating, whereas dynamic light scattering gives the
hydrodynamic diameter. However, the hydrodynamic diameter will vary depending on the concentration of the sample
and the buffer used.
Synthetic Strategies
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It is advantageous to be able to synthesize and coat (with a
biocompatible material) nanoparticles in a 1-step aqueous
process, as the U.S. Food and Drug Administration (USFDA)
approval process is easier in this case. The type of coating in
these situations is normally a water-soluble polymer. Nanoparticles such as liposomes or micelles do not need a coating
to be applied, as they are largely biocompatible as is. Organic
phase syntheses of solid nanoparticles lead to better control of
the size and hence contrast properties, in many cases.74,79,83
Nanoparticles synthesized in the organic phase are usually
insoluble in water and require a second coating step to render
them bioapplicable. Fortunately, there are several efficient
methods for doing this, normally using amphiphilic polymers
or molecules such as phospholipids.55,84,85
Another crucial consideration before synthesis is how
additional functionalities such as targeting groups or fluorophores will be attached to the surface. In the case of
cross-linked iron oxides, the dextran coating of these particles
is modified with amine groups,86 which can then be conjugated with targeting groups, fluorophores, or chelates for
metal ions.7 In the example featured before, where there is a
quantum dot at the core of a micelle (Figure 1B), the
biocompatible phospholipid/PEG coating is self-assembled
around the nanoparticle. To attach targeting moieties using
this type of strategy, a small amount of a phospholipid with a
reactive functionality is included. In that particular example
maleimide was used,15 but various others are available, such
as amines, carboxylic acids, pyridyl dithiopropionate (PDP),
and biotin.
Minimizing Toxicity
Nanoparticles are subject to the normal strictures with regards
to toxicity as other drugs or contrast agents applied to
humans. There may eventually be additional restrictions on
their use, however, as larger particles will remain in the body,
primarily in the liver and spleen.55 How these particles break
down and how the body reacts to them will determine
whether they can eventually be used in humans. The fact that
the USFDA has not issued guidelines or standards for
nanoparticles,87 despite calls for it to do so,88 is further
complicating the picture. Nevertheless, impressive efforts are
being made to determine the type of interactions nanoparticles will have in the body.89 For example, Shaw et al used
an array of 64 in vitro assays to elucidate the effect of
nanoparticles on a variety of cellular processes.90
Certain nanoparticles break down into nontoxic materials
and thus can be considered safe. Iron oxide particles are
injected at a dose of iron that, if injected as free ions, would
be toxic because of the limited ability of the body to process
iron. As the iron oxide is injected in a crystalline form, it is
nontoxic and breaks down slowly to release iron ions at a rate
that is manageable.1 Another option is that the injected
material will not break down, which could be the case for
gold nanoparticles as that element is quite inert. If the
particles break down into highly toxic substances, as is the
case for quantum dots (which release cadmium), it is unlikely
they can seriously be considered for clinical use.91 Particles
such as these are, however, very useful in preclinical settings.
Conclusions
Extremely effective molecular imaging contrast agents can be
synthesized using nanoparticles as their basis. The design of
these probes has improved significantly over the past decade,
with multifunctionality, more efficient targeting, better biocompatibility, and agents suited to each available imaging
modality being among these improvements. At the current
stage of development, generally speaking, a nanoparticle
contrast agent possessing the required attributes can be
synthesized for any desired application. However, there is
scope for significant enhancements in biocompatibility, efficacy, specificity, and detection of further disease targets.
Furthermore, standardized nanoparticle platforms that can be
used for preclinical/clinical disease investigations need to be
developed. Lastly, nanotechnology will continue to produce
new particles that possess novel and interesting properties
and will be exploited in medical imaging, as will medical
imaging continue to be developed, requiring new nanoparticle
formulations as contrast agents.
Acknowledgments
We thank the Danish Heart Association for studentship 07-10A1655-22406 (to T.S.). Partial support was provided by NIH/NHLBI
ROI HL71021, NIH/NHLBI HL78667 (to Z.A.F.).
Disclosures
None.
References
1. Corot C, Robert P, Idee J-M, Port M. Recent advances in iron oxide
nanocrystal technology for medical imaging. Adv Drug Deliv Rev. 2006;
58:1471–1504.
2. Mulder WJM, Griffioen AW, Strijkers GJ, Cormode DP, Nicolay K,
Fayad ZA. Magnetic and fluorescent nanoparticles for multimodality
imaging. Nanomedicine. 2007;2:307–324.
3. Huang X, Jain PK, El-Sayed IH, El-Sayed M. Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and
therapy. Nanomedicine. 2007;2:681– 693.
4. Azzazy HME, Mansour MMH, Kazmierczak SC. From diagnostics to
therapy: Prospects of quantum dots. Clin Biochem. 2007;40:917–927.
5. Wickline SA, Neubauer AM, Winter PM, Caruthers SD, Lanza GM.
Molecular imaging and therapy of atherosclerosis with targeted nanoparticles. J Magn Reson Imaging. 2007;25:667– 680.
6. Medintz IL, Uyeda HT, Goldman ER, Mattoussi H. Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater. 2005;4:
435– 446.
7. Nahrendorf M, Zhang H, Hembrador S, Panizzi P, Sosnovik DE, Aikawa
E, Libby P, Swirski FK, Weissleder R. Nanoparticle PET-CT imaging of
Cormode et al
8.
9.
10.
11.
12.
Downloaded from http://atvb.ahajournals.org/ by guest on May 5, 2017
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
macrophages in inflammatory atherosclerosis. Circulation. 2008;117:
379 –387.
Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipatic polyethyleneglycols effectively prolong the circulation time of liposomes.
FEBS Lett. 1990;268:235–238.
Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes
containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta. 1991;1066:
29 –36.
Schmieder AH, Winter PM, Caruthers SD, Harris TD, Williams TA,
Allen JS, Lacy EK, Zhang HY, Scott MJ, Hu G, Robertson JD, Wickline
SA, Lanza GM. Molecular MR imaging of melanoma angiogenesis with
alpha(nu)beta(3)-targeted paramagnetic nanoparticles. Magn Res Med.
2005;53:621– 627.
Winter PM, Neubauer AM, Caruthers SD, Harris TD, Robertson JD,
Williams TA, Schmieder AH, Hu G, Allen JS, Lacy EK, Zhang HY,
Wickline SA, Lanza GM Endothelial alpha (v) beta(3) integrin-targeted
fumagillin nanoparticles inhibit angiogenesis in atherosclerosis. Arterioscler
Thromb Vasc Biol. 2006;26:2103–2109.
Amirbekian V, Lipinski MJ, Briley-Saebo KC, Amirbekian S, Aguinaldo
JG, Weinreb DB, Vucic E, Frias JC, Hyafil F, Mani V, Fisher EA, Fayad
ZA. Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI. Proc Natl Acad Sci U S A.
2007;104:961–966.
Durr NJ, Larson T, Smith DK, Korgel BA, Sokolov K, Ben-Yakar A.
Two-photon luminescence imaging of cancer cells using molecularly
targeted gold nanorods. Nano Lett. 2007;7:941–945.
Del Vecchio S, Zanneti A, Fonti R, Pace L, Salvatore M. Nuclear imaging
in cancer theranostics. Quart J Nucl Med Mol Im. 2007;51:152–163.
Mulder WJM, Strijkers GJ, Briley-Saeboe KC, Frias JC, Aguinaldo JGS,
Vucic E, Amirbekian V, Tang C, Chin PTK, Nicolay K, Fayad ZA.
Molecular imaging of macrophages in atherosclerotic plaques using
bimodal PEG-micelles. Magn Res Med. 2007;58:1164 –1170.
Winter PM, Caruthers SD, Zhang H, Williams TA, Wickline SA, Lanza
GM. Antiangiogenic synergism of integrin-targeted fumagillin nanoparticles and atorvastatin in atherosclerosis. J Am Coll Cardiol: Cardiovasc
Im. 2008;1:624 – 634.
Winter PM, Cai K, Chen J, Adair CR, Kiefer GE, Athey PS, Gaffney PJ,
Buff CE, Robertson JD, Caruthers SD, Wickline SA, Lanza GM.
Targeted PARACEST nanoparticle contrast agent for the detection of
fibrin. Magn Reson Med. 2006;56:1384 –1388.
Cyrus T, Abendschein DR, Caruthers SD, Harris TD, Glattauer V, Werkmeister
JA, Ramshaw JAM, Wickline SA, Lanza GM. MR three-dimensional molecular
imaging of intramural biomarkers with targeted nanoparticles. J Cardiovasc
Magn Reson. 2006;8:1–7.
Ahrens ET, Flores R, Xu H, Morel PA. In vivo imaging platform for
tracking immunotherapeutic cells. Nat Biotech. 2005;23:983–987.
Bulte J. Hot spot MRI emerges from the background. Nat Biotech.
2005;23:945–946.
Medarova Z, Pham W, Farrar C, Petkova V, Moore A. In vivo imaging
of siRNA delivery and silencing in tumors. Nat Med. 2007;13:372–377.
Mulder WJM, Douma K, Koning GA, van Zandvoort MA, Lutgens E,
Daemen MJ, Nicolay K, Strijkers GJ. Liposome-enhanced MRI of neointimal lesions in the apoE-KO mouse. Magn Res Med. 2006;55:
1170 –1174.
Lanza GM, Winter PM, Caruthers SD, Hughes MS, Cyrus T, Marsh
JN, Neubauer AM, Partlow KC, Wickline SA. Nanomedicine opportunities for cardiovascular disease with perfluorocarbon nanoparticles.
Nanomedicine. 2006;1:321–329.
Frias JC, Williams KJ, Fisher EA, Fayad ZA. Recombinant HDL-like
nanoparticles: a specific contrast agent for MRI of atherosclerotic
plaques. J Am Chem Soc. 2004;126:16316 –16317.
Anderson EA, Isaacman S, Peadbody DS, Wang EY, Canary JW, Kirshenbaum K. Viral nanoparticles donning a paramagnetic coat: conjugation
of MRI contrast agents to the MS2 capsid. Nano Lett. 2006;6:1160 –1164.
Sitharaman B, Kissell KR, Hartman KB, Tran LA, Baikalov A, Rusakova
I, Sun Y, Khant HA, Ludtke SJ, Chiu W, Laus S, Toth W, Helm L,
Merbach AE Superparamagnetic gadonanotubes are high-performance
MRI contrast agents. Chem Commun. 2005;3915–3917.
van Tilborg GAF, Mulder WJM, Deckers N, Storm G, Reutelingsperger
CPM, Strijkers GJ, Nicolay K. Annexin A5-functionalized bimodal
lipid-based contrast agents for the detection of apoptosis. Bioconjate
Chem. 2006;17:741–749.
Lipinski MJ, Briley-Saebo KC, Mani V, Fayad ZA. “Positive contrast”
inversion-recovery with oxide nanoparticles-resonant water suppression
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
Nanotechnology and Probe Design
999
magnetic resonance imaging: a change for the better? J Am Coll Cardiol.
2008;52:492– 494.
Briley-Saebo KC, Mulder WJM, Mani V, Hyafil F, Amirbekian V,
Aguinaldo JGS, Fisher EA, Fayad ZA. Magnetic resonance imaging of
vulnerable atherosclerotic plaques: Current imaging strategies and
molecular imaging probes. J Magn Reson Imaging. 2007;26:460 – 479.
Walczak P, Zhang J, Gilad AA, Kedziorek DA, Ruiz-Cabello J, Young
RG, Pittenger MF, Van Zijl PCM, Huang J, Bulte JWM. Dual-modality
monitoring of targeted intraarterial delivery of mesenchymal stem cells
after transient ischemia. Stroke. 2008;39:1569 –1574.
Leatherdale CA, Woo W-K, Mikulec FV, Bawendi MG. On the absorption
cross section of CdSe nanocrystal quantum dots. J Phys Chem B. 2002;116:
7619–7622.
Bruchez M, Jr., Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor Nanocrystals as Fluorescent Biological Labels. Science. 1998;281:
2013–2016.
Sukhanova A, Devy M, Venteo L, Kaplan H, Artemyev M, Oleinikov V,
Klinov D, Pluot M, Cohen JHM, Nabiev I. Biocompatible fluorescent
nanocrystals for immunolabeling of membrane proteins and cells. Anal
Biochem. 2004;324:60 – 67.
Sosnovik D, Weissleder R. Magnetic resonance and fluorescence based
molecular imaging technologies. Progress Drug Res. 2005;62:86 –114.
Hyafil F, Cornily JC, Feig JE, Gordon R, Vucic E, Amirbekian V, Fisher
EA, Fuster V, Feldman LJ, Fayad ZA. Noninvasive detection of macrophages using a nanoparticulate contrast agent for computed tomography.
Nat Med. 2007;13:636 – 641.
Rabin O, Perez JM, Grimm J, Wojtkiewicz G, Weissleder R. An X-ray
computed tomography imaging agent based on long-circulating bismuth
sulphide nanoparticles. Nat Mater. 2006;5:118 –122.
Hainfeld JF, Slatkin DN, Focella TM, Smilowitz HM. Gold nanoparticles: a new X-ray contrast agent. Brit J Radiol. 2006;79:248 –253.
Mukundan S, Ghaghada KB, Badea CT, Kao C-Y, Hedlund LW,
Provanzale JM, Johnson GA, Chen E, Bellamkonda RV, Annapragada A.
A liposomal nanoscale contrast agent for preclinical CT in mice. AJR.
2006;186:300 –307.
Kim D, Park S, Lee JH, Jeong YY, Jon S. Antibiofouling polymer-coated
gold nanoparticles as a contrast agent for in vivo x-ray computed
tomography imaging. J Am Chem Soc. 2007;129:7661–7665.
Lumbroso P, Dick CE. X-ray attenuation properties of radiographic
contrast media. Med Phys. 1987;14:752–758.
Weissleder R, Mahmood U. Molecular Imaging. Radiology. 2001;219:
316 –333.
Villanueva FS, Jankowski RJ, Klibanov S, Pina ML, Alber SM, Watkins
SC, Brandenburger GH, Wagner WR. Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial
cells. Circulation. 1998;98:1–5.
Klibanov AL. Microbubble contrast agents targeted ultrasound imaging
and ultrasound-assisted drug-delivery applications. Invest Radiol. 2006;
41:354 –362.
Huang SL, Hamilton AJ, Pozharski E, Nagaraj A, Klegerman ME,
McPherson DD, MacDonald RC. Physical correlates of the ultrasonic
reflectivity of lipid dispersions suitable as diagnostic contrast agents.
Ultrasound Med Biol. 2002;28:339 –348.
Lanza GM, Wallace KD, Scott MJ, Cacheris WP, Abendschein DR,
Christy DH, Sharkey AM, Miller JG, Gaffney PJ, Wickline SA. A novel
site-targeted ultrasonic contrast agent with broad biomedical application.
Circulation. 1996;94:3334 –3340.
Schiffelers RM, Banciu M, Metselaar JM, Storm G. Therapeutic application of long circulation liposomal glucocorticoids in auto-immune
diseases and cancer. J Liposome Res. 2006;16:185–194.
Berry CC, Wells S, Charles S, Curtis ASG. Dextran and albumin derivatised iron oxide nanoparticles: influence on fibroblasts in vitro. Biomaterials. 2003;24:4551– 4557.
Liu HL, Ko SP, Wu JH, Jung MH, Min JH, Lee JH, An BH, Kim YK.
One-pot polyol synthesis of monosize PVP-coated sub-5nm Fe3O4 nanoparticles for biomedical applications. J Magnetism Magnetic Mater.
2007;310:E815–E817.
Zhang Y, Kohler N, Zhang M. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials. 2002;23:1553–1561.
Selvan ST, Tan TT, Ying JY. Robust, non-cytotoxic, silica-coated CdSe
quantum dots with efficient photoluminescence. Adv Mater. 2005;17:
1620 –1625.
1000
Arterioscler Thromb Vasc Biol
July 2009
Downloaded from http://atvb.ahajournals.org/ by guest on May 5, 2017
51. Otsuka H, Nagasaki Y, Kataoka K. PEGylated nanoparticles for biological and pharmaceutical applications. Adv Drug Deliv. 2003;55:
403– 419.
52. Vallet-Regi M, Balas F, Arcos D. Mesoporous materials for drug
delivery. Angew Chem Int Ed. 2007;46:7548 –7558.
53. Chowdhury EH, Akaike T. Bio-functional inorganic materials: An
attractive branch of gene-based nano-medicine delivery for 21st century.
Curr Gene Ther. 2005;5:669 – 676.
54. Slowing II, Trewyn BG, Lin VSY. Mesoporous silica nanoparticles for
intracellular delivery of membrane-impermeable proteins. J Am Chem
Soc. 2007;129:8845– 8849.
55. van Schooneveld MM, Vucic E, Koole R, Zhou Y, Stocks J, Cormode
DP, Tang CY, Gordon R, Nicolay K, Meijerink A, Fayad ZA, Mulder
WJM. Improved biocompatibility and pharmacokinetics of silica nanoparticles by means of a lipid coating: a multimodality investigation. Nano
Lett. 2008;8:2517–2525.
56. Cormode DP, Mulder WJM, Fisher EA, Fayad ZA. Modified lipoproteins
as contrast agents for molecular imaging. Future Lipidol. 2007;2:
587–590.
57. Huang X, Bronstein LM, Retrum J, Dufort C, Tsvetkova I, Aniagyei S,
Stein B, Stucky G, McKenna B, Rennes N, Baxter D, Kao CC, Dragnea
B. Self-assembled virus-like particles with magnetic cores. Nano Lett.
2007;7:2407–2416.
58. Sun J, DuFort C, Daniel M-C, Murali A, Chen C, Gopinath K, Stein B,
De M, Rotello VM, Holzenburg A, Kao CC, Dragnea B. Core-controlled
polymorphism in virus-like particles. Proc Natl Acad Sci U S A. 2007;
104:1354 –1359.
59. Cormode DP, Skajaa T, van Schooneveld MM, Koole R, Jarzyna P,
Lobatto ME, Calcagno C, Barazza A, Gordon RE, Zanzonico P, Fisher
EA, Fayad ZA, Mulder WJM. Nanocrystal core high-density lipoproteins:
A multimodal molecular imaging contrast agent platform. Nano Lett.
2008;8:3715–3723.
60. Sanz J, Fayad ZA. Imaging of atherosclerotic cardiovascular disease.
Nature. 2008;451:953–957.
61. Kwon GS, Kataoka K. Block copolymer micelles as long-circulating drug
vehicles. Adv Drug Deliv Rev. 1995;16:295–309.
62. Ruehm SG, Corot C, Vogt P, Kolb S, Debatin JF. Magnetic resonance
imaging of atherosclerotic plaque with ultrasmall superparamagnetic particles of iron oxide in hyperlipidemic rabbits. Circulation. 2001;103:
415– 422.
63. Kang HW, Weissleder R, Bogdanov A. Targeting of MPEG-protected
polyamino acid carrier to human E-selectin in vitro. Amino Acids. 2002;
23:301–308.
64. Schellenberger EA, Sosnovik D, Weissleder R, Josephson L. Magneto/
optical annexin V, a multimodal protein. Bioconjugate Chem. 2004;15:
1062–1067.
65. Nahrendorf M, Jaffer FA, Kelly KA, Sosnovik DE, Aikawa E, Libby P,
Weissleder R. Noninvasive vascular cell adhesion molecule-1 imaging
identifies inflammatory activation of cells in atherosclerosis. Circulation.
2006;114:1504 –1511.
66. Javier DJ, Nitin N, Levy M, Ellington A, Richards-Kortum R. Aptamertargeted gold nanoparticles as molecular-specific contrast agents for
reflectance imaging. Bioconjugate Chem. 2008;19:1309 –1312.
67. Villanueva FS, Lu EX, Bowry S, Kilic S, Tom E, Wang JJ, Gretton J,
Pacella JJ, Wagner WR. Myocardial ischemic memory imaging with
molecular echocardiography. Circulation. 2007;115:345–352.
68. Zheng G, Chen J, Li H, Glickson JD. Rerouting lipoprotein nanoparticles
to selected alternate receptors for the targeted delivery of cancer diagnostic and therapeutic agents. Proc Natl Acad Sci U S A. 2005;102:
17757–17762.
69. Frangioni JV. New technologies for human cancer imaging. J Clin Oncol.
2008;26:4012– 4021.
70. Weissleder R, Kelly K, Sun EY, Shtatland T, Josephson L. Cell-specific
targeting of nanoparticles by multivalent attachment of small nanoparticles. Nat Biotech. 2005;23:1418 –1423.
71. Serda RE, Adolphi NL, Bisoffi M, Sillerud LO. Targeting and cellular
trafficking of magnetic nanoparticles for prostate cancer imaging. Mol
Imaging. 2007;6:277–288.
72. Paganelli G, Chinol M, Maggiolo M, Sidoli A, Corti A, Baroni S, Siccardi
AG. The three-step pretargeting approach reduces the human anti-mouse
antibody response in patients submitted to radioimmunoscintigraphy and
radioimmunotherapy. Eur J Nucl Med. 1997;24:350 –351.
73. Torchilin VP. Recent advances with liposomes as pharmaceutical
carriers. Nat Rev Drug Discov. 2005;4:145–160.
74. Gupta AK, Gupta M. Synthesis and surface engineering of iron oxide
nanoparticles for biomedical applications. Biomaterials. 2005;26:
3995– 4021.
75. Hostetler MJ, Wingate JE, Zhong CJ, Harris JE, Vachet RW, Clark MR,
Londono JD, Green SJ, Stokes JJ, Wignall GD, Glish GL, Porter MD,
Evans ND, Murray RW. Alkanethiolate gold cluster molecules with core
diameters from 1.5 to 5.2 nm: core and monolayer properties as a function
of core size. Langmuir. 1998;14:17–30.
76. van Helden AK, Jansen JW, Vrij A. Preparation and characterization of
spherical monodisperse silica dispersions in nonaqueous solvents. J Colloid
Interface Sci. 1980;78:354–368.
77. Murray CB, Norris DJ, Bawendi MG. Synthesis and characterization of
nearly monodisperse CdE (E⫽S, Se, Te) semiconductor nanocrystallites.
J Am Chem Soc. 1993;115:8706 – 8715.
78. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, Bawendi MG,
Frangioni JV. Renal clearance of quantum dots. Nat Biotech. 2007;25:
1165–1170.
79. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ,
Sundaresan G, Wu AM, Gambhir SS, Weiss S. Quantum dots for live
cells, in vivo imaging, and diagnostics. Science. 2005;307:538 –544.
80. McAteer MA, Sibson NR, von zur Muhlen C, Schneider JE, Lowe AS,
Warrick N, Channon KM, Anthony DC, Choudhury RP. In vivo magnetic
resonance imaging of acute brain inflammation using microparticles of
iron oxide. Nat Med. 2007;13:1253–1258.
81. Gleich B, Weizenecker J. Tomographic imaging using the nonlinear
response of magnetic particles. Nat Biotech. 2005;435:1214 –1217.
82. Gaumet M, Vargas A, Gurny R, Delie F. Nanoparticles for drug delivery:
the need for precision in reporting particle size parameters. Eur J Pharm
Biopharm. 2008;69:1–9.
83. Brust M, Walker M, Bethell D, Schiffrin DJ, Whyman R Synthesis of
thiol-derivatised gold nanoparticles in a two-phase liquid–liquid system.
Chem Commun. 1994;801– 802.
84. Kim BS, Qiu JM, Wang JP, Taton TA. Magnetomicelles: composite
nanostructures from magnetic nanoparticles and cross-linked amphiphilic
block copolymers. Nano Lett. 2005;5:1987–1991.
85. Yu WW, Chang E, Falkner JC, Zhang J, Al-Somali AM, Sayes CM,
Johns J, Drezek R, Colvin VL. Forming biocompatible and nonaggregated nanocrystals in water using amphiphilic polymers. J Am Chem
Soc. 2007;129:2871–2879.
86. McCarthy JR, Kelly KA, Sun EY, Weissleder R. Targeted delivery of
multifunctional magnetic nanoparticles. Nanomedicine. 2007;2:153–167.
87. http://www.fda.gov/nanotechnology/.
88. Davies JC Nanotechnology Oversight: An Agenda for the New Administration. Woodrow Wilson International Center for Scholars, Project on
Emerging Nanotechnologies. 2008;PEN 13.
89. http://nanoehsalliance.org/.
90. Shaw SY, Westly EC, Pittet MJ, Subramanian A, Schreiber SL,
Weissleder R. Perturbational profiling of nanomaterial biologic activity.
Proc Natl Acad Sci U S A. 2008;105:7387–7392.
91. Mancini MC, Kairdolf BA, Smith AM, Nie S. Oxidative quenching and
degradation of polymer-encapsulated quantum dots: new insights into the
long-term fate and toxicity of nanocrystals in vivo. J Am Chem Soc.
2008;130:10836 –10837.
Downloaded from http://atvb.ahajournals.org/ by guest on May 5, 2017
Nanotechnology in Medical Imaging: Probe Design and Applications
David P. Cormode, Torjus Skajaa, Zahi A. Fayad and Willem J.M. Mulder
Arterioscler Thromb Vasc Biol. 2009;29:992-1000; originally published online December 4,
2008;
doi: 10.1161/ATVBAHA.108.165506
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